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--- howto:lifetime-fitting_using_the_flim_script [2020/05/13 09:38] – [Lifetime-Fitting using the FLIM Analysis (updated for SymPhoTime v2.5)] ruckelshausen
+++ howto:lifetime-fitting_using_the_flim_script [2023/08/03 14:16] (current) – lan
@@ Line 1: / Line 1: @@
-{{tag>howto tutorial FLIM analysis SPT MT LSM_upgrade}}
+{{tag>howto tutorial FLIM analysis SymPhoTime MicroTime LSM_upgrade}}
-====== Lifetime-Fitting Using the FLIM Analysis (updated for SymPhoTime v2.5) ======
+====== Lifetime-Fitting Using the FLIM Analysis (updated for SymPhoTime V 2.5 and above) ======
 ===== Introduction =====
-This tutorial shows step-by-step, how the FLIM script of SymPhoTime 64 (V2.5) can be used to perform a pixel-by-pixel lifetime fit of a FLIM image and how to extract and read the results. In detail, the FLIM script is started using an image of mouse kidney tissue in the "Tutorials" workspace, a suited fitting model is chosen and a pixel-by-pixel fit is performed.
+This tutorial shows step-by-step, how the FLIM script of SymPhoTime 64 (V2.5) can be used to perform a pixel-by-pixel lifetime fit of a FLIM image and how to extract and read 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.
@@ Line 92: / Line 92: @@
 **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.\\
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   * Set the same limit for A2 and A3.\\
   * Uncheck the boxes for the individual lifetime components, Bkgr(Decay), Bkgr(IRF) and Shift(IRF) in order to fix these values, IRF and Background. Then in the pixel-by-pixel fit, these values are kept constant. Do NOT click "Initial Fit" or "Fit" at this stage!\\
-{{ howto:parameterfix_roi0_kidney_2.png?400 |}}
+{{ :writingroom:3comp-exp-kidney.png?400 |}}
   * Click "FLIM Fit" in the upper panel.\\