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--- howto:flim-fret_calculation_for_single_exponential_donors [2014/06/18 09:17] – admin
+++ howto:flim-fret_calculation_for_single_exponential_donors [2020/05/06 09:38] (current) – [Step-by-Step Tutorial] ruckelshausen
@@ Line 26: / Line 26: @@
 ===== Step-by-Step Tutorial =====
+**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 "Lifetime FRET Image" analysis.
   * Start [[products:SymPhoTime64|SymPhoTime 64]].
@@ Line 38: / Line 39: @@
 **Response:** The files of the sample workspace are displayed in the workspace panel on the left side of the main window.
-{{ flim-fret_calculation_for_single_exponential_donors_Image_1.png }}
+{{ :howto:flim-fret-1expd_samples_ws.png |}}
-  * Highlight the file ''FRET_GFP.ptu and mRFP.ptu'' by a single mouse click.
+  * Highlight the file ''FRET_GFP and mRFP.ptu'' by a single mouse click.
-{{ flim-fret_calculation_for_single_exponential_donors_Image_2.png }}
+{{ :howto:flim-fret-1expd_gret_gfp_and_mrfp.png |}}
   * Select the "Analysis" tab and in there, open the drop down menu "Imaging".
-{{ flim-fret_calculation_for_single_exponential_donors_Image_3.png }}
+{{ :howto:flim-fret-1expd_analysis_imaging.png?500 |}}
 **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 "Lifetime FRET Image" script by clicking on "Start".
+  * Start the "Lifetime FRET Image" analysis by clicking on "Start".
 {{ flim-fret_calculation_for_single_exponential_donors_Image_4.png }}
-**Response:** The “Lifetime FRET Image” script is applied to the file ''FRET_GFP.ptu and mRFP.ptu''. Thereby, a new Window opens:
+**Response:** The “Lifetime FRET Image” analysis is applied to the file ''FRET_GFP.ptu and mRFP.ptu''. Thereby, a new Window opens:
-{{ flim-fret_calculation_for_single_exponential_donors_Image_5.png?600 }}
+{{ :howto:flim-fret-1expd_analysis_lifetime-fret.png |}}
 The window contains five different regions:
@@ Line 65: / Line 67: @@
 {{ flim-fret_calculation_for_single_exponential_donors_Image_6.png }}
-  * In the image parameter panel, open the "Threshold" drop down menu.
-  * Activate "Use Threshold" and enter a threshold of 500.
-    * To set the threshold one can also use the bars in the intensity histogram.
-{{ flim-fret_calculation_for_single_exponential_donors_Image_7.png }}
-**Response:** In the image, only pixels with higher photon counts are highlighted.
+  *  On top left, under Region of Interest, check "Use ROI" box and click on "ROI from Threshold".
-{{ flim-fret_calculation_for_single_exponential_donors_Image_8.png }}
+{{ :howto:flim-fret-1expd_roiactive.png?300 |}}
+**Response:**\\
+A large window with three sections will appear, where you can set a threshold for an image analysis visually.
+{{ :howto:flim-fret-1expd_roi_from_thresh_1.png |}}
+**Note:**\\
+Left to right: Preview FLIM Image, Intensity Histogram and Lifetime Histogram. For any ROI you can set each of these parameters individually.
+There are two ways to define the new threshold. You can use the edit-box at the lower side to type the threshold and press enter to check the result or use cursor keys or mouse wheel to increase/decrease the value. Another option is using the blue vertical bars on the sides of the intensity or lifetime histograms; click and drag the bars to set the threshold.
+  * In this example set the lower intensity threshold to 500 counts.
+{{ :howto:flim-fret-1expd_lowerthresh_500_bearb.png?400 |}}
+  * 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.
+{{ :howto:flim-fret-1expd_roi_from_thresh_2_v3.png?400 |}}
   * In the lifetime fitting panel, keep the default Fitting model "Mono-Exp. Donor".
-{{ flim-fret_calculation_for_single_exponential_donors_Image_9.png }}
+  * Choose the corresponding ROI as Decay (in this case "ROI 0").
+{{ :howto:flim-fret-1expd_fittingmodel.png?400 |}}
+  * Click "Initial Fit" below the fitting parameter.
+**Response:** The fit is performed to the ROI decay.
+{{ :howto:flim-fret-1expd_initialFit.png |}}
-  * Click "Initial Fit".
-**Response:** The fit is performed to the overall decay.
-{{ flim-fret_calculation_for_single_exponential_donors_Image_10.png?600px }}
   * In the Fitting panel, set the parameters τ<sub>D</sub>, Bkgr<sub>Dec</sub>, Shift <sub>IRF</sub> and Bkgr<sub> IRF</sub> constant by removing the mark. Set the background of the decay to 0.
-**Note:** In order to reduce statistical fluctuations, as many parameters as possible need to be set constant. In a single pixel, the background counts of the pixel are negligible, therefore it is valid to set this to 0 in this case. Don't press "Initial Fit again".
+**Note:** In order to reduce statistical fluctuations, as many parameters as possible need to be set constant.
+In this model, it is assumed that the long lifetime is caused by unbound donor molecules, and therefore this lifetime is labeled τ<sub>D</sub> and is considered constant, in contrast to the FRET-efficiency, which can vary in different regions, unless you know from the biological setup that it will mainly be the same in all e.g. complexes. In a single pixel, the background counts of the pixel are negligible, therefore it is valid to set this to 0 in this case. Don't press "Initial Fit again".
-{{ flim-fret_calculation_for_single_exponential_donors_Image_11.png }}
+{{ :howto:FLIM-FRET-1expD_fixFitParam.png?400 |}}
   * Press "Calculate FRET" in the upper panel on the left.
 **Note:** Depending on the screen resolution, one may have to use the scroll bar to access this button.
+{{ :howto:flim-fret-1expd_calcfret_bearb.png?400 |}}
-{{ flim-fret_calculation_for_single_exponential_donors_Image_12.png?600px }}
 **Response:**
@@ Line 97: / Line 112: @@
   * From the results, a "FRET Image" is calculated and plotted in the FLIM image area. The FLIM image is hidden behind in another tab.
   * On the right, the FRET efficiency histogram is plotted from the pixel values.
-    * This graph shows a single peak, which is in contrast to the lifetime variations in the FLIM image. The explanation can be found in the Binding histogram below showingtwo distinct peaks.
+    * This graph shows a single peak, which is in contrast to the lifetime variations in the FLIM image. The explanation can be found in the Binding histogram below showing two distinct peaks.
   * On the lower right, the Binding histogram is plotted. The Binding histogram plots the amplitude fraction of the donor lifetime in presence of FRET. As the amplitude of a donor dye is not changed by its proximity to a FRET acceptor, the binding ratio has a direct effect on the average lifetime.
-{{ flim-fret_calculation_for_single_exponential_donors_Image_13.png?600 }}
+{{ :howto:flim-fret-1expd_calcfret_response.png |}}
   * To plot an image of the binding, simply change the parameter in the color scale to "Binding %". It is obvious that the population in the nucleus undergoing FRET is very low.
-{{ flim-fret_calculation_for_single_exponential_donors_Image_14.png }}
+{{ :howto:flim-fret-1expd_bindingimage_bearb.png?400 |}}
   * Press "Save Result" to save the current analysis.
@@ Line 108: / Line 123: @@
 **Response:** A result file is generated and linked to the raw data file.
-{{ flim-fret_calculation_for_single_exponential_donors_Image_16.png }}
+{{ :howto:flim-fret-1expd_pqres.png?400 |}}
   * The image analysis is now finished. There are several possibilities to continue: