Time-Resolved Fluorescence Wiki

separation_of_2_species_with

Anonymous (anonymous@undisclosed.example.com) — 2014-06-18T12:22:45+00:00

2014/05/23 07:45		current
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	{{tag> howto tutorial FCS FLCS analysis SymPhoTime}}		{{tag> howto tutorial FCS FLCS analysis SymPhoTime}}

-	~~TOC~~	+	~~NOTOC~~

-	====== Separation of 2 Species with Different Lifetimes Using the FLCS Analysis ======	+	====== Separation of 2 Species with Different Lifetimes Using FLCS ======


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	This tutorial shows step-by-step, how the FLCS analysis can be used to calculate separate autocorrelation curves for the two components of a mixture of ATTO655 and Cy5 based on their different lifetimes.		This tutorial shows step-by-step, how the FLCS analysis can be used to calculate separate autocorrelation curves for the two components of a mixture of ATTO655 and Cy5 based on their different lifetimes.
		+
		+	{{ youtube>xx-WzT5TgqA?large }}

	===== Background Information =====		===== Background Information =====

-
-	Note:\\
	The two fluorophores ATTO655 (ATTO-Tec, Germany) and Cy5 (GE Healthcare) have almost identical spectral properties and are thus excited with the same wavelength (e.g. 635 nm) and detected at the same spectral range. As also their molecular weight is comparable, the diffusion constant is too similar to distinguish between both dyes in a mixture by means of standard FCS.\\		The two fluorophores ATTO655 (ATTO-Tec, Germany) and Cy5 (GE Healthcare) have almost identical spectral properties and are thus excited with the same wavelength (e.g. 635 nm) and detected at the same spectral range. As also their molecular weight is comparable, the diffusion constant is too similar to distinguish between both dyes in a mixture by means of standard FCS.\\
	Nevertheless, the autocorrelation curves of both dyes differ in the microsecond range. ATTO655 shows practically no photophysical fluctuations. Therefore, FCS curves of ATTO655 can be fitted with the simplest 3D diffusion model, making it very suited for calibration. Cy5 on the contrary, exhibits a distinct bunching term on its FCS trace originating from photophysical processes, i.e. cis/trans isomerization.\\		Nevertheless, the autocorrelation curves of both dyes differ in the microsecond range. ATTO655 shows practically no photophysical fluctuations. Therefore, FCS curves of ATTO655 can be fitted with the simplest 3D diffusion model, making it very suited for calibration. Cy5 on the contrary, exhibits a distinct bunching term on its FCS trace originating from photophysical processes, i.e. cis/trans isomerization.\\
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	* Click on "Save result" to save this curve. Rename the generated file as "Cy5-FLCS".		* Click on "Save result" to save this curve. Rename the generated file as "Cy5-FLCS".
	* These curves can now be fitted by selecting "Transfer to fit". The process of how to fit a FCS curve is explained in the tutorial [[howto:Calculate and Fit FCS Traces with the FCS Script]].		* These curves can now be fitted by selecting "Transfer to fit". The process of how to fit a FCS curve is explained in the tutorial [[howto:Calculate and Fit FCS Traces with the FCS Script]].
-

syntax

Anonymous (anonymous@undisclosed.example.com) — 2024-10-11T08:13:38+00:00

2026/04/11 16:45	current
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	+	====== Formatting Syntax ======
	+
	+	[[doku>DokuWiki]] supports some simple markup language, which tries to make the datafiles to be as readable as possible. This page contains all possible syntax you may use when editing the pages. Simply have a look at the source of this page by pressing "Edit this page". If you want to try something, just use the [[playground:playground\|playground]] page. The simpler markup is easily accessible via [[doku>toolbar\|quickbuttons]], too.
	+
	+	===== Basic Text Formatting =====
	+
	+	DokuWiki supports bold, //italic//, __underlined__ and ''monospaced'' texts. Of course you can __//''combine''//__ all these.
	+
	+	DokuWiki supports bold, //italic//, __underlined__ and ''monospaced'' texts.
	+	Of course you can __//''combine''//__ all these.
	+
	+	You can use <sub>subscript</sub> and <sup>superscript</sup>, too.
	+
	+	You can use <sub>subscript</sub> and <sup>superscript</sup>, too.
	+
	+	You can mark something as <del>deleted</del> as well.
	+
	+	You can mark something as <del>deleted</del> as well.
	+
	+	Paragraphs are created from blank lines. If you want to force a newline without a paragraph, you can use two backslashes followed by a whitespace or the end of line.
	+
	+	This is some text with some linebreaks\\ Note that the
	+	two backslashes are only recognized at the end of a line\\
	+	or followed by\\ a whitespace \\this happens without it.
	+
	+	This is some text with some linebreaks\\ Note that the
	+	two backslashes are only recognized at the end of a line\\
	+	or followed by\\ a whitespace \\this happens without it.
	+
	+	You should use forced newlines only if really needed.
	+
	+	===== Links =====
	+
	+	DokuWiki supports multiple ways of creating links.
	+
	+	==== External ====
	+
	+	External links are recognized automagically: http://www.google.com or simply www.google.com - You can set the link text as well: [[http://www.google.com\|This Link points to google]]. Email addresses like this one: <andi@splitbrain.org> are recognized, too.
	+
	+	DokuWiki supports multiple ways of creating links. External links are recognized
	+	automagically: http://www.google.com or simply www.google.com - You can set
	+	link text as well: [[http://www.google.com\|This Link points to google]]. Email
	+	addresses like this one: <andi@splitbrain.org> are recognized, too.
	+
	+	==== Internal ====
	+
	+	Internal links are created by using square brackets. You can either just give a [[pagename]] or use an additional [[pagename\|link text]].
	+
	+	Internal links are created by using square brackets. You can either just give
	+	a [[pagename]] or use an additional [[pagename\|link text]].
	+
	+	[[doku>pagename\|Wiki pagenames]] are converted to lowercase automatically, special characters are not allowed.
	+
	+	You can use [[some:namespaces]] by using a colon in the pagename.
	+
	+	You can use [[some:namespaces]] by using a colon in the pagename.
	+
	+	For details about namespaces see [[doku>namespaces]].
	+
	+	Linking to a specific section is possible, too. Just add the section name behind a hash character as known from HTML. This links to [[syntax#internal\|this Section]].
	+
	+	This links to [[syntax#internal\|this Section]].
	+
	+	Notes:
	+
	+	* Links to [[syntax\|existing pages]] are shown in a different style from [[nonexisting]] ones.
	+	* DokuWiki does not use [[wp>CamelCase]] to automatically create links by default, but this behavior can be enabled in the [[doku>config]] file. Hint: If DokuWiki is a link, then it's enabled.
	+	* When a section's heading is changed, its bookmark changes, too. So don't rely on section linking too much.
	+
	+	==== Interwiki ====
	+
	+	DokuWiki supports [[doku>Interwiki]] links. These are quick links to other Wikis. For example this is a link to Wikipedia's page about Wikis: [[wp>Wiki]].
	+
	+	DokuWiki supports [[doku>Interwiki]] links. These are quick links to other Wikis.
	+	For example this is a link to Wikipedia's page about Wikis: [[wp>Wiki]].
	+
	+	==== Windows Shares ====
	+
	+	Windows shares like [[\\server\share\|this]] are recognized, too. Please note that these only make sense in a homogeneous user group like a corporate [[wp>Intranet]].
	+
	+	Windows Shares like [[\\server\share\|this]] are recognized, too.
	+
	+	Notes:
	+
	+	* For security reasons direct browsing of windows shares only works in Microsoft Internet Explorer per default (and only in the "local zone").
	+	* For Mozilla and Firefox it can be enabled through different workaround mentioned in the [[http://kb.mozillazine.org/Links_to_local_pages_do_not_work\|Mozilla Knowledge Base]]. However, there will still be a JavaScript warning about trying to open a Windows Share. To remove this warning (for all users), put the following line in ''conf/lang/en/lang.php'' (more details at [[doku>localization#changing_some_localized_texts_and_strings_in_your_installation\|localization]]): <code - conf/lang/en/lang.php>
	+	<?php
	+	/**
	+	* Customization of the english language file
	+	* Copy only the strings that needs to be modified
	+	*/
	+	$lang['js']['nosmblinks'] = '';
	+	</code>
	+
	+	==== Image Links ====
	+
	+	You can also use an image to link to another internal or external page by combining the syntax for links and [[#images_and_other_files\|images]] (see below) like this:
	+
	+	[[http://php.net\|{{wiki:dokuwiki-128.png}}]]
	+
	+	[[http://php.net\|{{wiki:dokuwiki-128.png}}]]
	+
	+	Please note: The image formatting is the only formatting syntax accepted in link names.
	+
	+	The whole [[#images_and_other_files\|image]] and [[#links\|link]] syntax is supported (including image resizing, internal and external images and URLs and interwiki links).
	+
	+	===== Footnotes =====
	+
	+	You can add footnotes ((This is a footnote)) by using double parentheses.
	+
	+	You can add footnotes ((This is a footnote)) by using double parentheses.
	+
	+	===== Sectioning =====
	+
	+	You can use up to five different levels of headlines to structure your content. If you have more than three headlines, a table of contents is generated automatically -- this can be disabled by including the string ''<nowiki>~~NOTOC~~</nowiki>'' in the document.
	+
	+	==== Headline Level 3 ====
	+	=== Headline Level 4 ===
	+	== Headline Level 5 ==
	+
	+	==== Headline Level 3 ====
	+	=== Headline Level 4 ===
	+	== Headline Level 5 ==
	+
	+	By using four or more dashes, you can make a horizontal line:
	+
	+	----
	+
	+	===== Media Files =====
	+
	+	You can include external and internal [[doku>images\|images, videos and audio files]] with curly brackets. Optionally you can specify the size of them.
	+
	+	Real size: {{wiki:dokuwiki-128.png}}
	+
	+	Resize to given width: {{wiki:dokuwiki-128.png?50}}
	+
	+	Resize to given width and height((when the aspect ratio of the given width and height doesn't match that of the image, it will be cropped to the new ratio before resizing)): {{wiki:dokuwiki-128.png?200x50}}
	+
	+	Resized external image: {{https://www.php.net/images/php.gif?200x50}}
	+
	+	Real size: {{wiki:dokuwiki-128.png}}
	+	Resize to given width: {{wiki:dokuwiki-128.png?50}}
	+	Resize to given width and height: {{wiki:dokuwiki-128.png?200x50}}
	+	Resized external image: {{https://www.php.net/images/php.gif?200x50}}
	+
	+
	+	By using left or right whitespaces you can choose the alignment.
	+
	+	{{ wiki:dokuwiki-128.png}}
	+
	+	{{wiki:dokuwiki-128.png }}
	+
	+	{{ wiki:dokuwiki-128.png }}
	+
	+	{{ wiki:dokuwiki-128.png}}
	+	{{wiki:dokuwiki-128.png }}
	+	{{ wiki:dokuwiki-128.png }}
	+
	+	Of course, you can add a title (displayed as a tooltip by most browsers), too.
	+
	+	{{ wiki:dokuwiki-128.png \|This is the caption}}
	+
	+	{{ wiki:dokuwiki-128.png \|This is the caption}}
	+
	+	For linking an image to another page see [[#Image Links]] above.
	+
	+	==== Supported Media Formats ====
	+
	+	DokuWiki can embed the following media formats directly.
	+
	+	\| Image \| ''gif'', ''jpg'', ''png'' \|
	+	\| Video \| ''webm'', ''ogv'', ''mp4'' \|
	+	\| Audio \| ''ogg'', ''mp3'', ''wav'' \|
	+	\| Flash \| ''swf'' \|
	+
	+	If you specify a filename that is not a supported media format, then it will be displayed as a link instead.
	+
	+	By adding ''?linkonly'' you provide a link to the media without displaying it inline
	+
	+	{{wiki:dokuwiki-128.png?linkonly}}
	+
	+	{{wiki:dokuwiki-128.png?linkonly}} This is just a link to the image.
	+
	+	==== Fallback Formats ====
	+
	+	Unfortunately not all browsers understand all video and audio formats. To mitigate the problem, you can upload your file in different formats for maximum browser compatibility.
	+
	+	For example consider this embedded mp4 video:
	+
	+	{{video.mp4\|A funny video}}
	+
	+	When you upload a ''video.webm'' and ''video.ogv'' next to the referenced ''video.mp4'', DokuWiki will automatically add them as alternatives so that one of the three files is understood by your browser.
	+
	+	Additionally DokuWiki supports a "poster" image which will be shown before the video has started. That image needs to have the same filename as the video and be either a jpg or png file. In the example above a ''video.jpg'' file would work.
	+
	+	===== Lists =====
	+
	+	Dokuwiki supports ordered and unordered lists. To create a list item, indent your text by two spaces and use a ''*'' for unordered lists or a ''-'' for ordered ones.
	+
	+	* This is a list
	+	* The second item
	+	* You may have different levels
	+	* Another item
	+
	+	- The same list but ordered
	+	- Another item
	+	- Just use indention for deeper levels
	+	- That's it
	+
	+	<code>
	+	* This is a list
	+	* The second item
	+	* You may have different levels
	+	* Another item
	+
	+	- The same list but ordered
	+	- Another item
	+	- Just use indention for deeper levels
	+	- That's it
	+	</code>
	+
	+	Also take a look at the [[doku>faq:lists\|FAQ on list items]].
	+
	+	===== Text Conversions =====
	+
	+	DokuWiki can convert certain pre-defined characters or strings into images or other text or HTML.
	+
	+	The text to image conversion is mainly done for smileys. And the text to HTML conversion is used for typography replacements, but can be configured to use other HTML as well.
	+
	+	==== Text to Image Conversions ====
	+
	+	DokuWiki converts commonly used [[wp>emoticon]]s to their graphical equivalents. Those [[doku>Smileys]] and other images can be configured and extended. Here is an overview of Smileys included in DokuWiki:
	+
	+	* 8-) %% 8-) %%
	+	* 8-O %% 8-O %%
	+	* :-( %% :-( %%
	+	* :-) %% :-) %%
	+	* =) %% =) %%
	+	* :-/ %% :-/ %%
	+	* :-\ %% :-\ %%
	+	* :-? %% :-? %%
	+	* :-D %% :-D %%
	+	* :-P %% :-P %%
	+	* :-O %% :-O %%
	+	* :-X %% :-X %%
	+	* :-\| %% :-\| %%
	+	* ;-) %% ;-) %%
	+	* ^_^ %% ^_^ %%
	+	* m( %% m( %%
	+	* :?: %% :?: %%
	+	* :!: %% :!: %%
	+	* LOL %% LOL %%
	+	* FIXME %% FIXME %%
	+	* DELETEME %% DELETEME %%
	+
	+	==== Text to HTML Conversions ====
	+
	+	Typography: [[DokuWiki]] can convert simple text characters to their typographically correct entities. Here is an example of recognized characters.
	+
	+	-> <- <-> => <= <=> >> << -- --- 640x480 (c) (tm) (r)
	+	"He thought 'It's a man's world'..."
	+
	+	<code>
	+	-> <- <-> => <= <=> >> << -- --- 640x480 (c) (tm) (r)
	+	"He thought 'It's a man's world'..."
	+	</code>
	+
	+	The same can be done to produce any kind of HTML, it just needs to be added to the [[doku>entities\|pattern file]].
	+
	+	There are three exceptions which do not come from that pattern file: multiplication entity (640x480), 'single' and "double quotes". They can be turned off through a [[doku>config:typography\|config option]].
	+
	+	===== Quoting =====
	+
	+	Some times you want to mark some text to show it's a reply or comment. You can use the following syntax:
	+
	+	<code>
	+	I think we should do it
	+
	+	> No we shouldn't
	+
	+	>> Well, I say we should
	+
	+	> Really?
	+
	+	>> Yes!
	+
	+	>>> Then lets do it!
	+	</code>
	+
	+	I think we should do it
	+
	+	> No we shouldn't
	+
	+	>> Well, I say we should
	+
	+	> Really?
	+
	+	>> Yes!
	+
	+	>>> Then lets do it!
	+
	+	===== Tables =====
	+
	+	DokuWiki supports a simple syntax to create tables.
	+
	+	^ Heading 1 ^ Heading 2 ^ Heading 3 ^
	+	\| Row 1 Col 1 \| Row 1 Col 2 \| Row 1 Col 3 \|
	+	\| Row 2 Col 1 \| some colspan (note the double pipe) \|\|
	+	\| Row 3 Col 1 \| Row 3 Col 2 \| Row 3 Col 3 \|
	+
	+	Table rows have to start and end with a ''\|'' for normal rows or a ''^'' for headers.
	+
	+	^ Heading 1 ^ Heading 2 ^ Heading 3 ^
	+	\| Row 1 Col 1 \| Row 1 Col 2 \| Row 1 Col 3 \|
	+	\| Row 2 Col 1 \| some colspan (note the double pipe) \|\|
	+	\| Row 3 Col 1 \| Row 3 Col 2 \| Row 3 Col 3 \|
	+
	+	To connect cells horizontally, just make the next cell completely empty as shown above. Be sure to have always the same amount of cell separators!
	+
	+	Vertical tableheaders are possible, too.
	+
	+	\| ^ Heading 1 ^ Heading 2 ^
	+	^ Heading 3 \| Row 1 Col 2 \| Row 1 Col 3 \|
	+	^ Heading 4 \| no colspan this time \| \|
	+	^ Heading 5 \| Row 2 Col 2 \| Row 2 Col 3 \|
	+
	+	As you can see, it's the cell separator before a cell which decides about the formatting:
	+
	+	\| ^ Heading 1 ^ Heading 2 ^
	+	^ Heading 3 \| Row 1 Col 2 \| Row 1 Col 3 \|
	+	^ Heading 4 \| no colspan this time \| \|
	+	^ Heading 5 \| Row 2 Col 2 \| Row 2 Col 3 \|
	+
	+	You can have rowspans (vertically connected cells) by adding ''%%:::%%'' into the cells below the one to which they should connect.
	+
	+	^ Heading 1 ^ Heading 2 ^ Heading 3 ^
	+	\| Row 1 Col 1 \| this cell spans vertically \| Row 1 Col 3 \|
	+	\| Row 2 Col 1 \| ::: \| Row 2 Col 3 \|
	+	\| Row 3 Col 1 \| ::: \| Row 2 Col 3 \|
	+
	+	Apart from the rowspan syntax those cells should not contain anything else.
	+
	+	^ Heading 1 ^ Heading 2 ^ Heading 3 ^
	+	\| Row 1 Col 1 \| this cell spans vertically \| Row 1 Col 3 \|
	+	\| Row 2 Col 1 \| ::: \| Row 2 Col 3 \|
	+	\| Row 3 Col 1 \| ::: \| Row 2 Col 3 \|
	+
	+	You can align the table contents, too. Just add at least two whitespaces at the opposite end of your text: Add two spaces on the left to align right, two spaces on the right to align left and two spaces at least at both ends for centered text.
	+
	+	^ Table with alignment ^^^
	+	\| right\| center \|left \|
	+	\|left \| right\| center \|
	+	\| xxxxxxxxxxxx \| xxxxxxxxxxxx \| xxxxxxxxxxxx \|
	+
	+	This is how it looks in the source:
	+
	+	^ Table with alignment ^^^
	+	\| right\| center \|left \|
	+	\|left \| right\| center \|
	+	\| xxxxxxxxxxxx \| xxxxxxxxxxxx \| xxxxxxxxxxxx \|
	+
	+	Note: Vertical alignment is not supported.
	+
	+	===== No Formatting =====
	+
	+	If you need to display text exactly like it is typed (without any formatting), enclose the area either with ''%%<nowiki>%%'' tags or even simpler, with double percent signs ''<nowiki>%%</nowiki>''.
	+
	+	<nowiki>
	+	This is some text which contains addresses like this: http://www.splitbrain.org and formatting, but nothing is done with it.
	+	</nowiki>
	+	The same is true for %%//__this__ text// with a smiley ;-)%%.
	+
	+	<nowiki>
	+	This is some text which contains addresses like this: http://www.splitbrain.org and formatting, but nothing is done with it.
	+	</nowiki>
	+	The same is true for %%//__this__ text// with a smiley ;-)%%.
	+
	+	===== Code Blocks =====
	+
	+	You can include code blocks into your documents by either indenting them by at least two spaces (like used for the previous examples) or by using the tags ''%%<code>%%'' or ''%%<file>%%''.
	+
	+	This is text is indented by two spaces.
	+
	+	<code>
	+	This is preformatted code all spaces are preserved: like <-this
	+	</code>
	+
	+	<file>
	+	This is pretty much the same, but you could use it to show that you quoted a file.
	+	</file>
	+
	+	Those blocks were created by this source:
	+
	+	This is text is indented by two spaces.
	+
	+	<code>
	+	This is preformatted code all spaces are preserved: like <-this
	+	</code>
	+
	+	<file>
	+	This is pretty much the same, but you could use it to show that you quoted a file.
	+	</file>
	+
	+	==== Syntax Highlighting ====
	+
	+	[[wiki:DokuWiki]] can highlight sourcecode, which makes it easier to read. It uses the [[http://qbnz.com/highlighter/\|GeSHi]] Generic Syntax Highlighter -- so any language supported by GeSHi is supported. The syntax uses the same code and file blocks described in the previous section, but this time the name of the language syntax to be highlighted is included inside the tag, e.g. ''<nowiki><code java></nowiki>'' or ''<nowiki><file java></nowiki>''.
	+
	+	<code java>
	+	/**
	+	* The HelloWorldApp class implements an application that
	+	* simply displays "Hello World!" to the standard output.
	+	*/
	+	class HelloWorldApp {
	+	public static void main(String[] args) {
	+	System.out.println("Hello World!"); //Display the string.
	+	}
	+	}
	+	</code>
	+
	+	The following language strings are currently recognized: //4cs 6502acme 6502kickass 6502tasm 68000devpac abap actionscript3 actionscript ada aimms algol68 apache applescript apt_sources arm asm asp asymptote autoconf autohotkey autoit avisynth awk bascomavr bash basic4gl batch bf biblatex bibtex blitzbasic bnf boo caddcl cadlisp ceylon cfdg cfm chaiscript chapel cil c_loadrunner clojure c_mac cmake cobol coffeescript c cpp cpp-qt cpp-winapi csharp css cuesheet c_winapi dart dcl dcpu16 dcs delphi diff div dos dot d ecmascript eiffel email epc e erlang euphoria ezt f1 falcon fo fortran freebasic freeswitch fsharp gambas gdb genero genie gettext glsl gml gnuplot go groovy gwbasic haskell haxe hicest hq9plus html html4strict html5 icon idl ini inno intercal io ispfpanel java5 java javascript jcl j jquery julia kixtart klonec klonecpp kotlin latex lb ldif lisp llvm locobasic logtalk lolcode lotusformulas lotusscript lscript lsl2 lua m68k magiksf make mapbasic mathematica matlab mercury metapost mirc mk-61 mmix modula2 modula3 mpasm mxml mysql nagios netrexx newlisp nginx nimrod nsis oberon2 objc objeck ocaml-brief ocaml octave oobas oorexx oracle11 oracle8 oxygene oz parasail parigp pascal pcre perl6 perl per pf phix php-brief php pic16 pike pixelbender pli plsql postgresql postscript povray powerbuilder powershell proftpd progress prolog properties providex purebasic pycon pys60 python qbasic qml q racket rails rbs rebol reg rexx robots roff rpmspec rsplus ruby rust sas sass scala scheme scilab scl sdlbasic smalltalk smarty spark sparql sql sshconfig standardml stonescript swift systemverilog tclegg tcl teraterm texgraph text thinbasic tsql twig typoscript unicon upc urbi uscript vala vbnet vb vbscript vedit verilog vhdl vim visualfoxpro visualprolog whitespace whois winbatch wolfram xbasic xml xojo xorg_conf xpp yaml z80 zxbasic//
	+
	+	There are additional [[doku>syntax_highlighting\|advanced options]] available for syntax highlighting, such as highlighting lines or adding line numbers.
	+
	+	==== Downloadable Code Blocks ====
	+
	+	When you use the ''%%<code>%%'' or ''%%<file>%%'' syntax as above, you might want to make the shown code available for download as well. You can do this by specifying a file name after language code like this:
	+
	+	<code>
	+	<file php myexample.php>
	+	<?php echo "hello world!"; ?>
	+	</file>
	+	</code>
	+
	+	<file php myexample.php>
	+	<?php echo "hello world!"; ?>
	+	</file>
	+
	+	If you don't want any highlighting but want a downloadable file, specify a dash (''-'') as the language code: ''%%<code - myfile.foo>%%''.
	+
	+	===== RSS/ATOM Feed Aggregation =====
	+	[[DokuWiki]] can integrate data from external XML feeds. For parsing the XML feeds, [[http://simplepie.org/\|SimplePie]] is used. All formats understood by SimplePie can be used in DokuWiki as well. You can influence the rendering by multiple additional space separated parameters:
	+
	+	^ Parameter ^ Description ^
	+	\| any number \| will be used as maximum number items to show, defaults to 8 \|
	+	\| reverse \| display the last items in the feed first \|
	+	\| author \| show item authors names \|
	+	\| date \| show item dates \|
	+	\| description\| show the item description. All HTML tags will be stripped \|
	+	\| nosort \| do not sort the items in the feed \|
	+	\| //n//[dhm] \| refresh period, where d=days, h=hours, m=minutes. (e.g. 12h = 12 hours). \|
	+
	+	The refresh period defaults to 4 hours. Any value below 10 minutes will be treated as 10 minutes. [[wiki:DokuWiki]] will generally try to supply a cached version of a page, obviously this is inappropriate when the page contains dynamic external content. The parameter tells [[wiki:DokuWiki]] to re-render the page if it is more than //refresh period// since the page was last rendered.
	+
	+	By default the feed will be sorted by date, newest items first. You can sort it by oldest first using the ''reverse'' parameter, or display the feed as is with ''nosort''.
	+
	+	Example:
	+
	+	{{rss>http://slashdot.org/index.rss 5 author date 1h }}
	+
	+	{{rss>http://slashdot.org/index.rss 5 author date 1h }}
	+
	+
	+	===== Control Macros =====
	+
	+	Some syntax influences how DokuWiki renders a page without creating any output it self. The following control macros are availble:
	+
	+	^ Macro ^ Description \|
	+	\| %%~~NOTOC~~%% \| If this macro is found on the page, no table of contents will be created \|
	+	\| %%~~NOCACHE~~%% \| DokuWiki caches all output by default. Sometimes this might not be wanted (eg. when the %%<php>%% syntax above is used), adding this macro will force DokuWiki to rerender a page on every call \|
	+
	+	===== Syntax Plugins =====
	+
	+	DokuWiki's syntax can be extended by [[doku>plugins\|Plugins]]. How the installed plugins are used is described on their appropriate description pages. The following syntax plugins are available in this particular DokuWiki installation:
	+
	+	~~INFO:syntaxplugins~~

calculate_ratiometric_single_pair_fret_distributions_using_the_pie-fret_script

Anonymous (anonymous@undisclosed.example.com) — 2015-11-03T14:12:40+00:00

2014/06/18 15:58		current
Line 1:		Line 1:
	{{tag> tutorial howto FRET PIE analysis time-trace SymPhoTime}}		{{tag> tutorial howto FRET PIE analysis time-trace SymPhoTime}}
-	~~TOC~~	+	~~NOTOC~~

	====== Calculate Ratiometric Single Pair FRET Distributions Using PIE-FRET ======		====== Calculate Ratiometric Single Pair FRET Distributions Using PIE-FRET ======
Line 10:		Line 10:

	This tutorial shows step-by-step, how to calculate a FRET histogram from single [[glossary:fret\|FRET]]pairs detected under single molecule conditions using pulsed interleaved excitation (PIE). The script requires a spectrally resolved fluorescence time trace, in which donor and acceptor have been excited alternately. As a result, the distribution of FRET efficiencies of different molecules is shown which can be used to detect subpopulations.		This tutorial shows step-by-step, how to calculate a FRET histogram from single [[glossary:fret\|FRET]]pairs detected under single molecule conditions using pulsed interleaved excitation (PIE). The script requires a spectrally resolved fluorescence time trace, in which donor and acceptor have been excited alternately. As a result, the distribution of FRET efficiencies of different molecules is shown which can be used to detect subpopulations.
		+
		+	{{ youtube>cSAbgJpZhrY?large }}

	Note: The script requires a time trace containing the fluorescence of two spectrally separated channels, one channel mainly detecting the fluorescence of the donor dye and the other channel detecting mainly the fluorescence of the acceptor dye.\\		Note: The script requires a time trace containing the fluorescence of two spectrally separated channels, one channel mainly detecting the fluorescence of the donor dye and the other channel detecting mainly the fluorescence of the acceptor dye.\\
	The time traces can be acquired from single molecule events in a diluted solution, where the passages of single molecules are registered as "bursts". Alternatively, traces obtained from a stationary single FRET pair can be analyzed, e.g. to observe conformational changes.\\		The time traces can be acquired from single molecule events in a diluted solution, where the passages of single molecules are registered as "bursts". Alternatively, traces obtained from a stationary single FRET pair can be analyzed, e.g. to observe conformational changes.\\
-	To record such traces, usually single molecule sensitive detectors as [[glossary:spad\|SPAD]] or Hybrid-PMA are necessary to successfully detect single molecule fluorescence.\\	+	To record such traces, usually single molecule sensitive detectors as [[glossary:spad\|SPAD]] or [[glossary: Hybrid PMT]]s are necessary to successfully detect single molecule fluorescence.\\
	For the pulsed interleaved excitation ([[glossary:pie\|PIE]]) analysis of the script, pulsed excitation using two lasers is required, one laser exciting the donor, the other the acceptor dye. Usually, this is achieved using the multichannel laser driver PDL828 “Sepia II” in combination with two pulsed diode lasers.		For the pulsed interleaved excitation ([[glossary:pie\|PIE]]) analysis of the script, pulsed excitation using two lasers is required, one laser exciting the donor, the other the acceptor dye. Usually, this is achieved using the multichannel laser driver PDL828 “Sepia II” in combination with two pulsed diode lasers.

Line 162:		Line 164:

	Note: Further information about the sample used in this measurement can be found under: J.L. Fiore et. al., Enthalpy-driven RNA-folding: single-molecule thermodynamics of tetraloop-receptor tertiary interaction, Biochemistry (2009), 48(11), 2550-8. ([[http://pubs.acs.org/doi/abs/10.1021/bi8019788]]).		Note: Further information about the sample used in this measurement can be found under: J.L. Fiore et. al., Enthalpy-driven RNA-folding: single-molecule thermodynamics of tetraloop-receptor tertiary interaction, Biochemistry (2009), 48(11), 2550-8. ([[http://pubs.acs.org/doi/abs/10.1021/bi8019788]]).
-

pattern_matching

Anonymous (anonymous@undisclosed.example.com) — 2014-06-18T13:26:07+00:00

2014/05/06 14:41		current
Line 1:		Line 1:
-	{{tag> howto imaging FLIM pattern-matching tutorial analysis}}	+	{{tag> howto imaging FLIM pattern-matching tutorial analysis SymPhoTime}}

	~~NOTOC~~		~~NOTOC~~
Line 10:		Line 10:

	Pattern Matching Analysis by decomposing a recorded image into different user-defined patterns. Display of the calculated data using an RGB false color model. The calculated amplitudes of the first three defined patterns are depicted in three different colors for each pixel.		Pattern Matching Analysis by decomposing a recorded image into different user-defined patterns. Display of the calculated data using an RGB false color model. The calculated amplitudes of the first three defined patterns are depicted in three different colors for each pixel.
		+
		+	{{ youtube>L68IgRbNSLE?large }}

	==== Open an Image ====		==== Open an Image ====

flim_fret_calculation_for_multi_exponential_donors

Anonymous (anonymous@undisclosed.example.com) — 2023-11-21T10:33:47+00:00

2020/05/12 12:39		current
Line 3:		Line 3:
	~~NOTOC~~		~~NOTOC~~

-	====== FLIM FRET Calculation for Multi Exponential Donors ======	+	====== FLIM-FRET Calculation for Multi Exponential Donors ======


Line 25:		Line 25:

	Note: This Step-by-Step tutorial was written for SymPhoTime version 2.5. There might be slight differences compared to other software versions. For example the way of setting an intensity threshold has changed. In the video the former version is shown.		Note: This Step-by-Step tutorial was written for SymPhoTime version 2.5. There might be slight differences compared to other software versions. For example the way of setting an intensity threshold has changed. In the video the former version is shown.
-	==== Determine the donor only lifetime using the FLIM script ====	+	==== Determine the donor only lifetime using the FLIM analysis ====


Line 53:		Line 53:
	Response:\\		Response:\\
	The FLIM script is applied to the file ''CENP-labelled_cells_for_FRET.ptu''. Thereby, a new Window opens:		The FLIM script is applied to the file ''CENP-labelled_cells_for_FRET.ptu''. Thereby, a new Window opens:
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_5.png?600 }}	+	{{ :howto:flim-fret-multiexpd_analysis_flim.png \|}}

	Note:\\		Note:\\
Line 64:		Line 64:

	* As data channels, check only Channel 2, as in this channel the donor emission has been recorded.		* As data channels, check only Channel 2, as in this channel the donor emission has been recorded.
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_6.png }}	+	{{ :howto:flim-fret-multiexpd_ch2.png?400 \|}}

	* Press "Calculate Fast FLIM" to update the image.		* Press "Calculate Fast FLIM" to update the image.
	Response:\\		Response:\\
	The image and the graphs are recalculated, using photons only from channel 2.		The image and the graphs are recalculated, using photons only from channel 2.
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_7.png?600 }}	+	{{ :writingroom:flim-fret-multiexpd_fastflim.png \|}}

-	* To enhance the lifetime and intensity contrast, set in the image the intensity from 1 count to 30 counts and the lifetime from 0 to 5 ns.	+	* To enhance the lifetime and intensity contrast, set in the image the intensity from 1 count to 30 counts and the lifetime from 0 to 5 ns.
	Response:\\		Response:\\
	The image scales are adapted.		The image scales are adapted.
Line 78:		Line 78:
	Now four cells are visible, the two nuclei on the left have longer Fast lifetimes then the other two. These are the cells transfected only with the donor constructs.		Now four cells are visible, the two nuclei on the left have longer Fast lifetimes then the other two. These are the cells transfected only with the donor constructs.

-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_8.png }}	+	{{ :howto:flim-fret-multiexpd_enahncedcontrast.png?400 \|}}

	* Keep the mouse pointer over the image and open the context menu by a right mouse click. Select the "Free ROI" tool.		* Keep the mouse pointer over the image and open the context menu by a right mouse click. Select the "Free ROI" tool.
Line 86:		Line 86:
	For more information about the different ROI selection tools, check the tutorial [[howto:roi_fitting_using_the_flim_script\|ROI fitting using the FLIM script]].		For more information about the different ROI selection tools, check the tutorial [[howto:roi_fitting_using_the_flim_script\|ROI fitting using the FLIM script]].

-	* Draw a ROI around one of the left cells by maintaining the left mouse button activated while circling the selected part of the image. Then release the left mouse button, press "Shift" continuously and cycle the other left nucleus. This way, the donor only cells are marked as ROI 0.	+	* Draw an ROI around one of the left cells by maintaining the left mouse button activated while circling the selected part of the image. Then release the left mouse button, press "Shift" continuously and cycle the other left nucleus. This way, the donor only cells are marked as ROI 0.

	Response:\\		Response:\\
	* Only the selected area is now shown in color.		* Only the selected area is now shown in color.
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_10.png }}	+	{{ :howto:flim-fret-multiexpd_roi.png?400 \|}}

	* In the TCSPC window, the photons from ROI 0 are added in grey.		* In the TCSPC window, the photons from ROI 0 are added in grey.
	{{ flim-fret-calculation_for_multi-exponential_donors_Image_11.png?600 }}		{{ flim-fret-calculation_for_multi-exponential_donors_Image_11.png?600 }}
		+

	* In the decay form on the lower left, select n-exponential reconvolution as fitting model.		* In the decay form on the lower left, select n-exponential reconvolution as fitting model.
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_12.png }}	+	{{ :howto:flim-fret-multiexpd_reconvfit.png?400 \|}}

	Response:\\		Response:\\
	* In the TCSPC window, the IRF is displayed in bright red, the data fitting limits are moved to the border of the TCSPC window.		* In the TCSPC window, the IRF is displayed in bright red, the data fitting limits are moved to the border of the TCSPC window.
	* The new fitting parameters "Shift IRF" and "Bkgr IRF" (=background IRF) appear in the fitting parameter table.		* The new fitting parameters "Shift IRF" and "Bkgr IRF" (=background IRF) appear in the fitting parameter table.
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_13.png?600 }}	+	{{ :howto:flim-fret-multiexpd_reconvfit_response.png \|}}

	Note:\\		Note:\\
-	The software offers the possibility to fit the data using a n-exponential tailfit or a n-exponential reconvolution fit. A tailfit can be used when the fitted lifetimes are significantly longer than the instrument response function. Still a reconvolution fit is usually preferable, especially for FRET determinations, because the complete decay is fitted, while for a tailfit, the start of the fitting range is usually a bit arbitrary, and the short component can be underestimated.	+	The software offers the possibility to fit the data using a n-exponential tailfit, a n-exponential reconvolution fit or a rapid reconvolution fit. A tailfit can be used when the fitted lifetimes are significantly longer than the instrument response function. Still a reconvolution fit is usually preferable, especially for FRET determinations, because the complete decay is fitted, while for a tailfit, the start of the fitting range is usually a bit arbitrary, and the short component can be underestimated. The rapid reconvolution model is used for systems with rapid reconvolution electronics (e.g. hybrid PMA-detector + MultiHarp photon counting board), where the peak count rates have been significantly higher than the pile up limit, or in cases, where the repetition rate has been too high to fit the decay completely into the TCSPC-window, e.g. 2PE excitation with 80MHz and dyes with a lifetime above ~3ns.
	For explanation on the fitting model and the used equations, click on the "Help" button next to the selected model. This opens a help window containing the fitting equation and the explanation of the different parameters.		For explanation on the fitting model and the used equations, click on the "Help" button next to the selected model. This opens a help window containing the fitting equation and the explanation of the different parameters.

Line 111:		Line 112:
	Response:\\		Response:\\
	The TCSPC curve of ROI 0 is highlighted in green.		The TCSPC curve of ROI 0 is highlighted in green.
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_14.png?600 }}	+	{{ :howto:flim-fret-multiexpd_roi-graph.png \|}}

	* At the IRF, click on the "Import" button.		* At the IRF, click on the "Import" button.
Line 133:		Line 134:
	Response:\\		Response:\\
	The imported IRF is highlighted in red.		The imported IRF is highlighted in red.
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_18.png?600 }}	+	{{ :howto:flim-fret-multiexpd_irf-graph.png \|}}

	* As model parameters, select n="2".		* As model parameters, select n="2".
		+	{{ :howto:flim-fret-multiexpd_n2_bearb.png?400 \|}}

	Note:\\		Note:\\
	In this case, the decay is double exponential. As it is not the scope of the tutorial to explain standard lifetime fitting (see the tutorial "ROI fitting using the FLIM script"), we skip the steps of finding the correct fitting model here.		In this case, the decay is double exponential. As it is not the scope of the tutorial to explain standard lifetime fitting (see the tutorial "ROI fitting using the FLIM script"), we skip the steps of finding the correct fitting model here.
-
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_19.png }}

	* Click on "Initial Fit".		* Click on "Initial Fit".
	Response:The fit is calculated.		Response:The fit is calculated.
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_20.png?600 }}	+	{{ :howto:flim-fret-multiexpd_initialfit.png \|}}

-	* The software calculates the amplitude weighted average lifetime τ<sub>Av. Amp.</sub>of 2.675. This is the result of the average donor lifetime in absence of FRET.	+	* The software calculates the amplitude weighted average lifetime τ<sub>Av. Amp.</sub>of 2.66. This is the result of the average donor lifetime in absence of FRET.

	Note:\\		Note:\\
Line 153:		Line 153:

	* Save the result by clicking on "Save Result".		* Save the result by clicking on "Save Result".
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_21.png }}	+
		+	{{ :howto:flim-fret-multiexpd_saveresult_bearb.png?400 \|}}

	Response:\\		Response:\\
	A result file (''FLIM.pqres'') is stored under the raw data file (''CENP-labelled_cells_for_FRET.ptu'').		A result file (''FLIM.pqres'') is stored under the raw data file (''CENP-labelled_cells_for_FRET.ptu'').
-	{{ flim-fret-calculation_for_multi-exponential_donors_Image_22.png }}	+	{{ :howto:flim-fret-multiexpd_pqres.png?400 \|}}

	* Now the first part is finished and the FLIM window should be closed.		* Now the first part is finished and the FLIM window should be closed.

fluorescence_correlation_spectroscopy-_a_short_introduction

Anonymous (anonymous@undisclosed.example.com) — 2024-10-11T08:30:57+00:00

2023/02/17 12:39		current
Line 1:		Line 1:
-	{{tag>MT200 MircoTime LSM Theory SymPhoTime FCS Correlation}}	+	{{tag>MicroTime LSM Theory SymPhoTime FCS Correlation}}

-	~~TOC~~

	====== Introduction to Fluorescence Correlation Spectroscopy ======		====== Introduction to Fluorescence Correlation Spectroscopy ======

calculate_ratiometric_single_pair_fret_distributions

Anonymous (anonymous@undisclosed.example.com) — 2015-11-03T14:17:54+00:00

2014/06/18 15:55		current
Line 1:		Line 1:
	{{tag> tutorial howto time-trace analysis FRET SymPhoTime}}		{{tag> tutorial howto time-trace analysis FRET SymPhoTime}}
-	~~TOC~~	+	~~NOTOC~~

	====== Calculate Ratiometric Single Pair FRET Distributions ======		====== Calculate Ratiometric Single Pair FRET Distributions ======
Line 15:		Line 15:
	Note: The script requires a time trace containing the fluorescence of two spectrally separated channels, one channel mainly detecting the fluorescence of the donor dye and the other channel detecting mainly the fluorescence of the acceptor dye.\\		Note: The script requires a time trace containing the fluorescence of two spectrally separated channels, one channel mainly detecting the fluorescence of the donor dye and the other channel detecting mainly the fluorescence of the acceptor dye.\\
	The time traces can be acquired from single molecule events in a diluted solution, where the passages of single molecules are registered as "bursts". Alternatively, traces obtained from a stationary single FRET pair can be analyzed, e.g. to observe conformational changes.\\		The time traces can be acquired from single molecule events in a diluted solution, where the passages of single molecules are registered as "bursts". Alternatively, traces obtained from a stationary single FRET pair can be analyzed, e.g. to observe conformational changes.\\
-	To record such traces, usually single molecule sensitive detectors as SPAD or Hybrid PMA are necessary to successfully detect single molecule fluorescence.\\	+	To record such traces, usually single molecule sensitive detectors as [[glossary:SPAD]] or [[glossary:Hybrid PMT]]s are necessary to successfully detect single molecule fluorescence.\\

	\\		\\

lifetime-fitting_using_the_rapid_reconvolution_algorithm

Anonymous (anonymous@undisclosed.example.com) — 2023-02-17T12:40:42+00:00

2021/03/23 10:39		current
Line 1:		Line 1:
-		+	{{tag>howto tutorial FLIM analysis SymPhoTime MT LSM_upgrade reconvolution fitting}}
-	{{tag>howto tutorial FLIM analysis SPT SymPhoTime MT LSM_upgrade reconvolution fitting}}	+


Line 11:		Line 10:
	This tutorial shows step-by-step, how to use the Multi-Exponential Reconvolution fitting model with correction for rapidFLIM. This new approach allows better fitting for data acquired with rapidFLIM capable hardware with count-rates way beyond classical pile up limit.		This tutorial shows step-by-step, how to use the Multi-Exponential Reconvolution fitting model with correction for rapidFLIM. This new approach allows better fitting for data acquired with rapidFLIM capable hardware with count-rates way beyond classical pile up limit.

-	For the principle of rapidFLIM - recording, check the technical note available here: [[https://www.picoquant.com/images/uploads/page/files/16651/2016_soh_appnote_rapidflim.pdf\|Application Note: rapidFLIM]]	+	For the principle of rapidFLIM - recording, check the application note available here:
		+	[[https://www.picoquant.com/dl_technotes/AppNote_rapidFLIM_HiRes.pdf\|Application Note: rapidFLIM]].

	Here in detail the FLIM analysis of SymPhoTime 64 is used to perform a lifetime fit and how to extract and read the results. A mixture of Dragon Green and Nile Red fluorescent microspheres were imaged with more than three frames per second. Tutorials workspace can be downloaded from [[https://figshare.com/s/4957fcfa684daef86c23]].		Here in detail the FLIM analysis of SymPhoTime 64 is used to perform a lifetime fit and how to extract and read the results. A mixture of Dragon Green and Nile Red fluorescent microspheres were imaged with more than three frames per second. Tutorials workspace can be downloaded from [[https://figshare.com/s/4957fcfa684daef86c23]].

using_the_flcs_script_for_spectral_crosstalk_removal_via_flccs

Anonymous (anonymous@undisclosed.example.com) — 2015-02-25T14:00:13+00:00

2014/06/19 08:09		current
Line 1:		Line 1:
	{{tag> howto tutorial FCS FLCS crosstalk analysis SymPhoTime}}		{{tag> howto tutorial FCS FLCS crosstalk analysis SymPhoTime}}
-	~~TOC~~	+	~~NOTOC~~

-	====== Spectral crosstalk removal via FLCCS ======	+	====== Spectral Crosstalk Removal via FLCCS ======

data_file_import

Anonymous (anonymous@undisclosed.example.com) — 2022-08-04T13:24:27+00:00

2022/08/03 14:07		current
Line 3:		Line 3:
	Create a new workspace or open an existing one:		Create a new workspace or open an existing one:

-	{{ :howto:et2_createopen.png?370 \| File menu }}	+	{{ :howto:et2_createopen.png \| File menu }}


-	Then select Import Files. A new dialog opens where the file is selected:	+	Then select Import Files. A new dialog opens, where the data file is selected:

-	{{ :howto:et2_fileimport.png?1161 \| Selects the file to be imported }}	+	{{ :howto:et2_fileimport.png \| Selects the file to be imported }}

-	The file selected above is a sample data file, ''Decay_Coumarin_6.phu'' installed during the installation of the native TimeHarp260 software. This file contains two histograms: an Instrument Response Function (IRF) and a decay curve of Coumarin6 dissolved in ethanol. During file import, before pressing the Open button, it is possible to preview and step through the data content of selected file using the control elements on the right hand side of the dialog.	+	The file selected above is a sample data file, ''Decay_Coumarin_6.phu'' installed during the installation of the [[https://www.picoquant.com/dl_software/TimeHarp260/TimeHarp260_SW_and_DLL_V3_2.zip\|native TimeHarp260 software]]. This file contains two histograms: an [[howto:how_to_measure_the_instrument_response_function_irf\|Instrument Response Function (IRF)]] and a decay curve of Coumarin6 dissolved in ethanol. During file import, before pressing the __O__pen button, it is possible to preview and step through the data content of selected file using the control elements on the right hand side of the dialog.

	The same data file, when opened in the native software, looks like this:		The same data file, when opened in the native software, looks like this:

-	{{ :howto:et2_originalhistograms.png?1033 \| Original data file content }}	+	{{ :howto:et2_originalhistograms.png \| Original data file content }}

-	Trace #0 contains the IRF, Trace #2 is the decay curve. The other data blocks are empty. Both Trace#0 and #2 will be imported into a single ''.etc'' data container. This will make the data analysis easier. In general, such a ''.phu'' file may contain many more histograms. It is also possible that an IRF measurement and a decay measurement are stored in two separate ''.phu'' files. This is no problem, but then the file import procedure has to be repeated and the imported curves will end up in dedicated .etc data containers.	+	Trace #0 shows the IRF, Trace #2 is the decay curve. The other data blocks are empty. Both Trace #0 and #2 will be imported into a single ''.etc'' data container. This will make the data analysis easier. In general, such a ''.phu'' file may contain many more histograms.
		+
		+	It is also possible that an IRF measurement and a decay measurement are stored in two separate ''.phu'' files. This is no problem, but then the file import procedure has to be repeated for each file and the imported curves will end up in dedicated ''.etc'' data containers.

	After importing ''Decay_Coumarin_6.phu'', the workspace looks like this:		After importing ''Decay_Coumarin_6.phu'', the workspace looks like this:

-	{{ :howto:et2_workspace1.png?338 \|}}	+	{{ :howto:et2_workspace1.png \|}}
	\\		\\
	\\		\\
	After expanding the data container:		After expanding the data container:
-	{{ :howto:et2_workspace2.png?340 \|}}	+	{{ :howto:et2_workspace2.png \|}}

	Two histograms have been imported. They are shown as "Decay" because EasyTau does not know their assignment yet.		Two histograms have been imported. They are shown as "Decay" because EasyTau does not know their assignment yet.
		+	\\
		+	\\
		+
		+
		+	==== Multiple File Import ====
		+
		+	Importing multiple data files is easy. Use CTRL + Click to select multiple files:
		+
		+	{{ :howto:et2_fileimport2.png \|}}
		+	\\
		+	\\
		+	Each input ''.phu'' file has got a dedicated ''.etc'' data container:
		+	{{ :howto:et2_workspace3.png \|}}
		+	\\
		+	\\
		+	Unlike ''Decay_Coumarin_6.phu'' containing two histograms, both ''Anthracene_EtOH.phu'' and ''IRF375.phu'' contains only one histogram. These two measurements actually belong to each other. In order to perform reconvolution fitting of the Antracene decay, the corresponding IRF measured at 375nm excitation wavelength will be needed.
		+

intensity_time_trace_analysis

Anonymous (anonymous@undisclosed.example.com) — 2014-06-19T08:12:32+00:00

2014/05/07 16:14		current
Line 1:		Line 1:
	{{tag> tutorial howto time-trace analysis SymPhoTime}}		{{tag> tutorial howto time-trace analysis SymPhoTime}}

-	~~TOC~~	+	~~NOTOC~~

	======Intensity Time Trace Analysis======		======Intensity Time Trace Analysis======
Line 10:		Line 10:

	This tutorial shows step-by-step, how to draw intensity time traces from a single molecule. The script requires a point measurement of an immobilized fluorophore. When applied to images, it can be used to check for line and frame marker presence and assignment.		This tutorial shows step-by-step, how to draw intensity time traces from a single molecule. The script requires a point measurement of an immobilized fluorophore. When applied to images, it can be used to check for line and frame marker presence and assignment.
		+
		+	{{ youtube>D4BBTLBvanQ?large }}
		+

	\\		\\

measurement_hardware_instrumentation

Anonymous (anonymous@undisclosed.example.com) — 2018-06-25T07:18:07+00:00

2017/05/17 08:33		current
Line 32:		Line 32:
	[[video_tutorials#Kurt Thorn: Optical Sectioning and Confocal Microscopy\|Confocal microscoy and optical sectioning by Kurt Thorn]]		[[video_tutorials#Kurt Thorn: Optical Sectioning and Confocal Microscopy\|Confocal microscoy and optical sectioning by Kurt Thorn]]

-	[[technical docs:Beampath of the Zeiss LSM700]]	+	[[Beampath of the Zeiss LSM880]]

	[[howto:check_overlap_of_different_color_confocal_volumes\|How to check the overlap of different color confocal volumes]]		[[howto:check_overlap_of_different_color_confocal_volumes\|How to check the overlap of different color confocal volumes]]

video_tutorials

Anonymous (anonymous@undisclosed.example.com) — 2014-11-17T16:36:53+00:00

2014/06/18 09:58		current
Line 14:		Line 14:

	==== Confocal Microscopy ====		==== Confocal Microscopy ====
		+	Mark Armitage explains major components of confocal microscopes and their use.

-	{{ youtube>PLO0yDSkagDljzI0bdM5m1njrdLdVa80_N?large }}	+	{{ youtube>BUSZD4IVYSw?large }}
		+	This video is part of a series of videos. This is the related playlist: [[https://www.youtube.com/playlist?list=PLO0yDSkagDljzI0bdM5m1njrdLdVa80_N]]

		+
		+	\\

	Nikons website [[http://www.microscopyu.com/articles/confocal/index.html\|MicroscopyU.com]] has a very nice course on confocal microscopy.		Nikons website [[http://www.microscopyu.com/articles/confocal/index.html\|MicroscopyU.com]] has a very nice course on confocal microscopy.
Line 27:		Line 31:
	[[http://www.ibiology.org/ibioseminars/techniques/kurt-thorn-part-1.html]]		[[http://www.ibiology.org/ibioseminars/techniques/kurt-thorn-part-1.html]]

-	=== Kurt Thorn: Two Photon Microscopy ===	+	==== Kurt Thorn: Two Photon Microscopy ====
	Kurt Thorn of UCSF talks about two-photon microscopy which uses intense pulsed lasers to image deep into biological samples. It can be used for imaging thick tissue specimens or even imaging inside of live animals.		Kurt Thorn of UCSF talks about two-photon microscopy which uses intense pulsed lasers to image deep into biological samples. It can be used for imaging thick tissue specimens or even imaging inside of live animals.

Line 35:		Line 39:


-	=== Philippe Bastiaens: Fluorescence Lifetime Microscopy (FLIM) ===	+	==== Philippe Bastiaens: Fluorescence Lifetime Microscopy (FLIM) ====
	Fluorescence-lifetime imaging microscopy or [[glossary:flim\|FLIM]] is an imaging technique for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample. It can be used as an imaging technique in confocal microscopy, two-photon excitation microscopy, and multiphoton tomography.		Fluorescence-lifetime imaging microscopy or [[glossary:flim\|FLIM]] is an imaging technique for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample. It can be used as an imaging technique in confocal microscopy, two-photon excitation microscopy, and multiphoton tomography.
	The lifetime of the fluorophore signal, rather than its intensity, is used to create the image in FLIM. This has the advantage of minimizing the effect of photon scattering in thick layers of sample ([[wp>Fluorescence-lifetime_imaging_microscopy \| FLIM]]).		The lifetime of the fluorophore signal, rather than its intensity, is used to create the image in FLIM. This has the advantage of minimizing the effect of photon scattering in thick layers of sample ([[wp>Fluorescence-lifetime_imaging_microscopy \| FLIM]]).

calculate_ratiometric_fret-images

Anonymous (anonymous@undisclosed.example.com) — 2015-02-25T14:00:57+00:00

2014/06/18 09:24		current
Line 3:		Line 3:
	~~NOTOC~~		~~NOTOC~~

-	====== Calculate ratiometric FRET-Images ======	+	====== Calculate Ratiometric FRET Images ======


Line 41:		Line 41:
	{{ calculate_ratiometric_fret-images_with_the_fret-image-script_Image_3.png?348 }}		{{ calculate_ratiometric_fret-images_with_the_fret-image-script_Image_3.png?348 }}

-	Note:Comment: the drop down menu can be opened and closed by clicking on the grey button on the left side of the header of the drop down menu: {{grey_button.png}}	+	Note: the drop down menu can be opened and closed by clicking on the grey button on the left side of the header of the drop down menu: {{grey_button.png}}

	* Start the "FRET Image" script by clicking on "Start".		* Start the "FRET Image" script by clicking on "Start".

novaflim_calculated_irf

Anonymous (anonymous@undisclosed.example.com) — 2025-03-11T08:19:29+00:00

2025/02/18 13:31		current
Line 1:		Line 1:
	====== NovaFLIM Calculated IRF ======		====== NovaFLIM Calculated IRF ======
		+	{{tag> faq software novaflim IRF}}
		+

	In the Nova Flim the IRF is parameterized as a combination of two Gaussian curves (a total of 5 fit parameters). These parameters are adjusted during Initial Fit.		In the Nova Flim the IRF is parameterized as a combination of two Gaussian curves (a total of 5 fit parameters). These parameters are adjusted during Initial Fit.
Line 22:		Line 24:
	* Height of Gaussian curve 2 (limited to the height of Gaussian curve 1)		* Height of Gaussian curve 2 (limited to the height of Gaussian curve 1)

-	This model is the result of extensive internal development, testing, and refinement. By optimizing these parameters, the IRF model can accurately represent the measured response of the system.	+	The IRF is determined only during the initial TCSPC fit, which means it may vary depending on the different exponential components. During the pixelwise fitting in FLIM, this IRF is applied without any modifications. Consequently, all image points are fitted using the same fixed IRF established during the initial fit.
		+
		+	This model is the result of extensive internal development, testing, and refinement. By optimizing these parameters, the IRF model can accurately represent the measured response of the system.

2ffcs

Anonymous (anonymous@undisclosed.example.com) — 2015-11-04T09:41:00+00:00

2013/12/03 11:42		current
Line 1:		Line 1:
-	====== 2FFCS ======	+	{{tag> 2fFCS FCS}}
		+	~~TOC~~
		+	====== 2fFCS ======


Line 5:		Line 7:

	Correlation analysis that is applied to the fluctuations of the fluorescence intensity. The cross-correlation between the occurrence of events in two different detection volumes is used to introduce an absolute diffusion length into the analysis.		Correlation analysis that is applied to the fluctuations of the fluorescence intensity. The cross-correlation between the occurrence of events in two different detection volumes is used to introduce an absolute diffusion length into the analysis.
		+
		+	see [[glossary:FCS]] for a introduction on FCS.

	===== Experimental Setup =====		===== Experimental Setup =====
Line 38:		Line 42:
	Occasionally, pinhole size can be used to adjust amount of photons to change the signal intensity and increase SNR. In addition to the "optimal" 1 AU, Pinhole 1-3 AU is the range of choice. Bigger pinhole give you stronger signal but with the compromised confocal effects.		Occasionally, pinhole size can be used to adjust amount of photons to change the signal intensity and increase SNR. In addition to the "optimal" 1 AU, Pinhole 1-3 AU is the range of choice. Bigger pinhole give you stronger signal but with the compromised confocal effects.

-	PQ usually uses a 50 micron pinhole (1.4 AU) to get maximum detection efficiency, somewhat sacrificing background rejection.	+	PicoQuant usually uses a 50 micron pinhole (1.4 AU) to get maximum detection efficiency, somewhat sacrificing background rejection.

	=== Two Foci ===		=== Two Foci ===

calculate_fccs_trace_with_the_grouped_fcs_script

Anonymous (anonymous@undisclosed.example.com) — 2020-04-01T15:05:26+00:00

2014/06/18 09:21		current
Line 2:		Line 2:
	~~NOTOC~~		~~NOTOC~~

-	====== Calculate multiple FCCS Traces with the Grouped FCS Script ======	+	====== Calculate FCCS Traces with the Grouped FCS Script ======

	===== Summary =====		===== Summary =====

determination_of_the_focal_width_with_the_focal

Anonymous (anonymous@undisclosed.example.com) — 2015-01-22T14:48:12+00:00

2014/05/07 16:13		current
Line 11:		Line 11:
	This tutorial shows step-by-step, how to determine the Point Spread Function (PSF) of a microscopic system using the Focal Width Script. It requires the image of a subresolution bead, in which the center is marked in the image. The script applies then a Gaussian fit of the bead and calculates the Full Width Half Maximum (FWHM) in both scanning directions.		This tutorial shows step-by-step, how to determine the Point Spread Function (PSF) of a microscopic system using the Focal Width Script. It requires the image of a subresolution bead, in which the center is marked in the image. The script applies then a Gaussian fit of the bead and calculates the Full Width Half Maximum (FWHM) in both scanning directions.

-	\\	+	{{ youtube>WkSbWoVdw_g?large }}
	===== Step-by-Step Tutorial =====		===== Step-by-Step Tutorial =====

Line 51:		Line 52:

	Note: The data in the file is a bead that was excited via pulsed interleaved excitation (PIE) using two perpendicularly polarized lasers, which were guided through a DIC prism. This causes a displacement of the beams, which is used in dual focus FCS (2fFCS) measurements.\\		Note: The data in the file is a bead that was excited via pulsed interleaved excitation (PIE) using two perpendicularly polarized lasers, which were guided through a DIC prism. This causes a displacement of the beams, which is used in dual focus FCS (2fFCS) measurements.\\
-	If data are acquired with a scanner controlled by the SymPhoTime 64 software (i.e. the piezo scanner in the MicroTime 200), the image dimensions are automatically correct. If the image is acquired using an external scanner, the image size has to be defined or transferred before the measurement to make sure that the measured image dimensions are correct. Otherwise, one pixel is considered to a size of 1 µm.	+	If data are acquired with a scanner controlled by the SymPhoTime 64 software (i.e. the piezo scanner in the [[products:MicroTime#MicroTime 200\|MicroTime200]]), the image dimensions are automatically correct. If the image is acquired using an external scanner, the image size has to be defined or transferred before the measurement to make sure that the measured image dimensions are correct. Otherwise, one pixel is considered to a size of 1 µm.

	{{ determination_of_the_focal_width_with_the_focal_Image_6.png }}		{{ determination_of_the_focal_width_with_the_focal_Image_6.png }}
Line 63:		Line 64:

	* Optional: Apply the same script to the file ''TS-bead_immo_xz-scan.ptu'' to get an idea about the ratio of the focal width in xy vs. z.		* Optional: Apply the same script to the file ''TS-bead_immo_xz-scan.ptu'' to get an idea about the ratio of the focal width in xy vs. z.
-

lifetime_fitting_using_the_tcpsc_fitting_script

Anonymous (anonymous@undisclosed.example.com) — 2023-09-07T22:43:32+00:00

2014/06/18 16:11		current
Line 9:		Line 9:


-	This tutorial shows step-by-step, how to fit the lifetime of a measured sample. As an example, a single exponential reconvolution fit is used to determine the lifetime of ATTO655 diluted in water.	+	This tutorial shows step-by-step, how to fit a selected model to a decay curve in order to get the lifetime of a measured sample. As an example, a single exponential reconvolution fit is used to determine the lifetime of ATTO655 diluted in water.

	{{ youtube>WupJkKpUVV8?large }}		{{ youtube>WupJkKpUVV8?large }}
Line 50:		Line 50:
	{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_6.png?600 }}		{{ lifetime_fitting_using_the_tcpsc_fitting_script_Image_6.png?600 }}

-	Note: The software offers the possibility to fit the data using a n-exponential tailfit or a n-exponential reconvolution fit. A tailfit can be used when the fitted lifetimes are significantly longer than the instrument response function. Still a reconvolution fit is usually preferable, because the complete decay is fitted, while for a tailfit, the start of the fitting range is usually a bit arbitrary.\\	+	Note: The software offers the possibility to use a n-exponential tailfit or a n-exponential reconvolution model fit. A tailfit can be used when the expected lifetimes are significantly longer than the FWHM of instrument response function. Still a reconvolution fit is usually preferable, because the complete decay, including its rising edge is analyzed, while for a tailfit, the start of the fitting range is usually a bit arbitrary.\\
-	For explanation about the fitting model and the used equations, click on the "Help" button next to the selected model. This opens a help window containing the fitting equation and the explanation of the different parameters.	+	For explanation about the fitting model and the used equations, click on the "Help" button next to the selected model. This opens a help window containing the fitted model l equation and the explanation of the different parameters.

	* Click: "Initial Fit" (marked in orange).		* Click: "Initial Fit" (marked in orange).
Line 67:		Line 67:
	The χ²-value approaches 1.\\		The χ²-value approaches 1.\\
	The calculated fitting values are reasonable.\\		The calculated fitting values are reasonable.\\
-	Usually the fitting model with least parameters is selected.\\	+
		+	Usually the fitting model using the smallest amount of adjustable parameters is selected.\\
	=> In this example, the fit is already sufficient.		=> In this example, the fit is already sufficient.

update

Anonymous (anonymous@undisclosed.example.com) — 2023-02-23T06:37:29+00:00

2023/02/23 06:15		current
Line 1:		Line 1:
-	updating...	+	{{tag>howto tutorial FLIM analysis SymPhoTime MT LSM_upgrade}}
		+
		+
		+	====== ROI Fitting Using the FLIM Analysis (updated for SymPhoTime v2.5)======
		+	===== Introduction =====
		+	This tutorial shows step-by-step, how the FLIM script of SymPhoTime 64 can be used to fit several regions of interest (ROIs), and how to extract and interpret the results. In detail, the FLIM script is started using an image of mouse kidney tissue in the “Tutorials” workspace, [[https://figshare.com/s/4957fcfa684daef86c23]], a suited fitting model is chosen and a pixel-by-pixel fit is performed.\\
		+
		+
		+	===== Step-by-step Tutorial =====
		+
		+	==== Select a file and start the analysis ====
		+
		+	* Start SymPhoTime 64 software (this tutorial uses version 2.5, there might be differences when working with other versions).
		+	* Open the “Tutorials” workspace via File -> Open Workspace from the main menu.\\
		+
		+	Response:\\
		+	The files of the sample workspace are displayed in the workspace panel on the left side of the main window.
		+	* Highlight the file ''Kidney_Cells_FLIM.ptu'' by a single mouse click.
		+	* Select the "Analysis" tab and in there, open the "Imaging" drop-down menu.\\
		+	* Start the FLIM script by clicking on "Start".\\
		+
		+	{{ howto:startflim.png?200 \|}}
		+
		+
		+	Response:\\
		+	The FLIM analysis is applied to the file ''Kidney_Cells_FLIM.ptu''. Thereby, a new Window opens:
		+
		+	{{ howto:kidneyfullscreen3.png \|}}
		+
		+
		+	Note:\\
		+	The window contains five different sections:\\
		+	1: Setting panel\\
		+	Imaging analysis options can be defined in this section.\\
		+	2: [[glossary:Fast FLIM]] image\\
		+	The FLIM preview image displayed in a false color scale. The brightness encodes the intensity, while the color encodes the average "Fast FLIM" lifetime, i.e. the mean arrival times of the photons after the laser pulse. When not defined otherwise, intensity and color scale stretch from minimum to maximum. As the mean arrival time of the background photons in the areas where no fluorescence is detected, is randomly spread over the TCSPC window, the mean photon arrival time of the dark background is usually very long (with an average up to 1/2 of the TCSPC window), which makes the color scale loaded by default sometimes unsuited for displaying the lifetime contrast in the actual sample. In this case, the scale can be adapted.\\
		+	3: Fluorescence Lifetime Histogram\\
		+	Here the frequency of photon counts corresponding to the individual mean lifetimes is plotted. The settings of the plot can be adapted using the controls on the right of the plot. The histogram is photon weighted.\\
		+	4: Decay Fitting Parameters\\
		+	Here the lifetime fitting model can be selected. Fitting is then applied to the lifetime graph(s) on the lower center ([[glossary:TCSPC]] histogram).\\
		+	5: [[glossary:TCSPC]] Decay window\\
		+	By default, this set shows the [[glossary:TCSPC]] histogram of all photons in the image in green (= dataset 0) and the [[glossary:TCSPC]] histogram of a single pixel in grey (= dataset 1). In red an estimation of the instrument response function (=IRF) is shown. The IRF reconstruction is deducted from the rising edge of the [[glossary:TCSPC]] histogram.\\
		+
		+
		+	* To enhance the lifetime contrast, adapt the color scale by typing the limit values for the "Fast Lifetime [ns]" and "Events[cnts]". Additionally set the "Lifetime Histogram" range on the right hand.
		+	* Tick the checkbox of the detection "Data Channel" (i.e. detector), you want to use - in this case detector channel 4 - and press "Calculate FastFLIM".\\
		+
		+	{{ howto:detectionchannel.png?400 \|}}
		+
		+	Response:\\
		+	The FLIM Preview image will get updated. Optimize contrast by adapting "Min" and "Max" values.
		+
		+	==== Select a file and start the analysis ====
		+
		+	* In the FLIM preview image, ROIs can be selected using the context menu (Right Click).\\
		+
		+	{{ howto:selectmagicwand_w.png?300 \|}}
		+
		+	Note:\\
		+
		+	- The following ROI-selection tools are available: Rectangle ROI, Ellipse ROI for simple drawing and Paint ROI for marking the pixels the mouse passed over. The faster the mouse pointer moves, the broader the marked lines will get. Free ROI draws a free shape. The pixels within the drawn shape will be selected. Finally Magic Wand marks area with similar fast lifetime.
		+	- Using keyboard shortcuts ''<CTRL>+<Z>'' you can undo a selection. Redo also with ''<CTRL>+<Shift>+<Z>'' is possible.\\
		+	- You can add several areas to an existing ROI with the same or different selection tool by keeping the ''<Shift>'' key pressed while drawing the additional regions.\\
		+	- If you want to remove some of the pixels from the selected ROI, just keep the ''<CTRL>'' key pressed when using a ROI selection tool to exclude the unwanted parts.\\
		+	- Additionally you can zoom, move or Invert the ROI. Please see the shortcuts provided for each option in the context menu.\\
		+	- If you want to start again from scratch select "Select all as ROI".\\
		+
		+	* In this step, as an example, we mark an ROI with similar lifetime range using the "Magic Wand" tool. The sensitivity of the tool to lifetime differences can be adjusted in a box below the intensity and color scale options.\\
		+
		+	{{ howto:magicwand_w_thresh.png?300 \|}}
		+
		+	* Increase the threshold to 25.\\
		+
		+	{{ howto:inner_magicwand25_3.png \|}}
		+
		+	Response:\\
		+	The parts of the image with a similar fast lifetime range will be selected and the lifetime histogram will get updated accordingly. If you want to combine several spatially separated regions with a similar lifetime into one single ROI, as it's done here, you have to press "Shift" when selecting the spatial separated areas with the Magic wand tool.
		+
		+	Note:\\
		+	Since this function is based on the displayed image, the sensitivity of the selection is dependent on the scaling of the color-encoded component. Changing the color scale (in general the lifetime) can change the sensitivity of this tool.\\
		+
		+
		+	Response:\\
		+	An extra gray decay corresponding to ''ROI 0'' will appear under decay fit in "Decay Fitting" panel.\\
		+
		+
		+	Note:\\
		+	For fine adjustment, you can use any other ROI selection tools from context menu. In this case better to use ''<CTRL>'' or ''<Shift>'' buttons in order to remove pixel with a different or add pixels with similar fast lifetime. Ideally try to select an ROI with 10000 to 100000 counts in peak for getting the optimum statistics. ROIs with lower intensities can also be fitted, but in case of a more-exponential decay, some components might not be resolved.\\
		+
		+
		+	* In order to select an additional ROI, click "New" ROI.\\
		+
		+	{{ howto:addroi.png?400 \|}}
		+
		+
		+	Response:\\
		+	The software assigns sequential numbers to selected ROIs.The whole preview image will appear again.\\
		+
		+	* For the next ROI, first we use the same tool, "Magic Wand" but adapt its threshold to 20.
		+	{{ howto:tubul_magicwand20_3.png \|}}
		+
		+	* Repeat the last two steps in order to select the cell nuclei as the last ROI.\\
		+
		+	{{ howto:nuclei_magicwand30_3.png \|}}
		+
		+	Response:\\
		+	Now you can see fast FLIM (preview) images of "ROI 0", "ROI 1" and "ROI 2" when selecting them in the ROI-list In the Region of Interest-Menu, as well as their corresponding decays in TSCPC window in grey when selecting the different decays in the list.
		+
		+
		+	==== Perform an Initial Lifetime Fit on the Selected Regions (ROIs) ====
		+
		+
		+	* In the forth box, "Decay Fitting", from the Fitting Model drop-down list select "n-Exponential Reconvolution Fit".\\
		+
		+	{{ howto:nexpon_fitmodel.png?400 \|}}
		+
		+	Response:\\
		+	In the TCSPC window, the IRF is displayed in bright red, the data fitting limits are moved to the border of the TCSPC window.\\
		+	The new fitting parameters "Shift IRF" and "Bkgr IRF" (=background IRF) appear in the fitting parameter table.\\
		+
		+	Note:\\
		+	The software offers the possibility to fit the data using an n-exponential tailfit, n-exponential reconvolution or rapid reconvolution fit. A tailfit can be used when the fitted lifetimes are significantly longer than the instrument response function. A reconvolution fit is usually preferable, because the complete decay is fitted, while for a tailfit, the start of the fitting range is usually a bit arbitrary. A rapid reconvolution fit is suited, if the image was measured with a very high countrate and a system with a dead time significantly above the pile-up-limit or when the image has been acquired with a repetition rate, which is normally too high for the lifetime of the dye, so that the decay is not at the background level at the end of the decay window. The rapidFLIM-fitting is explained in another tutorial.\\
		+
		+	For explanation about the fitting model and the used equations, click on the "Help" button next to the selected model. This opens a help window containing the fitting equation and the explanation of the different parameters.\\
		+
		+
		+	* From "Decay" drop-down list select ''ROI 0'' and click "Initial Fit" (marked in orange).\\
		+	{{ howto:inner_monoexpo_3.png \|}}
		+
		+	Response:\\
		+	A single exponential reconvolution fit is performed. In the TCSPC window, the fit is displayed as a black line. Below, the residuals (= difference between raw data and fit values) are displayed.\\
		+	* Fit values appear in the fitting table. Check the fitting curve, residuals and χ².\\
		+
		+	Note:\\
		+	Usually, a decent fit is characterized by the following criteria:\\
		+	The fitted curve overlays well with the decay curve. In the residual window, the values spread randomly around 0.\\
		+	The χ²-value approaches 1.\\
		+	The calculated fitting values are reasonable.\\
		+	The fitting model with least parameters is preferred.\\
		+
		+
		+	* In this example it is obvious that the applied single exponential reconvolution fit is not suited to fit the decay curve. Increase the "Model Parameters" to n = 2. Again, click on "Initial fit".\\
		+
		+	{{ howto:inner_biexpo_3.png \|}}
		+
		+
		+	Response:\\
		+	The fit quality increases. But if it still doesn't look sufficient. One sees a trend in residuals and not a random distribution around 0. Increase number of "Model Parameters" to three.\\
		+
		+	{{ howto:inner_3expo_3.png \|}}
		+
		+	* Here the fitting model shows a satisfying result. A 3-exponential fitting model should be used for ROI 0.\\
		+
		+	* Try the last two steps with "ROI 1".\\
		+
		+	{{ howto:tubul_3expo_3.png \|}}
		+
		+
		+	* Find the best fit for "ROI 2" in a same manner. It is better always to start from monoexponential and try to find the fitting model with a lower number of components.\\
		+	{{ howto:nuclei_3expo_3.png \|}}
		+
		+	* Three components exponential decay seems to be suitable for all three ROIs. If all ROIs can be fitted with the same model, you can also click on "Fit All" to get all curves fitted with the same model.\\
		+	* Clicking on "Fit All" will fit all active data sets with the same model, using the start parameters of the active data set. If you do not want to fit the complete decay (dataset 1) and the decay of a single pixel (Dataset 2), press "Show All" button:
		+
		+
		+	{{ howto:showall.png \|}}
		+
		+	* Uncheck the first two datasets on top of the parameter table and press "Fit All" again.
		+
		+	{{ howto:deselectdataset1_3.png \|}}
		+
		+
		+
		+
		+	==== Compare the lifetimes and save results ====
		+
		+
		+
		+
		+	* In order to compare the lifetime of these three compartments, apart from fitting parameter table shown above, you can plot them by selecting the "Parameter Plot" on the right side of TCSPC window.\\
		+
		+	Note:\\
		+	The X-Axes is showing the "Dataset Number" corresponding to the dataset listed in Decay drop-down menu (in Dcay Fitting Panel). This means by default "Overall Decay" and "Pixel (0,0)", and then selected ROIs.\\
		+	For the Y-Axes one can select one or multiple fit parameters.\\
		+
		+	* Tick the "tau_Av_Int" checkbox for Y-Axes and zoom in to be able to see the lifetime differences better.\\
		+	{{ howto:parameterplot_3rois_3.png \|}}
		+
		+
		+
		+	* At the end, click on "Save Result" button in parameter panel.\\
		+
		+
		+	{{ howto:saveresultbutton.png?300 \|}}
		+
		+
		+	Response:\\
		+	A result file "FLIM.pqres" is stored under the raw data file ''Kidney_Cell_FLIM.ptu''.\\
		+
		+	* Later, clicking on the result file reopens the file in the same way as it was stored.

using_the_anisotropy_image_script

Anonymous (anonymous@undisclosed.example.com) — 2018-04-12T11:44:14+00:00

2018/04/12 10:26		current
Line 51:		Line 51:
	- Lower graph: Anisotropy histogram over the different pixels.		- Lower graph: Anisotropy histogram over the different pixels.

-	Note:	+	Note:
-	The anisotropy values can theoretically spread between -0.5 and 1 for parallel or vertical excitation and emission dipoles respectively. The anisotropy value of 0 is expected in case of very fast rotation or extremely efficient energy transfer processes (e.g. efficient HOMO-FRET). When measuring a randomly aligned ensemble due to photoselection the anisotropy varies with in the limits of -0.2 and 0.4 instead of -0.5 and 1.	+	The anisotropy values can theoretically spread between -0.5 and 1 for perpendicular and parallel excitation and emission dipoles respectively. The anisotropy value of 0 is expected in case of very fast rotation or extremely efficient energy transfer processes (e.g. efficient HOMO-FRET). When measuring a randomly aligned ensemble due to photoselection the anisotropy varies with in the limits of -0.2 and 0.4 instead of -0.5 and 1.

	==== Adapt the script to the image and the system parameters ====		==== Adapt the script to the image and the system parameters ====

using_the_antibunching_script

Anonymous (anonymous@undisclosed.example.com) — 2014-06-19T07:40:32+00:00

2014/05/23 07:39		current
Line 1:		Line 1:
	{{tag> antibunching howto tutorial correlation analysis SymPhoTime}}		{{tag> antibunching howto tutorial correlation analysis SymPhoTime}}
-	~~TOC~~	+	~~NOTOC~~

-	====== Using the Antibunching Analysis ======	+	====== Antibunching Analysis ======


Line 9:		Line 9:

	This tutorial shows step-by-step, how to calculate an antibunching curve from a fluorescence time trace of a single emitter, in this example a nanodiamond NV center, excited with a pulsed 530 nm laser.		This tutorial shows step-by-step, how to calculate an antibunching curve from a fluorescence time trace of a single emitter, in this example a nanodiamond NV center, excited with a pulsed 530 nm laser.
		+
		+	{{ youtube>bEyhuWnmFC4?large }}

	===== Background Information =====		===== Background Information =====
Line 77:		Line 79:

	* Here it is impossible to determine the number of emitters. In this measurement the luminescence stems from several emitters.		* Here it is impossible to determine the number of emitters. In this measurement the luminescence stems from several emitters.
-

burst_duration_and_fcs_diffusion_time

Anonymous (anonymous@undisclosed.example.com) — 2026-01-12T14:58:02+00:00

2026/01/12 10:06		current
Line 3:		Line 3:
	~~NOTOC~~		~~NOTOC~~

-	====== Burst Duration and FCS Diffusion Time ======	+	====== Burst duration and FCS diffusion time ======
		+	Diffusion, flow, burst selection, and photobleaching in confocal single-molecule fluorescence

-	===== Overview =====	+	===== 1. Introduction =====

-	In single-molecule fluorescence experiments, two closely related time scales are commonly discussed:	+	In confocal single-molecule fluorescence experiments, molecular transport through the detection volume is commonly characterized using two different observables: the diffusion time extracted from fluorescence correlation spectroscopy (FCS) and the burst duration obtained from single-molecule detection (SMD) or burst analysis. Both quantities probe molecular motion through the same optical volume, yet they originate from fundamentally different experimental procedures and statistical definitions.

-	* the burst duration obtained from single-molecule burst analysis	+	The apparent similarity of their numerical values often suggests a direct equivalence. However, this impression can be misleading, because each observable responds differently to transport mechanisms, detection geometry, selection criteria, and photophysical processes. As a consequence, diffusion time and burst duration must be treated as distinct quantities whose relationship depends on the underlying physical assumptions and on how the experiment is performed.
-	* the diffusion time extracted from fluorescence correlation spectroscopy (FCS)	+

-	Although both quantities probe molecular transport through the same confocal detection volume and often have comparable numerical values, they differ fundamentally in their physical meaning, statistical definition, and sensitivity to experimental parameters.	+	In this article, we develop a unified theoretical framework to analyze the diffusion time and burst duration within a three-dimensional Gaussian detection model. We first derive both quantities independently using their full mathematical formalism. We then compare the two observables and establish their relationship under ideal diffusion conditions. Finally, we investigate how burst-selection procedures, directed flow, and photobleaching modify each observable and how these effects alter their mutual relation.

-	This article summarizes their relationship within the framework of a three-dimensional Gaussian detection model, explains the origin of the frequently cited relation between the two time scales, and discusses the influence of burst-selection thresholds and directed flow.	+	===== 2. Three-dimensional Gaussian detection model =====
-		+
-	===== Three-dimensional Gaussian detection model =====	+

	Both FCS and burst-based single-molecule experiments rely on a confocal detection volume that is well approximated by a three-dimensional Gaussian molecular detection function (MDF):		Both FCS and burst-based single-molecule experiments rely on a confocal detection volume that is well approximated by a three-dimensional Gaussian molecular detection function (MDF):

	$$		$$
-	\mathrm{MDF}(x,y,z) \propto	+	W(\mathbf r)
-	\exp\left(-2\frac{x^2+y^2}{w_0^2}\right)	+	=
-	\exp\left(-2\frac{z^2}{z_0^2}\right)	+
		+	\exp\left(
		+	-\frac{2(x^2+y^2)}{w_0^2}
		+	-\frac{2z^2}{z_0^2}
		+	\right)
	$$		$$

-	Here, $w_0$ and $z_0$ denote the lateral and axial $1/e^2$ radii of the detection volume.	+	where $w_0$ and $z_0$ denote the lateral and axial $1/e^2$ radii of the detection volume.

-	This model forms the basis of classical FCS theory and has been validated extensively in both theoretical and experimental studies.	+	This Gaussian MDF constitutes the standard model underlying classical FCS theory and most analytical treatments of single-molecule residence times. It provides a consistent geometric framework for comparing correlation-based and trajectory-based observables.

-	===== Diffusion and transit-time distributions =====	+	===== 3. Diffusive transport and transit-time distributions =====

-	For freely diffusing molecules, transport is governed by the three-dimensional diffusion propagator:	+	For freely diffusing molecules, transport is governed by the three-dimensional diffusion propagator

	$$		$$
-	P(\mathbf{r},t) = (4\pi D t)^{-3/2}	+	G(\mathbf r,t \mid \mathbf r_0,0)
-	\exp\left(-\frac{\|\mathbf{r}\|^2}{4Dt}\right)	+	=
		+	\frac{1}{(4\pi Dt)^{3/2}}
		+	\exp\left(
		+	-\frac{\|\mathbf r-\mathbf r_0\|^2}{4Dt}
		+	\right)
	$$		$$

-	A crucial point is that the transit time through the confocal volume is not a single well-defined quantity, but a random variable with a broad distribution.	+	where $D$ is the diffusion coefficient.

-	This distribution depends on the diffusion coefficient $D$, the molecular entry position, and the spatial weighting imposed by the MDF.	+	A crucial point is that the time a molecule spends in the detection volume is not a single well-defined quantity but a stochastic variable with a broad distribution. This distribution depends on the diffusion coefficient, the entry position of the molecule, and the spatial weighting imposed by the MDF. Any experimentally extracted time scale therefore corresponds to a specific statistical functional of this distribution rather than to a unique physical residence time.

-	===== FCS diffusion time $t_D$ =====	+	===== 4. FCS diffusion time $t_D$ =====

-	==== Definition ====	+	==== 4.1 Definition ====

-	In FCS, the fluorescence intensity autocorrelation function for pure diffusion can be written as:	+	In FCS, the normalized fluorescence intensity autocorrelation function is defined as

	$$		$$
-	G(\tau) =	+	G(\tau)
		+	=
		+
		+	\frac{\langle \delta I(t)\,\delta I(t+\tau)\rangle}{\langle I\rangle^2}
		+	$$
		+
		+	For pure diffusion, this can be expressed as
		+
		+	$$
		+	G(\tau)
		+	\propto
		+	\int d^3r \int d^3r_0\,
		+	W(\mathbf r)\,W(\mathbf r_0)\,
		+	G(\mathbf r,\tau \mid \mathbf r_0,0)
		+	$$
		+
		+	For a three-dimensional Gaussian MDF, this integral yields
		+
		+	$$
		+	G(\tau)
		+	=
	\frac{1}{N}		\frac{1}{N}
-	\frac{1}{(1+\tau/t_D)\sqrt{1+\tau/(S^2 t_D)}}	+	\frac{1}{(1+\tau/t_D)\sqrt{1+\tau/(\kappa^2 t_D)}}
	$$		$$

-	where $S = z_0 / w_0$.	+	with

-	==== Physical meaning ====	+	$$
		+	t_D = \frac{w_0^2}{4D},\qquad
		+	\kappa = \frac{z_0}{w_0}
		+	$$

-	The diffusion time $t_D$ is not the mean residence time of an individual molecule.	+	==== 4.2 Physical interpretation ====

-	Instead, it is a correlation-derived time scale determined by the spatial extent of the detection function. Formally, it can be related to the second spatial moment of the MDF.	+	The diffusion time $t_D$ is not the mean residence time of an individual molecule. Instead, it is a correlation-derived geometry time scale governed by the quadratic spatial weighting of the MDF in the autocorrelation function.

-	Thus, $t_D$ reflects ensemble-averaged transport properties rather than individual molecular trajectories.	+	Formally,

-	===== Burst duration $t_B$ =====	+	$$
		+	t_D
		+	\approx
		+	\frac{\langle r^2\rangle_{W^2}}{6D},\qquad
		+	\langle r^2\rangle_{W^2}
		+	=

-	==== Definition ====	+	\frac{\int r^2 W(\mathbf r)^2\,d^3r}
		+	{\int W(\mathbf r)^2\,d^3r}
		+	$$

-	In burst analysis, individual molecular events are identified by applying an intensity threshold.	+	For a Gaussian MDF, the lateral contribution evaluates to

-	A burst is defined as the time interval during which the detected signal remains above this threshold.	+	$$
		+	\langle r^2\rangle_{W^2} = \frac{3}{4}w_0^2
		+	$$

-	As a consequence, bursts begin and end on iso-intensity surfaces of the MDF rather than at the geometric boundary of the confocal detection volume.	+	which directly leads to the expression $t_D = w_0^2/(4D)$.

-	For a Gaussian detection function, this surface is an ellipsoid whose effective radius depends on the chosen threshold.	+	===== 5. Burst duration $t_B$ =====

-	==== Statistical meaning ====	+	==== 5.1 Definition ====

-	The mean burst duration is defined as:	+	In burst analysis, individual molecular events are identified by applying an intensity threshold $W_{th}$. A burst is defined as

	$$		$$
-	\langle t_B \rangle = \int_0^\infty t\,p_B(t)\,dt	+	t_B = t_{exit} - t_{entry},\qquad
		+	W[\mathbf r(t)] > W_{th}
	$$		$$

-	Because short, peripheral trajectories often remain below threshold, the burst-time distribution differs from the full transit-time distribution.	+	Bursts therefore begin and end on iso-intensity surfaces of the MDF rather than at a geometric boundary of the confocal volume.

-	Burst analysis therefore yields a threshold-selected first moment of molecular residence times.	+	For a Gaussian detection function, the effective threshold radius scales as

-	===== Relation between diffusion time and burst duration =====	+	$$
		+	r_{th}
		+	\propto
		+	w_0\sqrt{\ln(1/W_{th})}
		+	$$

-	For a three-dimensional Gaussian detection volume, pure diffusion, and moderate threshold conditions, analytical treatments show:	+	==== 5.2 Statistical interpretation ====
		+
		+	The mean burst duration is

	$$		$$
-	\langle t_B \rangle \approx \frac{4}{3}\,t_D	+	\langle t_B\rangle
		+	=
		+
		+	\int_0^\infty t\,p_{burst}(t)\,dt
	$$		$$

-	===== Origin of the factor 4/3 =====	+	where $p_{burst}(t)$ denotes the threshold-conditioned residence-time distribution.
		+
		+	Burst analysis measures a first moment of residence times with linear spatial weighting, modified by explicit event selection. Short, peripheral trajectories often do not exceed the detection threshold and therefore do not contribute to the measured distribution.

-	The appearance of this constant factor reflects the fundamentally different nature of the two observables:	+	==== 5.3 Burst selection procedure ====

-	* the FCS diffusion time is obtained from an autocorrelation function and therefore involves a quadratic weighting of the MDF	+	In practice, burst detection is a multi-step selection process.
-	* the mean burst duration corresponds to a linearly weighted residence time conditioned on being above a detection threshold	+

-	For a Gaussian detection profile, this mismatch in statistical weighting leads, under ideal conditions, to the universal ratio:	+	Intensity thresholding.
		+	Photon arrival times are binned into short intervals $\Delta t$ or processed using a sliding window. A burst is initiated when the photon count rate exceeds a predefined threshold

	$$		$$
-	\frac{\langle t_B \rangle}{t_D} = \frac{4}{3}	+	R(t)=\frac{N_\gamma(t,\Delta t)}{\Delta t}>R_{th}
	$$		$$

-	This interpretation is consistent with theoretical treatments of single-molecule photon trajectories and residence-time statistics and with early analyses of diffusion in confocal volumes.	+	which is equivalent, up to proportionality factors, to $W[\mathbf r(t)]>W_{th}$.

-	===== Influence of burst-selection thresholds =====	+	Temporal continuity and photon-number criteria.
		+	To suppress spurious threshold crossings due to shot noise, bursts are often required to satisfy

-	In experimental practice, the ratio $\langle t_B \rangle / t_D$ is not universal.	+	$$
		+	t_B \ge t_{min},\qquad
		+	N_\gamma \ge N_{\gamma,min}
		+	$$

-	Increasing the burst-selection threshold preferentially selects long, central trajectories, thereby increasing the mean burst duration relative to the FCS diffusion time.	+	Selection functional.
		+	The burst-selection procedure can be written formally as

-	Conversely, photophysical effects such as blinking or inappropriate binning can shorten apparent burst durations without significantly affecting the FCS diffusion time, provided these effects are properly modeled in the correlation analysis.	+	$$
		+	\mathcal S[\mathbf r(t)]
		+	=
		+	\Theta(W[\mathbf r(t)]-W_{th})\,
		+	\Theta(t_B-t_{min})\,
		+	\Theta(N_\gamma-N_{\gamma,min})
		+	$$

-	===== Flow effects: decoupling of burst duration and FCS diffusion time =====	+	leading to an observed distribution

-	==== Physical background ====	+	$$
		+	p_{burst}(t)
		+	\propto
		+	\left\langle
		+	\delta(t-t_B)\,
		+	\mathcal S[\mathbf r(t)]
		+	\right\rangle
		+	$$

-	In the presence of flow, molecular transport through the confocal detection volume is no longer governed by diffusion alone.	+	==== 5.4 Consequences of burst selection ====

-	Directed motion introduces an additional transport mechanism that modifies single-molecule trajectories.	+	Burst selection introduces intrinsic and controllable biases. The mean burst duration depends explicitly on the chosen thresholds, increasing thresholds bias toward central trajectories and increase $\langle t_B\rangle$, and bursts are defined by iso-intensity surfaces rather than geometric boundaries. Burst statistics are also sensitive to photophysical processes such as blinking and photobleaching.

-	==== Effect on burst duration ====	+	===== 6. Comparison of diffusion time and burst duration =====

-	Burst analysis directly reflects single-molecule residence times.	+	Although both observables probe molecular motion through the same confocal detection volume, their statistical definitions differ fundamentally. The FCS diffusion time $t_D$ is determined by quadratic spatial weighting of the MDF and reflects the decay of concentration correlations. In contrast, the mean burst duration $\langle t_B\rangle$ is governed by linear spatial weighting combined with explicit selection rules.

-	Directed flow shortens these residence times by accelerating molecular passage through the detection volume.	+	Because of burst-selection thresholds, $\langle t_B\rangle$ can be smaller than, comparable to, or larger than $t_D$. There is no universal ordering between the two quantities, whereas $t_D$ is largely insensitive to burst-selection parameters.

-	Consequently, burst duration distributions shift toward shorter times even when the diffusion coefficient and optical geometry remain unchanged.	+	===== 7. Mathematical comparison and conditions for a fixed ratio =====

-	==== Effect on FCS diffusion time ====	+	Under the assumptions of pure diffusion, Gaussian detection geometry, moderate burst-selection thresholds, and negligible photophysical interruptions, a closed analytical relation can be derived.

-	In contrast, the FCS diffusion time remains essentially constant under moderate flow, as it is primarily determined by the optical geometry and diffusion coefficient.	+	For a Gaussian MDF,

-	==== Relation to the 4/3 factor under flow ====	+	$$
		+	\langle r^2\rangle_{W^2} = \frac{3}{4}w_0^2,\qquad
		+	\langle r^2\rangle_W = w_0^2
		+	$$

-	The relation above is derived under the assumption of pure diffusion.	+	Using $\langle r^2\rangle \approx 6Dt$,

-	The presence of flow violates these assumptions by introducing directed transport.	+	$$
		+	t_D = \frac{w_0^2}{4D},\qquad
		+	\langle t_B\rangle = \frac{w_0^2}{3D}
		+	$$

-	Deviations from the 4/3 ratio under flow conditions therefore reflect the fundamentally different sensitivity of burst-based and correlation-based observables rather than a breakdown of the Gaussian detection model.	+	Thus,

-	===== Summary =====	+	$$
		+	\frac{\langle t_B\rangle}{t_D} = \frac{4}{3}
		+	$$

-	Although burst duration and FCS diffusion time often appear numerically similar, they represent fundamentally different quantities.	+	This ratio represents a diffusion-limited reference case and is not universal.

-	The diffusion time is a correlation-derived, geometry-dependent time scale linked to the second spatial moment of the detection function, whereas the mean burst duration is a threshold-selected first moment of individual molecular residence times.	+	===== 8. Flow: advection--diffusion effects =====

-	Under ideal diffusion conditions, this distinction naturally leads to:	+	==== 8.1 Transport equation and time scales ====

	$$		$$
-	\langle t_B \rangle \approx \frac{4}{3}\,t_D	+	\frac{\partial c}{\partial t}
		+	+
		+	\mathbf v\cdot\nabla c
		+	=
		+
		+	D\nabla^2 c,\qquad
		+	\tau_v=\frac{w_0}{v},\qquad
		+	\mathrm{Pe}=\frac{vw_0}{D}
	$$		$$

-	Threshold selection and directed flow introduce systematic deviations from this relation, further emphasizing the conceptual difference between burst-based and correlation-based observables.	+	==== 8.2 Effect of flow on FCS ====
		+
		+	With advection--diffusion propagator
		+
		+	$$
		+	G(\mathbf r,\tau\mid\mathbf r_0,0)
		+	=
		+
		+	\frac{1}{(4\pi D\tau)^{3/2}}
		+	\exp\left(
		+	-\frac{\|\mathbf r-\mathbf r_0-\mathbf v\tau\|^2}{4D\tau}
		+	\right)
		+	$$
		+
		+	The autocorrelation function factorizes as
		+
		+	$$
		+	G(\tau)
		+	=
		+
		+	G_{diff}(\tau)
		+	\exp\left(
		+	-\frac{(v\tau)^2}{w_0^2(1+\tau/t_D)}
		+	\right)
		+	$$
		+
		+	When flow is modeled explicitly, $t_D$ remains unchanged.
		+
		+	==== 8.3 Effect of flow on burst duration ====
		+
		+	Burst durations scale approximately as
		+
		+	$$
		+	\langle t_B\rangle \sim \frac{L_{eff}}{v}
		+	\qquad (\mathrm{Pe}\gg1)
		+	$$
		+
		+	and therefore decrease continuously with increasing flow velocity.
		+
		+	==== 8.4 Consequences ====
		+
		+	Flow selectively affects burst durations while leaving the FCS diffusion time invariant when modeled correctly.
		+
		+	===== 9. Photobleaching effects =====
		+
		+	==== 9.1 General model ====
		+
		+	$$
		+	P_{survival}(t)=e^{-k_{bl}t},\qquad
		+	\tau_{bl}=\frac{1}{k_{bl}}
		+	$$
		+
		+	==== 9.2 Effect on FCS ====
		+
		+	$$
		+	G(\tau)=G_{diff}(\tau)e^{-\tau/\tau_{bl}},\qquad
		+	\tau_{bl}\gg t_D
		+	$$
		+
		+	Bleaching primarily affects amplitude and long-delay behavior.
		+
		+	==== 9.3 Effect on burst duration ====
		+
		+	$$
		+	t_{B,obs}=\min(t_{transport},t_{bleach}),\qquad
		+	p_{obs}(t)=p_{trans}(t)e^{-k_{bl}t}
		+	$$
		+
		+	Burst durations are systematically shortened.
		+
		+	===== 10. Conclusion =====
		+
		+	The diffusion time extracted from fluorescence correlation spectroscopy and the burst duration obtained from single-molecule detection probe molecular transport through the same confocal detection volume, yet they represent fundamentally different observables.
		+
		+	The FCS diffusion time $t_D$ is a correlation-derived geometry time scale governed by quadratic spatial weighting of the molecular detection function, whereas the mean burst duration $\langle t_B\rangle$ is a residence-time observable governed by linear weighting and explicit burst-selection criteria.
		+
		+	Because of these differences, there is no general reason for $t_D$ and $\langle t_B\rangle$ to coincide. Depending on burst-selection thresholds, the mean burst duration can be smaller than, comparable to, or larger than the diffusion time.
		+
		+	Under ideal diffusion conditions--Gaussian detection geometry, moderate thresholding, and negligible photophysical interruptions--a fixed relation can be derived,
		+
		+	$$
		+	\frac{\langle t_B\rangle}{t_D}=\frac{4}{3}
		+	$$
		+
		+	This factor is a geometric consequence of Gaussian detection combined with diffusion statistics and specific selection conditions. It is not universal.
		+
		+	Directed flow and photobleaching systematically break this reference relation. Flow introduces an additional transport time scale that leaves the FCS diffusion time unchanged when modeled explicitly, while directly shortening burst durations. Photobleaching truncates bursts and biases burst statistics strongly, whereas its influence on FCS is largely confined to slow non-stationarity.
		+
		+	Diffusion time and burst duration should therefore be regarded as complementary rather than interchangeable observables. Deviations from the diffusion-limited reference ratio provide direct physical insight into burst-selection effects, directed transport, and photophysical limitations in confocal single-molecule fluorescence experiments.

	===== References =====		===== References =====

-	* Elson, E. L.; Magde, D. Biopolymers 1974, 13, 1--27.	+	Elson, E. L.; Magde, D. Biopolymers 1974, 13, 1--27.
-	* Rigler, R.; Elson, E. L. (eds.) Fluorescence Correlation Spectroscopy, Springer.	+
-	* Rigler, R.; Widengren, J. Bioscience Reports 1990, 10, 395--403.	+	Rigler, R.; Elson, E. L. (eds.) Fluorescence Correlation Spectroscopy, Springer.
-	* Gopich, I. V.; Szabo, A. Journal of Chemical Physics 2005, 122, 014707.	+
-	* Rüttinger, S. Dissertation, 2006.	+	Rigler, R.; Widengren, J. Bioscience Reports 1990, 10, 395--403.
-	* Dörr, D. Dissertation, 2014.	+
		+	Widengren, J.; Rigler, R. Bioimaging 1996, 4, 149--157.
		+
		+	Gopich, I. V.; Szabo, A. Journal of Chemical Physics 2005, 122, 014707.
		+
		+	Ruettinger, S. Dissertation, 2006.
		+
		+	Doerr, D. Dissertation, 2014.

start

Anonymous (anonymous@undisclosed.example.com) — 2025-02-14T09:43:52+00:00

2025/02/14 09:36		current
Line 8:		Line 8:
	==== Why do I not get the same count rates on both SPAD detectors? ====		==== Why do I not get the same count rates on both SPAD detectors? ====

-	In this article we assume that both detectors are SPADs of the same kind (both Excelitas SPADs, or both PDM-SPADs, or both tau-SPADs).	+	In this article we assume that both detectors are SPADs of the same kind (both Excelitas SPADs, or both PDM-SPADs, or both tau-SPADs).

-	If you are using either a proper (see below) 50/50 cube beamsplitter to split the signal between two detectors or a 100% mirror to direct the signal to one detector and then the other, but still observe very different count rates on the two detectors, this discrepancy could be due to the following reasons:	+	If you are using either a proper (see below) 50/50 beamsplitter to split the signal between two detectors or a 100% mirror to direct the signal to one detector and then the other, but still observe very different count rates on the two detectors, this discrepancy could be due to the following reasons:

	1. Misalignment of the Lens in Front of the SPAD:		1. Misalignment of the Lens in Front of the SPAD:
Line 33:		Line 33:

	- Use a concentrated dye solution and ensure the same emission filter is mounted in front of each detector, or place the emission filter before the splitter (holder before the pinhole).		- Use a concentrated dye solution and ensure the same emission filter is mounted in front of each detector, or place the emission filter before the splitter (holder before the pinhole).
-	- Direct all light to Detector 1 and record the count rate (the count rate should be between 100 kcps and 700 kcps - adjust the laser power accordingly).	+	- Direct all light to Detector 1 and record the count rate (the count rate should be between 100 kcps and 700 kcps - adjust the laser power accordingly).
	- Use a 100% mirror to redirect all light to Detector 2 and record the count rate.		- Use a 100% mirror to redirect all light to Detector 2 and record the count rate.
	- Compare the count rates of Detector 1 and Detector 2. If the detectors are of the same type, similar count rates are expected.		- Compare the count rates of Detector 1 and Detector 2. If the detectors are of the same type, similar count rates are expected.
-	- If the detectors are the same type, and the count rate difference is more than 10%, the alignment on the detector with the lower countrate should be checked.	+	* If the count rate difference is more than 10%, the alignment on the detector with the lower countrate should be checked and maximized.
		+	* Follow the "Everyday Alignment" Procedure from the MicroTime 200 manual to correctly align the lens. It might be that also "Fundamental Alignment" is necessary.
	- Introduce the beamsplitter and measure the combined count rate from both detectors.		- Introduce the beamsplitter and measure the combined count rate from both detectors.
		+	* The sum of the signal on both detectors should be the same as from the previous measurement (i.e. 100% going to Detector 1 or 2).
		+	* If it is not (again, allow for 10% error), check the alignment.

-	If the sum	+	If you have followed these instructions, and the light is not split 50/50, then this is likely a property of the beamsplitter. Make sure that is it the correct kind of beamsplitter (see above), and/or contact us for advice.
-		+
-		+
-	By following these steps, you can determine if the discrepancy in count rates is due to misalignment or the characteristics of the beam-splitter.	+
	===== SymPhoTime =====		===== SymPhoTime =====

why_do_i_not_get_the_same_count_rates_on_both_spad_detectors

Anonymous (anonymous@undisclosed.example.com) — 2025-02-14T15:47:18+00:00

2025/02/14 15:42		current
Line 1:		Line 1:
	====== Why do I not get the same count rates on both SPAD detectors? ======		====== Why do I not get the same count rates on both SPAD detectors? ======
		+	{{tag> detectors faq SPAD MT200}}

	In this article we assume that both detectors are SPADs of the same kind (both Excelitas SPADs, or both PDM-SPADs, or both tau-SPADs).		In this article we assume that both detectors are SPADs of the same kind (both Excelitas SPADs, or both PDM-SPADs, or both tau-SPADs).

advantages_and_disadvantages_of_two_photon_excitation_tpe

Anonymous (anonymous@undisclosed.example.com) — 2014-01-28T13:07:58+00:00

2014/01/23 11:03		current
Line 1:		Line 1:
-	'Comment
	====== Advantages and disadvantages of two photon excitation (TPE) ======		====== Advantages and disadvantages of two photon excitation (TPE) ======

antibunching_measurements

Anonymous (anonymous@undisclosed.example.com) — 2025-04-09T14:01:09+00:00

2023/09/07 11:54		current
Line 5:		Line 5:
	====== How to Perform Antibunching Measurements ======		====== How to Perform Antibunching Measurements ======

		+	Antibunching Measurements are (normally) measured in T2 mode. In T2 mode, all input channels of the TCSPC electronics are treated the same, also the "Sync" channel. Therefore the sync has to be disabled either by disconneting the cable, or by changing the CFD level or trigger level to such a value that the sync is not recognized any more. i.e. Sync rate = 0 . Directly in the Sepia software, the sync out can also be disabled.
		+
		+	The descriptions below only mention physically disconnecting the sync. This is strictly necessary only for the PicoHarp 300 because it only has two channels.
	===== Emission Filters =====		===== Emission Filters =====

check_overlap_of_different_color_confocal_volumes

Anonymous (anonymous@undisclosed.example.com) — 2015-02-25T13:55:02+00:00

2013/11/27 10:04		current
Line 1:		Line 1:
-	====== How to check the overlap of different color confocal volumes ======	+	{{tag>howto confocal_volume}}
		+	====== How to Check the Overlap of Different Color Confocal Volumes ======

	Because both lasers are fed through the same fiber, there cannot be any optical beam displacement. According to my records, your system has the latest apochromatic fiber outcoupler, therefore there is practically nothing you can improve: lasers mounted in the [[products:LCU]] are always well overlapped. This is a simple consequence of the [[Products:MicroTime 200]] design.		Because both lasers are fed through the same fiber, there cannot be any optical beam displacement. According to my records, your system has the latest apochromatic fiber outcoupler, therefore there is practically nothing you can improve: lasers mounted in the [[products:LCU]] are always well overlapped. This is a simple consequence of the [[Products:MicroTime 200]] design.
Line 10:		Line 11:
	You need an appropriate dual band major dichroic and PIE excitation with the Sepia. In this case there is no need for detection color splitting. One detector is sufficient. Use a single long pass filter, target and image a single bead. Of course, you have to set the laser intensities so that both laser excitations contribute with approximately half of the final detected sum count rate.		You need an appropriate dual band major dichroic and PIE excitation with the Sepia. In this case there is no need for detection color splitting. One detector is sufficient. Use a single long pass filter, target and image a single bead. Of course, you have to set the laser intensities so that both laser excitations contribute with approximately half of the final detected sum count rate.

-	In [[Products:SymPhoTime]], calculate two images: one for each PIE time window, that is, one for each excitation laser. (It is easy with the [[Products:SymPhoTime]], use the graphical tool for setting the time-gates.) By comparing the two images, you should see no significant displacement.	+	In [[Products:SymPhoTime64]], calculate two images: one for each PIE time window, that is, one for each excitation laser. (It is easy with the [[Products:SymPhoTime64]], use the graphical tool for setting the time-gates.) By comparing the two images, you should see no significant displacement.

flim-fret_calculation_for_single_exponential_donors

Anonymous (anonymous@undisclosed.example.com) — 2020-05-06T09:38:57+00:00

2020/05/05 16:06		current
Line 81:		Line 81:

	* In this example set the lower intensity threshold to 500 counts.		* In this example set the lower intensity threshold to 500 counts.
		+	{{ :howto:flim-fret-1expd_lowerthresh_500_bearb.png?400 \|}}
	* Click "OK"		* Click "OK"

	Response: In the image, only pixels with higher photon counts are highlighted. Adapt the intensity maximum to see the highlighted pixels more clearly.		Response: In the image, only pixels with higher photon counts are highlighted. Adapt the intensity maximum to see the highlighted pixels more clearly.
-	{{ :howto:flim-fret-1expd_roi_from_thresh_2_v2.png?400 \|}}	+	{{ :howto:flim-fret-1expd_roi_from_thresh_2_v3.png?400 \|}}

	* In the lifetime fitting panel, keep the default Fitting model "Mono-Exp. Donor".		* In the lifetime fitting panel, keep the default Fitting model "Mono-Exp. Donor".

how_to_measure_the_instrument_response_function_irf

Anonymous (anonymous@undisclosed.example.com) — 2023-09-07T22:55:03+00:00

2023/09/07 22:49		current
Line 13:		Line 13:


-	=== Low count rate during IRF measurements is important ===	+	==== Low count rate during IRF measurements is important! ====

-	Note: Make sure that the detection count rate is much lower than the count rate used for fluorescence decay measurement.Diluting the scattering solution is better than using grey filters. Ideal is when the decay and the IRF are recorded at the same [[glossary:differential count rate]] (and NOT at the same average count rates).	+	Make sure that the detection count rate is much lower than the count rate during a fluorescence decay measurement. Diluting the scattering solution is better than using grey (ND) filters. Ideal is when the decay and the IRF are recorded at the same [[glossary:differential count rate]] (and NOT at the same average count rate).

-	If the IRF should be measured on a microscope system with SPAD detectors, in the UV range also the Raman-scattering of water can be used. E.g. the Raman scattering can be recorded with a HQ480/40 bandpass filter, if a 405nm diode is used. This method is less suited for long wavelengths, as the Raman efficiency decreases. The water must be very pure in order not to capture any fluorescence.	+	If the IRF should be measured in the UV range on a microscope system with SPAD detectors, the Raman scattering of water can be used, too. E.g. the Raman scattering can be recorded with a HQ480/40 bandpass filter, if a 405nm laser diode is used. This method is less suited for long wavelengths, as the Raman scattering decreases. In order to avoid signal contamination by any fluorescence, the water must be very pure.


	===== Using samples with ultrafast decay =====		===== Using samples with ultrafast decay =====

-	Some detectors (particularly SPADs) have wavelength dependent timing response. In this case an IRF recorded at the excitation wavelength may not be useful for precise reconvolution. The solution is to acquire the IRF at the fluorescence wavelength, or at least spectrally closer to the fluorescence emission.	+	Some detectors (particularly MPD SPADs) have a wavelength dependent timing response. In this case an IRF recorded at the excitation wavelength may not be useful for precise reconvolution. The solution is to acquire the IRF at the fluorescence wavelength, or at least spectrally closer to the fluorescence emission wavelength.

	==== General recipe ====		==== General recipe ====

lifetime_fitting_using_the_flim_analysis

Anonymous (anonymous@undisclosed.example.com) — 2015-02-25T13:59:23+00:00

2014/06/18 12:19		current
Line 1:		Line 1:
	{{tag>howto analysis FLIM imaging tutorial SymPhoTime}}		{{tag>howto analysis FLIM imaging tutorial SymPhoTime}}

-	~~TOC~~	+	~~NOTOC~~

-	====== Lifetime fitting Using the FLIM-analysis ======	+	====== Lifetime Fitting Using the FLIM Analysis ======

visualizing_dynamics_using_the_multiframe-flim_script

Anonymous (anonymous@undisclosed.example.com) — 2023-02-17T12:41:10+00:00

2021/03/23 10:44		current
Line 1:		Line 1:
-	{{tag>howto FLIM multi-frame imaging analysis dynamics tutorial LSM_upgrade SPT SymPhoTime}}	+	{{tag>howto FLIM multi-frame imaging analysis dynamics tutorial LSM_upgrade SymPhoTime}}

-	====== Visualizing Dynamics Using the Multi Frame FLIM Analysis (updated for SymPhoTime v2.5) ======	+	====== Visualizing Dynamics Using the Multi Frame FLIM Analysis (updated for SymPhoTime v2.5 and above) ======

	===== Introduction =====		===== Introduction =====

lsm710

Anonymous (anonymous@undisclosed.example.com) — 2016-07-21T13:31:38+00:00

2016/07/20 12:02		current
Line 2:		Line 2:


-	====== FLIM Measurement Using a Zeiss LSM710 with a FLIM and FCS Upgrade ======	+	====== FLIM Measurement Using a Zeiss LSM710/LSM780/LSM880 with a FLIM and FCS Upgrade ======

	This tutorial shows the recording of FLIM images using an LSM upgrade kit, in this case a Zeiss LSM710.		This tutorial shows the recording of FLIM images using an LSM upgrade kit, in this case a Zeiss LSM710.
Line 11:		Line 11:
	* to monitor concentration changes using a reporter dye		* to monitor concentration changes using a reporter dye

-	* To monitor interactions between two molecules via FRET.	+	* To monitor interactions between two molecules via FRET. \\

-	The data acquisition is general principles of the data acquisition are analogous in all LSM upgrade kits.

-	{{ youtube>QXGqA2IsivM?large }}	+	\\
		+	{{ youtube>QXGqA2IsivM?large }} \\
		+
		+
		+	The data acquisition is analogous also for upgrades on an LSM780 or LSM880, and the general principles of the data acquisition are analogous in all LSM upgrade kits.
		+	\\ \\
		+	\\
		+	{{ youtube>vofLPLVmq-Q?large }} \\

some_origins_of_multiexponetial_decays_for_single_dyes

Anonymous (anonymous@undisclosed.example.com) — 2019-03-19T12:31:43+00:00

community_news

Anonymous (anonymous@undisclosed.example.com) — 2020-05-04T12:11:21+00:00

2020/04/14 11:40		current
Line 2:		Line 2:

	===== News from the community=====		===== News from the community=====
		+	* 29.04.20 [[https://www.fret.community/announcement-protein-folding-and-dynamics-webinar/\|Protein folding and dynamics Webinar-Series.]] \\ \\
	* 14.04.20 [[https://analyticalscience.wiley.com/do/10.1002/was.00050003/full/bioimagedataanalysis.pdf\|Bioimage Data Analysis.]] New Expanded version of a nice open-source introduction. \\ \\		* 14.04.20 [[https://analyticalscience.wiley.com/do/10.1002/was.00050003/full/bioimagedataanalysis.pdf\|Bioimage Data Analysis.]] New Expanded version of a nice open-source introduction. \\ \\
-	* 14.04.20 [[https://science.sciencemag.org/content/368/6486/78\|De novo design of protein logic gates.]] Fundamendal enzymology work. Chen et al. describe the design of logic gates that can regulate protein association. The gates were built from small, designed proteins that all have a similar structure but where one module can be designed to interact specifically with another module. Using monomers and covalently connected monomers as inputs and encoding specificity through designed hydrogen-bond networks allowed the construction of two-input or three-input gates based on competitive binding. \\ \\	+	* 14.04.20 [[https://science.sciencemag.org/content/368/6486/78\|De novo design of protein logic gates.]] Fundamental enzymology work. Chen et al. describe the design of logic gates that can regulate protein association. The gates were built from small, designed proteins that all have a similar structure but where one module can be designed to interact specifically with another module. Using monomers and covalently connected monomers as inputs and encoding specificity through designed hydrogen-bond networks allowed the construction of two-input or three-input gates based on competitive binding. \\ \\
	* 31.03.20 FRET community has a new advisory board, [[https://www.fret.community/\|FRET community]]. Happy that Dr. Marcelle König , a senior scientist in PicoQuant was elected. \\ \\		* 31.03.20 FRET community has a new advisory board, [[https://www.fret.community/\|FRET community]]. Happy that Dr. Marcelle König , a senior scientist in PicoQuant was elected. \\ \\
	* 31.03.20 Extremely helpful reference paper about confocal microscopy. [[https://www.nature.com/articles/s41596-020-0313-9\|Tutorial: guidance for quantitative confocal microscopy]]. Available as a poster [[https://www.nature.com/articles/s41596-020-0307-7\|here.]] \\ \\		* 31.03.20 Extremely helpful reference paper about confocal microscopy. [[https://www.nature.com/articles/s41596-020-0313-9\|Tutorial: guidance for quantitative confocal microscopy]]. Available as a poster [[https://www.nature.com/articles/s41596-020-0307-7\|here.]] \\ \\

dead_time

Anonymous (anonymous@undisclosed.example.com) — 2015-06-03T15:04:41+00:00

2026/04/11 16:45	current
Line 1:	Line 1:
	+	====== Dead Time ======
	+	In TCSPC the term dead time refers to the time the TCSPC system needs armed again after detecting an event. During the dead time the TCSPC system is blind.

	+	If for example two photons are detected with the dead time of the TCSPC device the second photon will be lost. This leads to the so called [[glossary:Pile-up Effect]].
	+
	+	Note that not only TCSPC devices but also photon counting devices exhibit dead times.

differential_count_rate

Anonymous (anonymous@undisclosed.example.com) — 2017-09-26T11:46:03+00:00

2017/09/23 22:43		current
Line 15:		Line 15:
	A frequently asked question is: "//Why we have to consider some differential count rate if we are detecting rarely one photon (count) per excitation cycle, anyway? Detection probability of two or more photons in one cycle is completely negligible!//"		A frequently asked question is: "//Why we have to consider some differential count rate if we are detecting rarely one photon (count) per excitation cycle, anyway? Detection probability of two or more photons in one cycle is completely negligible!//"

-	Not really. In case of pulsed signals the average count rate is a misleading quantity. An average count rate value does not take into account when and how those photons are emitted and detected. Interpreting a 100 kcps intensity as a constant emission rate (Poisson mean rate, in maths terms) is a misconception. The physics of the measurement is completely different. These photons are obviously not emitted evenly, one by one over the whole one second period. They arrive to the detector bunched, as flashes. These are short time intervals with huge photon density (rate), separated by long "dark", quiet periods. Getting a count at "1% of SYNC rate" merely means a sampling rate of a signal with very inhomogeneous time distribution.	+	Not really. In case of pulsed signals the average count rate is a misleading quantity. An average count rate value does not take into account when and how those photons are emitted and detected. Interpreting a 100 kcps intensity as a constant emission rate (Poisson mean rate, in math terms) is a misconception. The physics of the measurement is completely different. These photons are obviously not emitted evenly, one by one over the whole one second period. They arrive to the detector bunched, as flashes. These are short time intervals with huge photon density (rate), separated by long "dark", quiet periods.

-	In mathematical terms, "average count rate of 1..2% of SYNC rate" means the overall detection probability, integrated over the whole duration of a measurement. The concept of //differential count rate// is related to the //probability density function//.	+	In yet another words, achieving a final count rate at "1% of SYNC rate" is a result of sparse sampling with dead time. The sampled signal features much higher photon density, but lasts only for a short time.
		+
		+	In mathematical terms, "average count rate of 1..2% of SYNC rate" means the overall detection probability, integrated over the whole duration of a measurement. The concept of //differential count rate// is related to the //probability density function//. The detected signal in TCSPC has a very inhomogeneous time distribution.

	Note: for detectors that exhibit count rate dependent shifting of the IRF, extra care has to be taken when measuring the IRF and directly using it for decay analysis. ((Takuhiro Otosu, Kunihiko Ishii and Tahei Tahara, Note: Simple calibration of the counting-rate dependence of the timing shift of single photon avalanche diodes by photon interval analysis, Rev. Sci. Instrum. 84, 036105 (2013); [[http://dx.doi.org/10.1063/1.4794769]]))		Note: for detectors that exhibit count rate dependent shifting of the IRF, extra care has to be taken when measuring the IRF and directly using it for decay analysis. ((Takuhiro Otosu, Kunihiko Ishii and Tahei Tahara, Note: Simple calibration of the counting-rate dependence of the timing shift of single photon avalanche diodes by photon interval analysis, Rev. Sci. Instrum. 84, 036105 (2013); [[http://dx.doi.org/10.1063/1.4794769]]))

pile-up_effect

Anonymous (anonymous@undisclosed.example.com) — 2015-06-03T15:09:55+00:00

2015/03/26 12:10		current
Line 1:		Line 1:
-	{{tag>pile-up TCSPC}}	+	{{tag> TCSPC pile-up dead_time}}
		+	~~TOC~~

	====== Pile-Up Effect ======		====== Pile-Up Effect ======
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	{{ :glossary:pile-up_example.png?600 \|}}		{{ :glossary:pile-up_example.png?600 \|}}
-

t2-mode

Anonymous (anonymous@undisclosed.example.com) — 2015-02-10T16:24:41+00:00

2013/08/06 14:59		current
Line 1:		Line 1:
	====== T2-mode ======		====== T2-mode ======

-	A special measurement mode of the [[Products:PicoHarp 300]]	+	A special measurement mode of the [[Products:PicoHarp 300]], [[Products:HydraHarp 400]] and [[Products:TimeHarp 260]]



-	In T2 Mode both signal inputs of the PicoHarp 300 are functionally identical. There is no dedication of input channel 0 to a [[SYNC]] signal. Usually both inputs are used to connect photon detectors. The events from both channels are recorded independently and treated equally. In each case an event record is generated that contains information about the channel it came from and the arrival time of the event with respect to the overall measurement start. The timing is recorded with 4 ps resolution. Each T2 Mode event record consists of 32 bits. There are 4 bits for the channel number and 28 bits for the time-tag. Routing is not supported. If the time tag overflows, a special overflow marker record is inserted in the data stream, so that upon processing of the data stream a theoretically infinite time span can be recovered at full resolution. Dead times exist only within each channel (95 ns typ.) but not across the channels. Therefore, cross correlations can be calculated down to zero lag time. This allows powerful new application such as [[FCS]] with lag times from picoseconds to hours. Autocorrelations can also be calculated at the full resolution but of course only starting from lag times larger than the deadtime.	+	In T2 Mode both signal inputs of the PicoHarp 300 are functionally identical. There is no dedication of input channel 0 to a [[SYNC]] signal. Usually both inputs are used to connect photon detectors. The events from both channels are recorded independently and treated equally. In each case an event record is generated that contains information about the channel it came from and the arrival time of the event with respect to the overall measurement start. The timing is recorded with 4 ps resolution. Each T2 Mode event record consists of 32 bits. There are 4 bits for the channel number and 28 bits for the time-tag. Routing is not supported. If the time tag overflows, a special overflow marker record is inserted in the data stream, so that upon processing of the data stream a theoretically infinite time span can be recovered at full resolution. [[Dead time]]s exist only within each channel (95 ns typ.) but not across the channels. Therefore, cross correlations can be calculated down to zero lag time. This allows powerful new application such as [[FCS]] with lag times from picoseconds to hours. Autocorrelations can also be calculated at the full resolution but of course only starting from lag times larger than the [[dead time]].

-	The 32 bit event records are queued in a [[FIFO]] (First In First Out) buffer capable of holding up to 256 k event records. The FIFO input is fast enough to accept records at the full speed of the time-to-digital converters (up to 10 Mcps each). This means, even during a fast burst no events will be dropped except those lost in th dead time anyhow. The FIFO output is continuously read by the host PC, thereby making room for fresh incoming events. Even if the average read rate of the host PC is limited, bursts with much higher rate can be recorded for some time. Only if the average count rate over a long period of time exceeds the readout speed of the PC, a FIFO overrun could occur. In case of a FIFO overrun the measurement must be aborted because data integrity cannot be maintained. However, on a modern and well configured PC with Windows 2000 or XP a sustained average count rate over 4 Mcps is possible. This total transfer rate must be shared by the two input channels. For all practically relevant fluorescence detection applications the effective rate per channel is more than sufficient.	+	The 32 bit event records are queued in a [[FIFO]] (First In First Out) buffer capable of holding up to 256 k event records. The FIFO input is fast enough to accept records at the full speed of the time-to-digital converters (up to 10 Mcps each). This means, even during a fast burst no events will be dropped except those lost in the [[dead time]] anyhow. The FIFO output is continuously read by the host PC, thereby making room for fresh incoming events. Even if the average read rate of the host PC is limited, bursts with much higher rate can be recorded for some time. Only if the average count rate over a long period of time exceeds the readout speed of the PC, a FIFO overrun could occur. In case of a FIFO overrun the measurement must be aborted because data integrity cannot be maintained. However, on a modern and well configured PC a sustained average count rate over 4 Mcps is possible. This total transfer rate must be shared by the two input channels. For all practically relevant fluorescence detection applications the effective rate per channel is more than sufficient.

-	For maximum throughput, T2 Mode data streams are normally written directly to disk. The current PicoHarp software 1.2 does not provide any immediate data visualization during a T2 Mode measurement, except count rate and progress display. However, using custom software it is also possible to analyze incoming data "on the fly". Even on-line correlation can be implemented. Obviously this requires efficient processing and possible restrictions in average count rate. The PicoHarp software installation CD contains demo programs to show how T2 Mode files can be read by custom software. The implementation of custom measurement programs requires the PicoHarp programming library, which is available as a separate option.	+	For maximum throughput, T2 Mode data streams are normally written directly to disk. The current Harp software does not provide any immediate data visualization during a T2 Mode measurement, except count rate and progress display. However, using custom software it is also possible to analyze incoming data "on the fly". Even on-line correlation can be implemented. Obviously this requires efficient processing and possible restrictions in average count rate. The PicoHarp software installation CD contains demo programs to show how T2 Mode files can be read by custom software. The implementation of custom measurement programs requires the Harp programming library, which is available as a separate option.

	Note: From a mathematical point of view, the T2 mode should be able to store 28 bit = 228 = 268435456 time bin data before need to "wrap around". However, the real wrap around time as shown in the demo code is only 210698240. This discrepancy is due to the design of the TDCs. Briefly, the [[TDCs]] include a coarse scaler and an interpolator for the the short times. This interpolator is not working in a binary fashion, which finally leads to the fact that not the full available memory is used. This does not lead to any data loss. To reconstruct the full temporal time trace one only needs to follow the procedures shown in the demo code		Note: From a mathematical point of view, the T2 mode should be able to store 28 bit = 228 = 268435456 time bin data before need to "wrap around". However, the real wrap around time as shown in the demo code is only 210698240. This discrepancy is due to the design of the TDCs. Briefly, the [[TDCs]] include a coarse scaler and an interpolator for the the short times. This interpolator is not working in a binary fashion, which finally leads to the fact that not the full available memory is used. This does not lead to any data loss. To reconstruct the full temporal time trace one only needs to follow the procedures shown in the demo code

	see also [[.:t3-mode]]		see also [[.:t3-mode]]
-

t3-mode

Anonymous (anonymous@undisclosed.example.com) — 2015-02-10T16:27:57+00:00

2015/02/10 16:26		current
Line 12:		Line 12:
	- the arrival time of the event pair on the overall experiment time scale (the time tag).		- the arrival time of the event pair on the overall experiment time scale (the time tag).

-	The latter was originally obtained from an independent asynchronous clock. This made it difficult to combine the start-stop timing with the time tag or to know the sync period the event belonged to. For the PicoHarp's T3 Mode a smarter approach was chosen:\\	+	The latter was originally obtained from an independent asynchronous clock. This made it difficult to combine the start-stop timing with the time tag or to know the sync period the event belonged to. For the *Harp's T3 Mode a smarter approach was chosen:\\
	The time tag is now obtained by simply counting sync pulses. From the T3 Mode event records it is therefore possible to precisely determine which sync period a photon event belongs to. Since the sync period is also known precisely, this furthermore allows to reconstruct the arrival time of the photon with respect to the overall experiment time.		The time tag is now obtained by simply counting sync pulses. From the T3 Mode event records it is therefore possible to precisely determine which sync period a photon event belongs to. Since the sync period is also known precisely, this furthermore allows to reconstruct the arrival time of the photon with respect to the overall experiment time.

Line 23:		Line 23:
	If the counter overflows, a special overflow marker record is inserted in the data stream, so that upon processing of the data stream a theoretically infinite time span can be recovered. The 12 bits for the start-stop time difference cover a time span of 4096*R where R is the chosen resolution. At the highest possible resolution of 4 ps this results in a span of 16 ns. If the time difference between photon and the last sync event is larger, the photon event cannot be recorded. This is the same as in histogramming mode, where the number of bins is also finite. However, by choosing a suitable sync rate and a compatible resolution R, it should be possible to reasonably accommodate all relevant experiment scenarios. R can be chosen in doubling steps between 4 ps and 512 ps.		If the counter overflows, a special overflow marker record is inserted in the data stream, so that upon processing of the data stream a theoretically infinite time span can be recovered. The 12 bits for the start-stop time difference cover a time span of 4096*R where R is the chosen resolution. At the highest possible resolution of 4 ps this results in a span of 16 ns. If the time difference between photon and the last sync event is larger, the photon event cannot be recorded. This is the same as in histogramming mode, where the number of bins is also finite. However, by choosing a suitable sync rate and a compatible resolution R, it should be possible to reasonably accommodate all relevant experiment scenarios. R can be chosen in doubling steps between 4 ps and 512 ps.

-	Dead time in T3 Mode is the same as in the other modes (eg. typ. 95 ns for PicoHarp) Since there is only one photon channel, only autocorrelations can be calculated (unless a router is used). This can be done at the resolution R but of course only starting from lag times larger than the deadtime.	+	[[Dead time]] in T3 Mode is the same as in the other modes (eg. typ. 95 ns for PicoHarp) Since there is only one photon channel, only autocorrelations can be calculated (unless a router is used). This can be done at the resolution R but of course only starting from lag times larger than the [[dead time]].

-	The 32 bit event records are queued in a [[FIFO]] (First In First Out) buffer capable of holding up to 256 k event records. The FIFO input is fast enough to accept records at the full speed of the time-to-digital converters (up to 10 Mcps each). This means, even during a fast burst no events will be dropped except those lost in th dead time anyhow. The FIFO output is continuously read by the host PC, thereby making room for fresh incoming events. Even if the average read rate of the host PC is limited, bursts with much higher rate can be recorded for some time. Only if the average count rate over a long period of time exceeds the readout speed of the PC, a FIFO overrun could occur. In case of a FIFO overrun the measurement must be aborted because data integrity cannot be maintained. However, on a modern and well configured PC with Windows 2000 or XP a sustained average count rate over 4 Mcps is possible. This total transfer rate is fully available for input channel 1. For all practically relevant fluorescence detection applications this is more than sufficient.	+	The 32 bit event records are queued in a [[FIFO]] (First In First Out) buffer capable of holding up to 256 k event records. The FIFO input is fast enough to accept records at the full speed of the time-to-digital converters (up to 10 Mcps each). This means, even during a fast burst no events will be dropped except those lost in the [[dead time]] anyhow. The FIFO output is continuously read by the host PC, thereby making room for fresh incoming events. Even if the average read rate of the host PC is limited, bursts with much higher rate can be recorded for some time. Only if the average count rate over a long period of time exceeds the readout speed of the PC, a FIFO overrun could occur. In case of a FIFO overrun the measurement must be aborted because data integrity cannot be maintained. However, on a modern and well configured PC with Windows 2000 or XP a sustained average count rate over 4 Mcps is possible. This total transfer rate is fully available for input channel 1. For all practically relevant fluorescence detection applications this is more than sufficient.

	For maximum throughput, T3 Mode data streams are normally written directly to disk. The current Harp software does not provide any immediate data visualization during a T3 Mode measurement, except count rate and progress display. However, using custom software it is also possible to analyze incoming data "on the fly". Even on-line correlation can be implemented. Obviously this requires efficient processing and possible restrictions in average count rate. The Harp software installation CD contains demo programs showing how T3 Mode files can be read by custom software. The implementation of custom measurement programs requires the *Harp programming library, which is available as a separate option.		For maximum throughput, T3 Mode data streams are normally written directly to disk. The current Harp software does not provide any immediate data visualization during a T3 Mode measurement, except count rate and progress display. However, using custom software it is also possible to analyze incoming data "on the fly". Even on-line correlation can be implemented. Obviously this requires efficient processing and possible restrictions in average count rate. The Harp software installation CD contains demo programs showing how T3 Mode files can be read by custom software. The implementation of custom measurement programs requires the *Harp programming library, which is available as a separate option.

	see also [[t2-mode]]		see also [[t2-mode]]

tttr

Anonymous (anonymous@undisclosed.example.com) — 2015-02-10T16:42:50+00:00

2015/02/10 16:41		current
Line 6:		Line 6:
	The [[Products:TimeHarp 200]] (also the [[Products:TimeHarp 100]]), the [[Products:PicoHarp 300]] [[Products:HydraHarp 400]] support TTTR mode measurements. With the the [[Products:PicoHarp 300]] a slight shift in the nomenclature was introduced. Due to its capability of simultaneously recording both signal inputs (in interactive mode used as 'Start' and 'Stop') as two separate TTTR traces with a time resolution of 4ps, a new TTTR mode was introduced, the so called T2-mode. The traditional recording as with the [[Products:TimeHarp 200]] / [[Products:TimeHarp 100]] is called [[T3-mode]]. In [[T2-mode]] both signal inputs are functionally identical.		The [[Products:TimeHarp 200]] (also the [[Products:TimeHarp 100]]), the [[Products:PicoHarp 300]] [[Products:HydraHarp 400]] support TTTR mode measurements. With the the [[Products:PicoHarp 300]] a slight shift in the nomenclature was introduced. Due to its capability of simultaneously recording both signal inputs (in interactive mode used as 'Start' and 'Stop') as two separate TTTR traces with a time resolution of 4ps, a new TTTR mode was introduced, the so called T2-mode. The traditional recording as with the [[Products:TimeHarp 200]] / [[Products:TimeHarp 100]] is called [[T3-mode]]. In [[T2-mode]] both signal inputs are functionally identical.

-	Today recording of T3 and T2 mode files is possible with all recent PicoQuant TCSPC devices ([[Products:TimeHarp 260]], [[Products:PicoHarp 300]] and [[Products:HydraHarp 400]] and their included software, analysis is supported mainly by the [[software:SymPhoTime]] software package.	+	Today recording of [[glossary:T3-mode]] and [[glossary:T2-mode]] files is possible with all recent PicoQuant TCSPC devices ([[Products:TimeHarp 260]], [[Products:PicoHarp 300]] and [[Products:HydraHarp 400]] and their included software, analysis is supported mainly by the [[software:SymPhoTime]] software package.

	Due to its capability of including external TTL pulses as markers in the stream of photon events TTTR mode is suitable for [[FLIM]] measurements. The markers are used to synchronise the photon events with a scanning process allowing to sort the photons into image pixels.		Due to its capability of including external TTL pulses as markers in the stream of photon events TTTR mode is suitable for [[FLIM]] measurements. The markers are used to synchronise the photon events with a scanning process allowing to sort the photons into image pixels.

calculate_and_fit_fcs_traces_with_the_fcs_script

Anonymous (anonymous@undisclosed.example.com) — 2015-11-03T14:11:17+00:00

2014/06/18 09:21		current
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	===== Background Information =====		===== Background Information =====

-	ATTO488 (ATTO-Tec, Germany) is a suited calibration dye to calibrate the confocal microscope at ~470 – 490 nm excitation and detection ~500 – 540 nm.\\	+	* ATTO488 (ATTO-Tec, Germany) is a suited calibration dye to calibrate the confocal microscope at ~470 – 490 nm excitation and detection ~500 – 540 nm.
-	The diffusion coefficient of ATTO488 carboxilic acid has been measured to be 400 µm²/s at 25°C. Published diffusion coefficients of various fluorophores are summarized in the Technical Note "Absolute Diffusion Coefficients: Compilation of Reference Data for FCS Calibration" available from PicoQuant [see http://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf ]. This list is extensive, but does not claim to be complete.\\	+	* The diffusion coefficient of ATTO488 carboxilic acid has been measured to be 400 µm²/s at 25°C. Published diffusion coefficients of various fluorophores are summarized in the Technical Note "Absolute Diffusion Coefficients: Compilation of Reference Data for FCS Calibration" available from PicoQuant [see http://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf ]. This list is extensive, but does not claim to be complete.
-	Note that the diffusion coefficients of all dyes are temperature dependent.\\	+	* Note that the diffusion coefficients of all dyes are temperature dependent.
-	FCS measurements are usually point measurements acquired with very sensitive detectors (typically SPAD or Hybrid PMT detectors).\\	+	* FCS measurements are usually point measurements acquired with very sensitive detectors (typically [[glossary:SPAD]] or [[glossary:Hybrid PMT]] detectors).
-	FCS is a technique that can be used over a wide range of concentrations, but ideal FCS concentrations are usually between 1 nM and 20 nM.\\	+	* FCS is a technique that can be used over a wide range of concentrations, but ideal FCS concentrations are usually between 1 nM and 20 nM.

	===== Step-by-Step Tutorial =====		===== Step-by-Step Tutorial =====
Line 65:		Line 65:
	{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_7.png?600 }}		{{ calculate_and_fit_fcs_traces_with_the_fcs_script_Image_7.png?600 }}

-	Note: A possibility to remove the afterpulsing when working with cw excitation is splitting the light onto two detectors and calculate a cross correlation. Some detectors (mainly Hybrid PMTs) also have so low afterpulsing that the problem does not occur.	+	Note: A possibility to remove the afterpulsing when working with cw excitation is splitting the light onto two detectors and calculate a cross correlation. Some detectors (mainly [[glossary:Hybrid PMT]]s) also have so low afterpulsing that the problem does not occur.

	* If the sample has beenexcited with cw light, we would now save the data by clicking "Save Result" and press afterwards "Transfer to Fit" (both in the "File" dropdown panel). As our sample has been excited with a pulsed laser, we can apply one additional step to increase data quality.		* If the sample has beenexcited with cw light, we would now save the data by clicking "Save Result" and press afterwards "Transfer to Fit" (both in the "File" dropdown panel). As our sample has been excited with a pulsed laser, we can apply one additional step to increase data quality.

calibrate_the_confocal_volume_for_fcs_using_the_fcs_calibration_script

Anonymous (anonymous@undisclosed.example.com) — 2019-02-07T12:16:52+00:00

2018/09/14 14:34		current
Line 17:		Line 17:
	Note: Calibration is a necessary task for FCS measurements. In a calibration procedure, the confocal volume VEff as well as the structural parameter κ (the ratio of z to xy extension) is determined using a reference dye. This is the prerequisite to determine correct diffusion coefficients and concentrations for the sample of interest.\\		Note: Calibration is a necessary task for FCS measurements. In a calibration procedure, the confocal volume VEff as well as the structural parameter κ (the ratio of z to xy extension) is determined using a reference dye. This is the prerequisite to determine correct diffusion coefficients and concentrations for the sample of interest.\\
	ATTO488 (ATTO-Tec, Germany) is a suited dye to calibrate the confocal microscope at ~470 – 490 nm excitation and detection around 500 – 540 nm.\\		ATTO488 (ATTO-Tec, Germany) is a suited dye to calibrate the confocal microscope at ~470 – 490 nm excitation and detection around 500 – 540 nm.\\
-	The diffusion coefficient of ATTO488 carboxilic acid has been measured to be 400 µm²/s at 25°C. Published diffusion coefficients of various fluorophores are summarized in the Technical Note "Absolute Diffusion Coefficients: Compilation of Reference Data for FCS Calibration" available from PicoQuant [see [[http://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf]]]. This list is extensive, but does not claim to be complete.\\	+	The diffusion coefficient of ATTO488 carboxilic acid has been measured to be 400 µm²/s at 25°C. Published diffusion coefficients of various fluorophores are summarized in the Technical Note "Absolute Diffusion Coefficients: Compilation of Reference Data for FCS Calibration" available from PicoQuant (see [[https://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf]]). This list is extensive, but does not claim to be complete.\\
	Note that the diffusion coefficients of all dyes are temperature dependent.\\		Note that the diffusion coefficients of all dyes are temperature dependent.\\
	FCS measurements are usually point measurements acquired with very sensitive detectors ([[glossary:spad\|SPAD]] or [[glossary:Hybrid PMT]] detectors).\\		FCS measurements are usually point measurements acquired with very sensitive detectors ([[glossary:spad\|SPAD]] or [[glossary:Hybrid PMT]] detectors).\\
	FCS is a technique applicable over a wide range of concentrations, but ideal FCS concentrations are usually between 1 and 20 nM.		FCS is a technique applicable over a wide range of concentrations, but ideal FCS concentrations are usually between 1 and 20 nM.
-
	===== Step-by-Step Tutorial =====		===== Step-by-Step Tutorial =====

flim_measurement_using_a_nikon_a1_with_a_flim_and_fcs_upgrade

Anonymous (anonymous@undisclosed.example.com) — 2015-06-30T13:07:15+00:00

2015/06/16 11:46		current
Line 15:		Line 15:

	The general principles of the data acquisition are analogous in all LSM upgrade kits.		The general principles of the data acquisition are analogous in all LSM upgrade kits.
-	The movie was recorded in 02/2014; the recording procedure may be somewhat different in older or newer	+	The movie was recorded in 02/2014 and shows the integration with Nikon A1 system with a NIS version lower than 4.3; the recording procedure may be somewhat different in higher or very old versions.
-	models.	+

	{{ youtube>1Tr9DZ-SkSQ?large }}		{{ youtube>1Tr9DZ-SkSQ?large }}
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	{{ youtube>d_-4MSWznkU?large }}		{{ youtube>d_-4MSWznkU?large }}

		+	The movie was recorded in 02/2015; the recording procedure may be somewhat different in older or newer
		+	models.

	\\		\\

flim-fret_measurement_using_an_olympus_fv1200_with_a_flim_and_fcs_upgrade

Anonymous (anonymous@undisclosed.example.com) — 2020-05-18T12:53:40+00:00

2015/02/06 11:55		current
Line 2:		Line 2:


-	====== FLIM-FRET Measurement using an Olympus FV1200 with a FLIM and FCS upgrade ======	+	====== FLIM-FRET Measurement Using an Olympus FV1200 with a FLIM and FCS Upgrade ======

	This tutorial shows the recording of FLIM-FRET images using an LSM upgrade kit, in this case connected to an Olympus FluoView FV1200 laser scanning microscope.		This tutorial shows the recording of FLIM-FRET images using an LSM upgrade kit, in this case connected to an Olympus FluoView FV1200 laser scanning microscope.
Line 11:		Line 11:

	The general principles of the data acquisition are analogous in all LSM upgrade kits.		The general principles of the data acquisition are analogous in all LSM upgrade kits.
-	The movie was recorded in 02/2014; the recording procedure may be slightly different in older or newer	+	The movie was recorded in 02/2018; the recording procedure may be slightly different in older or newer
	models.		models.

Line 20:		Line 20:
	{{topic>howto +IRF &nodate&nouser}}		{{topic>howto +IRF &nodate&nouser}}
	{{topic>howto +FLIM +FRET +analysis &nodate&nouser}}		{{topic>howto +FLIM +FRET +analysis &nodate&nouser}}
-	{{topic>howto +FLIM +Nikon}}

lifetime-fitting_using_the_flim_script

Anonymous (anonymous@undisclosed.example.com) — 2023-08-03T14:16:00+00:00

2023/02/17 12:40		current
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-	{{tag>howto tutorial FLIM analysis SymPhoTime MT LSM_upgrade}}	+	{{tag>howto tutorial FLIM analysis SymPhoTime MicroTime LSM_upgrade}}

	====== Lifetime-Fitting Using the FLIM Analysis (updated for SymPhoTime V 2.5 and above) ======		====== Lifetime-Fitting Using the FLIM Analysis (updated for SymPhoTime V 2.5 and above) ======
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	Note:\\		Note:\\
-	The software offers the possibility to fit the data using a n-exponential tailfit, n-exponential reconvolution or rapid reconvolution fit. A tailfit can be used when the fitted lifetimes are significantly longer than the instrument response function. A reconvolution fit is usually preferable, because the complete decay is fitted, while for a tailfit, the start of the fitting range is usually a bit arbitrary. A rapidReconvolution fit is needed, if the count rates, with which the image was acquired are significantly above the Pile Up limit. It is also needed, if the pulse frequency is too high for the fluorescence decay to decrease to the background level before the end of the decay window (often, this is the case for 2-Photon-Excitation data acquired with a TiSa-laser with 80MHz repetition rate). Check out the rapidFLIM-analysis tutorial for this case.\\	+	The software offers the possibility to fit the data using a n-exponential tailfit, n-exponential reconvolution or rapid reconvolution fit. A tailfit can be used when the fitted lifetimes are significantly longer than the instrument response function. A reconvolution fit is usually preferable, because the complete decay is fitted, while for a tailfit, the start of the fitting range is usually a bit arbitrary. A rapidReconvolution fit is needed, if the count rates, with which the image was acquired are significantly above the Pile Up limit. It is also needed, if the pulse frequency is too high for the fluorescence decay to decrease to the background level before the end of the decay window (often, this is the case for 2-Photon-Excitation data acquired with a TiSa-laser with 80MHz repetition rate). Check out the rapidReconvolution tutorial for this case (https://www.tcspc.com/doku.php/howto:lifetime-fitting_using_the_rapid_reconvolution_algorithm).\\

	For explanations about the fitting model and the used equations, click on the "Help" button next to the selected model. This opens a help window containing the fitting equation and the explanation of the different parameters.\\		For explanations about the fitting model and the used equations, click on the "Help" button next to the selected model. This opens a help window containing the fitting equation and the explanation of the different parameters.\\

measuring_quantum_yield

Anonymous (anonymous@undisclosed.example.com) — 2017-07-12T13:16:44+00:00

2015/02/25 13:52		current
Line 3:		Line 3:
	====== Measuring the Quantum Yield with the Integrating Sphere Assembly for the FluoTime 300 ======		====== Measuring the Quantum Yield with the Integrating Sphere Assembly for the FluoTime 300 ======

-	Note: The integrating Sphere is made from Spectralon™. While this material provides excellent spectral reflectivity and spectral flatness it is also very sensitive to contamination and due to the porous nature of its surface collects impurities easily. Any impurities will change the reflectiveness of the integrating sphere and thus might influence the measurements.	+	Note: The integrating Sphere is made from Spectralon™. While this material provides excellent spectral reflectivity and spectral flatness it is also very sensitive to contamination. Due to the porous nature of its surface, it collects impurities easily. Any impurity will change the reflectivity of the inner walls of the integrating sphere and thus will influence the measurements.
-	Therefore, handle the integrating sphere carefully avoiding any exposure to dust or other impurities. Avoid spilling sample into the sphere or the sample holder.	+	Therefore, handle the integrating sphere carefully avoiding any exposure to dust or other impurities. Avoid spilling sample into the sphere or contamination of the sample holder.

	{{ youtube>JLiDnlS5BS8?large }}		{{ youtube>JLiDnlS5BS8?large }}

-	===== Setp-by-Step =====	+	===== Step-by-Step =====
-	* Insert the integrating sphere with caution in order not to hit any optical elements in the FluoTime 300. The integrating sphere assembly has an indexed lid to ensure proper orientation.	+	* Mount the integrating sphere carefully. Make sure you do not hit any optical elements inside the FluoTime 300. The integrating sphere assembly has an indexed lid to ensure proper orientation.

	{{ :howto:2013-12-19_at_14-30-03.jpg?600 \|}}		{{ :howto:2013-12-19_at_14-30-03.jpg?600 \|}}

roi_fitting_using_the_flim_script

Anonymous (anonymous@undisclosed.example.com) — 2023-02-23T07:08:46+00:00

2015/02/25 13:58		current
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	~~NOTOC~~		~~NOTOC~~

-	====== FLIM ROI Fitting ======	+	====== ROI Fitting Using the FLIM Analysis ======


Line 9:		Line 9:


-	This tutorial shows step-by-step, how the [[glossary:flim\|FLIM]] script of [[products:SymPhoTime64\|SymPhoTime 64]] can be used to fit several regions of interest (ROIs), and how to extract and interpret the results. In detail, the FLIM script is started using a daisy pollen image from the "Samples" – workspace, then three regions of interest are defined which are finally fitted.	+	This tutorial shows step-by-step, how the [[glossary:flim\|FLIM]] script of [[products:SymPhoTime64\|SymPhoTime 64]] can be used to fit several regions of interest (ROIs), and how to extract and interpret the results. In details, the FLIM script is started using a daisy pollen image from the "Samples" – workspace, then three regions of interest are defined which are finally fitted.

	{{ youtube>g6uyvLQW4nY?large }}		{{ youtube>g6uyvLQW4nY?large }}

visualizing_dynamics_with_the_multi_frame_flim_analysis

Anonymous (anonymous@undisclosed.example.com) — 2020-05-13T09:39:06+00:00

2015/11/03 14:32		current
Line 3:		Line 3:
	~~NOTOC~~		~~NOTOC~~

-	====== Visualizing Dynamics with the Multi Frame FLIM Analysis ======	+	====== Visualizing Dynamics Using the Multi Frame FLIM Analysis ======

	===== Summary =====		===== Summary =====
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	- Upper right: Fluorescence Lifetime Histogram. Here the frequency of photon counts corresponding to the individual mean lifetimes is plotted. The settings of the plot can be adapted using the controls on the right of the plot.\\		- Upper right: Fluorescence Lifetime Histogram. Here the frequency of photon counts corresponding to the individual mean lifetimes is plotted. The settings of the plot can be adapted using the controls on the right of the plot.\\
	- Lower left: Lifetime fitting parameters allowing to define the lifetime fitting models. Fitting is then applied to the lifetime graph(s) in the [[glossary:TCSPC]] histogram.\\		- Lower left: Lifetime fitting parameters allowing to define the lifetime fitting models. Fitting is then applied to the lifetime graph(s) in the [[glossary:TCSPC]] histogram.\\
-	- Lower center/left: TCSPC histogram of all photons in the image in green (= dataset 0) and the [[glossary:TCSPC]] histogram of a single pixel in grey (= dataset 1). In red an estimation of the instrument response function (= IRF) is shown. The IRF reconstruction is deducted from the rising edge of the TCSPC histogram.	+	- Lower center/left: [[glossary:TCSPC]] histogram of all photons in the image in green (= dataset 0) and the [[glossary:TCSPC]] histogram of a single pixel in grey (= dataset 1). In red an estimation of the instrument response function (= [[glossary:IRF]]) is shown. The [[glossary:IRF]] reconstruction is deducted from the rising edge of the [[glossary:TCSPC]] histogram.

	* To enhance the lifetime contrast within the image, adapt the color scale from 0 to 15 ns by typing these values as limits for the Fast Lifetime color scale. Also adjust the intensity scale to 1 – 200 counts.		* To enhance the lifetime contrast within the image, adapt the color scale from 0 to 15 ns by typing these values as limits for the Fast Lifetime color scale. Also adjust the intensity scale to 1 – 200 counts.

microtime

Anonymous (anonymous@undisclosed.example.com) — 2015-11-03T14:24:05+00:00

2015/11/03 14:22		current
Line 18:		Line 18:
	* Complete confocal system with laser combining unit, inverted microscope body and multichannel detection unit		* Complete confocal system with laser combining unit, inverted microscope body and multichannel detection unit
	* Turn-key diode lasers for multicolor excitation from 375 nm to 900 nm		* Turn-key diode lasers for multicolor excitation from 375 nm to 900 nm
-	* Up to 4 truly parallel detection channels using application-optimized detection with [[glossray:SPAD]]s, [[glossary:PMT]]s or [[glossary:Hybrid PMT]]s	+	* Up to 4 truly parallel detection channels using application-optimized detection with [[glossary:SPAD]]s, [[glossary:PMT]]s or [[glossary:Hybrid PMT]]s
-	* Time-Correlated-Single Photon Counting (TCSPC) and TTTR mode for investigation of fast dynamics in FCS and FLIM	+	* Time-Correlated-Single Photon Counting ([[glossary:TCSPC]]) and [[glossary:TTTR]] mode for investigation of fast dynamics in [[glossary:FCS]] and [[glossary:FLIM]]
	* Piezo scanning for 2D- and 3D-lifetime imaging and accurate point positioning		* Piezo scanning for 2D- and 3D-lifetime imaging and accurate point positioning
	* Two optional exit ports for additional hardware, e.g. spectrographs		* Two optional exit ports for additional hardware, e.g. spectrographs

fluofit

Anonymous (anonymous@undisclosed.example.com) — 2015-11-03T14:07:12+00:00

2026/04/11 16:45	current
Line 1:	Line 1:
	+	====== FluoFit ======
	+	The FluoFit software package is a powerful global analysis software for fluorescence decay and anisotropy measurements. Tail fitting as well as a numerical reconvolution algorithm to account for the finite Instrument Response Function ([[glossary:IRF]]) can be applied. The FluoFit software is available in two versions (Basic and Professional) with different functionalities to meet individual needs.

	+	see [[https://www.picoquant.com/products/category/software/fluofit-global-fluorescence-decay-data-analysis-software]]

ax_flim_examples

Anonymous (anonymous@undisclosed.example.com) — 2025-06-16T08:53:39+00:00

2025/06/16 08:48		current
Line 4:		Line 4:

	1. How to automatically acquire both confocal and FLIM channels		1. How to automatically acquire both confocal and FLIM channels
		+
		+	{{ :howto:ax_flim_examples:nis-twochannel_publish.mp4?1000 \|}}

	2. How to perform FLIM stitching to visualize a large sample		2. How to perform FLIM stitching to visualize a large sample
		+
		+	{{ :howto:ax_flim_examples:nis_stiching_publish.mp4?1000 \|}}
		+
		+

2020/04/14 11:40		current
Line 2:		Line 2:

	===== News from the community=====		===== News from the community=====
		+	* 29.04.20 [[https://www.fret.community/announcement-protein-folding-and-dynamics-webinar/\|Protein folding and dynamics Webinar-Series.]] \\ \\
	* 14.04.20 [[https://analyticalscience.wiley.com/do/10.1002/was.00050003/full/bioimagedataanalysis.pdf\|Bioimage Data Analysis.]] New Expanded version of a nice open-source introduction. \\ \\		* 14.04.20 [[https://analyticalscience.wiley.com/do/10.1002/was.00050003/full/bioimagedataanalysis.pdf\|Bioimage Data Analysis.]] New Expanded version of a nice open-source introduction. \\ \\
-	* 14.04.20 [[https://science.sciencemag.org/content/368/6486/78\|De novo design of protein logic gates.]] Fundamendal enzymology work. Chen et al. describe the design of logic gates that can regulate protein association. The gates were built from small, designed proteins that all have a similar structure but where one module can be designed to interact specifically with another module. Using monomers and covalently connected monomers as inputs and encoding specificity through designed hydrogen-bond networks allowed the construction of two-input or three-input gates based on competitive binding. \\ \\	+	* 14.04.20 [[https://science.sciencemag.org/content/368/6486/78\|De novo design of protein logic gates.]] Fundamental enzymology work. Chen et al. describe the design of logic gates that can regulate protein association. The gates were built from small, designed proteins that all have a similar structure but where one module can be designed to interact specifically with another module. Using monomers and covalently connected monomers as inputs and encoding specificity through designed hydrogen-bond networks allowed the construction of two-input or three-input gates based on competitive binding. \\ \\
	* 31.03.20 FRET community has a new advisory board, [[https://www.fret.community/\|FRET community]]. Happy that Dr. Marcelle König , a senior scientist in PicoQuant was elected. \\ \\		* 31.03.20 FRET community has a new advisory board, [[https://www.fret.community/\|FRET community]]. Happy that Dr. Marcelle König , a senior scientist in PicoQuant was elected. \\ \\
	* 31.03.20 Extremely helpful reference paper about confocal microscopy. [[https://www.nature.com/articles/s41596-020-0313-9\|Tutorial: guidance for quantitative confocal microscopy]]. Available as a poster [[https://www.nature.com/articles/s41596-020-0307-7\|here.]] \\ \\		* 31.03.20 Extremely helpful reference paper about confocal microscopy. [[https://www.nature.com/articles/s41596-020-0313-9\|Tutorial: guidance for quantitative confocal microscopy]]. Available as a poster [[https://www.nature.com/articles/s41596-020-0307-7\|here.]] \\ \\

2025/06/16 08:48		current
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	1. How to automatically acquire both confocal and FLIM channels		1. How to automatically acquire both confocal and FLIM channels
		+
		+	{{ :howto:ax_flim_examples:nis-twochannel_publish.mp4?1000 \|}}

	2. How to perform FLIM stitching to visualize a large sample		2. How to perform FLIM stitching to visualize a large sample
		+
		+	{{ :howto:ax_flim_examples:nis_stiching_publish.mp4?1000 \|}}
		+
		+

2019/03/06 12:44		current
Line 6:		Line 6:


-	The fluorescence lifetime of a dye measured with a TCSPC spectrometer can be multiexponential due to many reasons. The most obvious cases are due to scattering or presence of impurities. Although less obvious, it is also widely known that in an inhomogeneous media a pure dye will also exhibit a multiexponential decay. Advanced users know that if the decay is not measured with polarizers at magic angle, the rotation correlation time shows up as a second exponential in the decay (the second exponential is in fact the product of the rotation correlation time by the fluorescence lifetime, divided by their sum). And they also know that measuring without polarizers is not equivalent to measure at magic angle... But suspicion may arise when even a pure dye measured at magic angle in a homogeneous media exhibits a multiexponential decay. Is the spectrometer properly adjusted? Are the polarizers properly calibrated? A typical mistake is to measure the [[glossary:IRF]] at the nominal laser wavelength instead of measuring at the ideal wavelength for that specific laser head. Note that all diode lasers heads emit at slightly different wavelengths and each of them have an optimum at which the IRF should be measured. As little as 0.5 nm displacement from their optimum may induce a "non perfect" [[glossary:deconvolution]] fit.	+	The fluorescence lifetime of a dye measured with a TCSPC spectrometer can be multiexponential due to many reasons. The most obvious cases are due to scattering or presence of impurities. Although less obvious, it is also widely known that in an inhomogeneous medium a pure dye will also exhibit a multiexponential decay. Advanced users know that if the decay is not measured with polarizers at magic angle, the rotation correlation time shows up as a second exponential in the decay (the second exponential is in fact the product of the rotation correlation time and the fluorescence lifetime, divided by their sum). And they also know that measuring without polarizers is not equivalent to measuring at magic angle... But suspicion may arise when even a pure dye measured at magic angle in a homogeneous medium exhibits a multiexponential decay. Is the spectrometer properly adjusted? Are the polarizers properly calibrated? A typical mistake is to measure the [[glossary:IRF]] at the nominal laser wavelength instead of measuring at the ideal wavelength for that specific laser head. Note that all diode lasers heads emit at slightly different wavelengths and each of them have an optimum at which the IRF should be measured. As little as 0.5 nm displacement from their optimum may induce a "non perfect" [[glossary:deconvolution]] fit.

	But it is worth noting that even at magic angle in a perfectly aligned spectrometer pure dyes in homogeneous media may exhibit a multiexponential decay. The origin may be physical, like solvent relaxation, or chemical, when the fluorescent molecule undergoes a ground or excited state reaction. In this brief article a few examples are described.		But it is worth noting that even at magic angle in a perfectly aligned spectrometer pure dyes in homogeneous media may exhibit a multiexponential decay. The origin may be physical, like solvent relaxation, or chemical, when the fluorescent molecule undergoes a ground or excited state reaction. In this brief article a few examples are described.
Line 25:		Line 25:


-	The electronic redistribution of electrons due to optical excitation leads in many cases to a different reactivity in the ground and excited states. In other words, a fluorescent molecule which is boring under the dark may become reactive upon excitation. Common excited state reactions are redox (electron transfer) and acid-base (proton transfer) reactions. Depending on the rate of the excited-state rection relative the the original fluorescence lifetime, the observed decay time measured with a TCSPC spectrometer may be single- or multiexponential.	+	The electronic redistribution of electrons due to optical excitation leads in many cases to a different reactivity in the ground and excited states. In other words, a fluorescent molecule which is mostly inert in the dark may become reactive upon excitation. Common excited state reactions are redox (electron transfer) and acid-base (proton transfer) reactions. Depending on the rate of the excited-state reaction relative the the original fluorescence lifetime, the observed decay time measured with a TCSPC spectrometer may be single- or multiexponential.

	Let us consider the reaction in Scheme 2 in different situations:		Let us consider the reaction in Scheme 2 in different situations:
Line 34:		Line 34:
	Scheme 2		Scheme 2

-	//Starting point: The molecule A is promoted to the excited-state where it can react with a molecule X to form the compound B, through a rate constant $k_{AB}$. Once the compound B is formed the back-reaction can occur, with a rate constant $k_{BA}$. Compounds A and B are fluorescent with original fluorescence lifetimes $\tau_A$ and $\tau_B$. Once B decays to the ground state the back-reaction takes place. Hence the system is always in its starting position $(A + X)$ prior to any excitation pulse.//	+	//Starting point: The molecule A is promoted to the excited-state where it can react with a molecule X to form the compound B, through a rate constant $k_{AB}$. Once the compound B is formed, the back-reaction can occur with a rate constant $k_{BA}$. Compounds A and B are fluorescent with original fluorescence lifetimes $\tau_A$ and $\tau_B$. Once B decays to the ground state, the back-reaction takes place. Hence the system is always in its starting position $(A + X)$ prior to any excitation pulse.//

	//Case A) The constant kAB is too slow with respect to $\tau_A$ and $\tau_B$.// In this case the compound A would decay to the ground-sate before the excited-state reaction could take place. The decay measured would be single exponential and coincident with $\tau_A$.		//Case A) The constant kAB is too slow with respect to $\tau_A$ and $\tau_B$.// In this case the compound A would decay to the ground-sate before the excited-state reaction could take place. The decay measured would be single exponential and coincident with $\tau_A$.
Line 60:		Line 60:
	being $Z = \sqrt{(M-Y)^2 + 4 k_{AB} k_{BA}[x]}$		being $Z = \sqrt{(M-Y)^2 + 4 k_{AB} k_{BA}[x]}$

-	$A_1= A_0 [M- (1/\tau_2)] / [(1/\tau_1 – 1/\tau_2]$	+	$A_1= A_0 [M- (1/\tau_2)] / [1/\tau_1 – 1/\tau_2]$

-	$A_2= A_0 [(1/\tau_1)- M] / [(1/\tau_1 – 1/\tau_2]$	+	$A_2= A_0 [(1/\tau_1)- M] / [1/\tau_1 – 1/\tau_2]$

-	$B_1= -A_0 k_{AB}[x] / [(1/\tau_1 – 1/\tau_2]$	+	$B_1= -A_0 k_{AB}[x] / [1/\tau_1 – 1/\tau_2]$

-	$B_2= A_0 k_{AB} [x] / [(1/\tau_1 – 1/\tau_2]$	+	$B_2= A_0 k_{AB} [x] / [1/\tau_1 – 1/\tau_2]$

	being $A_0$ the concentration of $A$ at $t=0$.		being $A_0$ the concentration of $A$ at $t=0$.
Line 72:		Line 72:
	Note from $A(t)$ that, even if $B$ would not be fluorescent, the decay of $A$ will still be biexponential!		Note from $A(t)$ that, even if $B$ would not be fluorescent, the decay of $A$ will still be biexponential!

-	A situation like this may occur with molecules dissolved in an aprotic but hygroscopic media, like acetonirile. Water molecules may diffuse (diffusion happens in the nanosecond time scale) and react with fluorophores bearing proton-transfer groups like -OH or -NH2.	+	A situation like this may occur with molecules dissolved in an aprotic but hygroscopic medium, like acetonitrile. Water molecules may diffuse (diffusion happens in the nanosecond time scale) and react with fluorophores bearing proton-transfer groups like -OH or -NH2.

-	//Case D) Interconversion rate constants kAB and kBA are very quick compared to the intrinsic lifetimes $\tau_A$ and $\tau_B$.// In this case the equations of case C would still apply. But in practice, a quick equilibrium between reactants and product would be established. This means that the concentrations of A and B with respect to each other would always be constant prior to their decay, and hence the whole system could be treated as a single dye. The decay would be single exponential, being an average of $\tau_A$ and $\tau_B$ weighted by their fraction in the equilibrium.	+	//Case D) Interconversion rate constants $k_{AB}$ and $k_{BA}$ are very quick compared to the intrinsic lifetimes $\tau_A$ and $\tau_B$.// In this case the equations of case C would still apply. But in practice, a quick equilibrium between reactants and product would be established. This means that the concentrations of A and B with respect to each other would always be constant prior to their decay, and hence the whole system could be treated as a single dye. The decay would be single exponential, being an average of $\tau_A$ and $\tau_B$ weighted by their fraction in the equilibrium.

-	This situation may happen if compounds A and X were directly in contact prior to excitation, for example throgh ground-state interactions.	+	This situation may happen if compounds A and X were directly in contact prior to excitation, for example through ground-state interactions.
	===== 3) Solvation dynamics =====		===== 3) Solvation dynamics =====


-	Even pure fluorophores in pure solvents may lead to multiexponential decays. This is the case of molecules with strong charge transfer character in polar solvents when undergoing solvation dynamics. In such cases the fluorescence spectra shifts to longer wavelengths in time. Measuring the decay in the blue edge of the steady-state spectrum will lead to a multiexponential decay. The ns lifetimes corresponds to the intrinsic fluorescence lifetime of the fluorophore, whereas the ultrafast components (ps) are related to the rate of the shift. Measuring in the red-flank of the steady-sate spectrum leads to multiexponential decays with positive (intrinsic lifetime, ns) an negative (rate of shift, ps) pre-exponential factors. This is depicted in Scheme 3.	+	Even pure fluorophores in pure solvents may lead to multiexponential decays. This is the case of molecules with strong charge transfer character in polar solvents when undergoing solvation dynamics. In such cases the fluorescence spectrum shifts to longer wavelengths on a picosecond time-scale. Measuring the decay in the blue edge of the steady-state spectrum will lead to a multiexponential decay. The ns lifetime corresponds to the intrinsic fluorescence lifetime of the fluorophore, whereas the ultrafast components (ps) are related to the rate of the shift. Measuring in the red-flank of the steady-sate spectrum leads to multiexponential decays with positive (intrinsic lifetime, ns) and negative (rate of shift, ps) pre-exponential factors. This is depicted in Scheme 3.


	{{:solvation_dynamics.png?800\|}}		{{:solvation_dynamics.png?800\|}}

-	Scheme 3. Left: energetic representation of solvation dynamics. The fluorophore is represented as a sphere with a pointing dipole. Solvent molecules are represented in gray around the fluorophore. Right up : Spectral consequence of solvation dynamics. The fluorescence spectrum shifts in time to lower energies. Right bottom: Decay traces measured in different spectral regions. Blue flanks are multiexponential with positive pre-exponentila factors, red flanks are multiexponential with rising components.	+	Scheme 3. Left : energetic representation of solvation dynamics. The fluorophore is represented as a sphere with a pointing dipole. Solvent molecules are represented in gray around the fluorophore. Right, top : Spectral consequence of solvation dynamics. The fluorescence spectrum shifts in time to lower energies. Right, bottom: Decay traces measured in different spectral regions. Blue curves are multiexponential with positive pre-exponentila factors, red curves are multiexponential with rising components.

-	Before the excitation, the fluorophore is in the ground state S0 , which has a characteristic dipole moment. Solvent molecules, which also have their characteristic dipole moment are oriented in such a way that the interactions dipole-dipole with the fluorophore are as favorable possible. When the fluorophore is prompted to the excited state, its electronic distribution switches almost instantly. At time zero after excitation the solvent molecules remain in their "original" orientation to solvate ground state . The resulting dipole -dipole interactions with the fluorophore are hence less favorable. As a result , the solvent begins to relax to solvate the S1 state and brings the system to a more favorable position. Spectroscopically, this is manifested by the time-shift of the emission spectrum to longer wavelengths. Consider that the Steady-State spectrum is the time integral of all those shifting spectra. Measuring in the blue flank (λ1) will lead to a multiexponential decay: the signal decreases because of the shift and the intrinsic decay. Measuring in the red flank (λ3) will lead to a multiexponential decay with a negative pre-exponential factor: the signal first rises due to the increase in signal due to the displacement, and then decays due to the fluorescence lifetime.	+	Before the excitation, the fluorophore is in the ground state S0 , which has a characteristic dipole moment. Solvent molecules, which also have their characteristic dipole moment are oriented in such a way that the dipole-dipole interactions with the fluorophore are as favorable as possible. When the fluorophore is prompted to the excited state, its electronic distribution switches almost instantly. At time zero after excitation the solvent molecules remain in their "original" orientation. The resulting dipole -dipole interactions with the fluorophore are hence less favorable. As a result, the solvent begins to relax to solvate the S1 state and brings the system to a more favorable position. Spectroscopically, this is manifested by the time-shift of the emission spectrum to longer wavelengths. Consider that the Steady-State spectrum is the time integral of all those shifting spectra. Measuring in the blue flank (λ1) will lead to a multiexponential decay: the signal decreases because of the shift and the intrinsic decay. Measuring in the red flank (λ3) will lead to a multiexponential decay with a negative pre-exponential factor: the signal first rises due to the increase in signal due to the displacement, and then decays due to the fluorescence lifetime.

	In fluid media, solvation dynamics can be described with a multiexponential function spanning from the femtosecond time-scale to tens of picoseconds. Hence, the tail of this process can be monitored with a TCSPC spectrometer equipped with fast detectors such as a [[glossary:MCP]] or a Hybrid-PMT. In viscous media or at low temperatures, the ps tail component slows down to ns, and the process can be monitored with slower detectors, such as standard [[glossary:PMT]].		In fluid media, solvation dynamics can be described with a multiexponential function spanning from the femtosecond time-scale to tens of picoseconds. Hence, the tail of this process can be monitored with a TCSPC spectrometer equipped with fast detectors such as a [[glossary:MCP]] or a Hybrid-PMT. In viscous media or at low temperatures, the ps tail component slows down to ns, and the process can be monitored with slower detectors, such as standard [[glossary:PMT]].