Ch.0.
-Disconnect SPAD 1 Signal from PHR800 Router.
-Connect SPAD 1 Signal to [[Products:Pico... is NIM compatible. If the SPAD provides only TTL output pulses, you need to use the SIA400 adapter.
... ors directly to the [[Products:PicoHarp 300]] without passing SPAD 2 through the router. **In this case, however, the router cable (DVI-cable) needs to be d
which can be used to detect subpopulations.
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**Note:** The script req... ion is used to distinguish molecules with and without acceptor dyes and to filter the events for signals without acceptor to calculate the stoichiometry parameter... n intensity threshold is calculated for filtering out events without acceptor present. Four the fourth
cy using the "Lifetime FRET Image" analysis.
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===== Background informa... mage_9.png }}
**Note:**\\
For more information about the different ROI selection tools, check the tuto... of importance. Therefore exclude the bright dots outside the nucleus of the cell on the lower right by... ously "CRTL" and draw rectangles around the spots outside the nucleus.
* **Response:** The marked reg
phere or contamination of the sample holder.
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===== Step-by-Step =====... _at_14-35-37.jpg?600 |}}
* Select either IN or OUT position
**Note:** The sample holder has two inde... on beam will directly hit the sample, when in the OUT position the sample will only be illuminated indi... der into the IN-position.
* Select either IN or OUT position.
**Note:** The position should be the s
ontains manually entered text (e.g. information about the experiment)|Menu: **File → Create Comment**|D... ns a file viewer, in which you find information about the file type and acquisition settings
<imgcapti... line-analysis will contain relevant information about the measurement. To check the values calculated d... r above the scale, right click and select **"Clip Outliners"**. This feature ignores outliners of the p
ed in three different colors for each pixel.
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==== Open an Image ====
... iversity Map//. This new feature tells you more about the different populations in the picture. The //D... e second therefore is the non-FRET population without any FRET performing molecules.
{{ pattern_matchin... the residuals of the initial fit show no trend or outliers, it is safe to assume that the selected patt
erest are defined which are finally fitted.
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===== Step-by-Step =====... ry selection, the corresponding red dot is greyed out.
{{ roi_fitting_using_the_flim_script_Image_14.pn... image are highlighted, everything else is greyed out. The decay corresponding to ROI 2 contains now th... e is usually a bit arbitrary.\\
For explanation about the fitting model and the used equations, click o
calculated and fitted using the FCS script.
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===== Background Informa... not necessary to calculate first a FCS curve without the photon filter. You can also directly activate... the FCS Calibration Script]] for a description
about how to calibrate the confocal volume.\\
If no cal... the fitting parameter. If you
need information about how the fitting models are defined and what is th
ligonucleotide using the Grouped FCS script.
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===== Background Informa... Traces with the FCS Script]]". Look there to find out, how to proceed.
==== Need for calibration to re
at different in higher or very old versions.
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===== Update: NIS Elemen... .3 integrates control for FLIM and FCS =====
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The movie was recorded i
ow to measure na IRF on confocal microscopes.
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\\
==== Low count rate ... s, and it is very likely to produce artefacts without allowing to tell apart artefacts and effects.
==... lso a remarkably short fluorescence lifetime of about 10 ps. This short lifetime does not depend on the
e]]).
* It is important that the “Laser Power” output is not changed at the PDL or SEPIA laser drive... scribed here can also be used for IRF-fitting without time gating. However, as these decays contain the... ly. Please check the different FLIM-related tutorials for more information about the fitting.
ne and frame marker presence and assignment.
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\\
===== Step-by-Step... ine stop markers, respectively.
* You can zoom out again by activating the context menu with a right
hosen and a pixel-by-pixel fit is performed.
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===== Step-by-Step Tutor... e is usually a bit arbitrary.\\
For explanation about the fitting model and the used equations, click o... he photon numbers. Inside the pollen, there are about 5000 - 7000 counts, outside the pollen the image intensity is below 100.
* Therefore, we activate th