Time-Resolved Fluorescence Wiki

User Tools

Site Tools

Trace:
howto:flim-fret_calculation_for_single_exponential_donors

Differences

This shows you the differences between two versions of the page.

Link to this comparison view

Both sides previous revisionPrevious revision
Next revisionBoth sides next revision
howto:flim-fret_calculation_for_single_exponential_donors [2020/04/22 07:02] – buschmannhowto:flim-fret_calculation_for_single_exponential_donors [2020/05/05 15:24] – [Step-by-Step Tutorial] ruckelshausen
Line 27: Line 27:
  
  
-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]].   * Start [[products:SymPhoTime64|SymPhoTime 64]].
Line 38: Line 38:
  
 **Response:** The files of the sample workspace are displayed in the workspace panel on the left side of the main window. **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 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".   * 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}} **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 }} {{ 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:   The window contains five different regions:  
  
Line 65: Line 66:
 {{ flim-fret_calculation_for_single_exponential_donors_Image_6.png }} {{ 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 }}+ 
 +{{ :writingroom: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 threshold to 500 counts. 
 +  * 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_v2.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".
 {{ flim-fret_calculation_for_single_exponential_donors_Image_9.png }} {{ flim-fret_calculation_for_single_exponential_donors_Image_9.png }}
 +  * Choose the corresponding ROI as Decay (in this case "ROI 0").
   * Click "Initial Fit".   * 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.+**Response:** The fit is performed to the ROI decay. 
 +{{ :howto:flim-fret-1expd_initialFit.png |}}
  
  
-**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".+  * 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. 
  
-{{ flim-fret_calculation_for_single_exponential_donors_Image_11.png }}+**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". 
 +{{ :howto:FLIM-FRET-1expD_fixFitParam.png?400 |}}
  
   * Press "Calculate FRET" in the upper panel on the left.   * 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. **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:** **Response:**
Line 97: Line 111:
   * From the results, a "FRET Image" is calculated and plotted in the FLIM image area. The FLIM image is hidden behind in another tab.   * 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.    * 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.   * 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.   * 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.   * Press "Save Result" to save the current analysis.
Line 108: Line 122:
  
 **Response:** A result file is generated and linked to the raw data file. **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:   * The image analysis is now finished. There are several possibilities to continue:
howto/flim-fret_calculation_for_single_exponential_donors.txt · Last modified: 2020/05/06 09:38 by ruckelshausen