Time-Resolved Fluorescence Wiki

symphotime_tips_and_tricks

Anonymous (anonymous@undisclosed.example.com) — 2020-04-03T14:54:26+00:00

2020/04/03 13:52		current
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	{{tag>Howto Analysis Tutorial SymPhoTime Tips&Tricks}}		{{tag>Howto Analysis Tutorial SymPhoTime Tips&Tricks}}
-	~~TOC~~



	====== SymPhoTime 64 Analysis Tips and Tricks ======		====== SymPhoTime 64 Analysis Tips and Tricks ======
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		+	~~TOC~~
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	===== Summary =====		===== Summary =====

phasor_analysis

Anonymous (anonymous@undisclosed.example.com) — 2019-08-28T16:15:15+00:00

2018/01/24 14:50		current
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-	{{tag> FLIM howto phasor bin-export analysis tutorial SymPhoTime Globals software}}	+	{{tag> FLIM howto phasor bin-export bin_export analysis tutorial SymPhoTime Globals software}}

	~~TOC~~		~~TOC~~

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
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-	{{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 =====

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
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	{{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 ======
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	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.
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		+	{{ 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.

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	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]]).
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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 ======


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-	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 }}

start

Anonymous (anonymous@undisclosed.example.com) — 2026-02-11T14:19:03+00:00

2024/12/19 15:56		current
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	{{topic>howto -FT300 -sample -analysis -draft &nodate&nouser}}		{{topic>howto -FT300 -sample -analysis -draft &nodate&nouser}}

-	===== NovaFLIM / Analysis =====

-	{{topic>howto +tutorial +novaflim}}
	===== SymPhoTime64 / Analysis =====		===== SymPhoTime64 / Analysis =====

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
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	~~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.