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writingroom:visualizing_dynamics_using_the_multiframe-flim_script [2020/05/12 15:56] – ruckelshausenhowto:visualizing_dynamics_using_the_multiframe-flim_script [2020/05/12 16:09] – ↷ Page moved from writingroom:visualizing_dynamics_using_the_multiframe-flim_script to howto:visualizing_dynamics_using_the_multiframe-flim_script ruckelshausen
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   * Start the "multi Frame FLIM" analysis by clicking on "Start".\\   * Start the "multi Frame FLIM" analysis by clicking on "Start".\\
  
-{{ :writingroom:mflimgui.png?300 |}}+{{ howto:mflimgui.png?300 |}}
  
 **Response:​**\\ ​ **Response:​**\\ ​
 The FLIM analysis is applied to the file ''Hyperosmotic_Shock_MDCK_Cells.ptu''. Thereby, a new Window opens: The FLIM analysis is applied to the file ''Hyperosmotic_Shock_MDCK_Cells.ptu''. Thereby, a new Window opens:
  
-{{ :writingroom:fullscreen_tensionsensor_2.png |}}+{{ howto:fullscreen_tensionsensor_2.png |}}
  
 **Note:**\\ **Note:**\\
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   * In parameter panel, enter a frame binning of 10 in order to decrease processing time and increase photon numbers per pixel, while still visualizing the temporal changes. Then start the recalculation by clicking on "Calculate FastFLIM".\\   * In parameter panel, enter a frame binning of 10 in order to decrease processing time and increase photon numbers per pixel, while still visualizing the temporal changes. Then start the recalculation by clicking on "Calculate FastFLIM".\\
  
-{{ :writingroom:framebin_10_3.png?300 |}}+{{ howto:framebin_10_3.png?300 |}}
  
 **Response:**\\ **Response:**\\
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   * To enhance the lifetime contrast, adapt the color scale by typing the limit values for the "Fast Lifetime [ns]" and "Events[cnts]". Additionally set the "Lifetime Histogram" range on the right hand.\\   * To enhance the lifetime contrast, adapt the color scale by typing the limit values for the "Fast Lifetime [ns]" and "Events[cnts]". Additionally set the "Lifetime Histogram" range on the right hand.\\
  
-{{ :writingroom:fastlifetimerange_2.png?300 |}}+{{ howto:fastlifetimerange_2.png?300 |}}
  
  
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   * A few images of the series is plotted here; images 1 [Frame 1-10], 3 [Frame 21-30], 4, 6, 10:\\   * A few images of the series is plotted here; images 1 [Frame 1-10], 3 [Frame 21-30], 4, 6, 10:\\
  
-{{ :writingroom:1st_10frame_2.png?400 |}} +{{ howto:1st_10frame_2.png?400 |}} 
-{{ :writingroom:3rd_10frame_2.png?400 |}} +{{ howto:3rd_10frame_2.png?400 |}} 
-{{ :writingroom:4th_10frame_2.png?400 |}} +{{ howto:4th_10frame_2.png?400 |}} 
-{{ :writingroom:6th_10frame_2.png?400 |}} +{{ howto:6th_10frame_2.png?400 |}} 
-{{ :writingroom:10th_10frame_2.png?400 |}}+{{ howto:10th_10frame_2.png?400 |}}
  
  
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   * In order to quantify tension changes in cell membrane, draw a ROI on the image where the bigger cells are located.    * In order to quantify tension changes in cell membrane, draw a ROI on the image where the bigger cells are located. 
-{{ :writingroom:roi0_10framebinning_2.png |}}+{{ howto:roi0_10framebinning_2.png |}}
  
  
   * Tick the "Select in all Frames" checkbox. Click "ROI per Frame" to select an ROI with the same marked pixels in each frame.  \\   * Tick the "Select in all Frames" checkbox. Click "ROI per Frame" to select an ROI with the same marked pixels in each frame.  \\
  
-{{ :writingroom:select_in_all_frames.png?300 |}}+{{ howto:select_in_all_frames.png?300 |}}
    
 **Response:**\\ **Response:**\\
 When the ROI in each image is created, also the corresponding decay appears in grey in the TCSPC decay window. When the ROI in each image is created, also the corresponding decay appears in grey in the TCSPC decay window.
-{{ :writingroom:rois_and_decays_afterbinning_2.png |}}+{{ howto:rois_and_decays_afterbinning_2.png |}}
  
 ==== Perform an Initial Lifetime Fit on the Selected Regions (ROIs) ==== ==== Perform an Initial Lifetime Fit on the Selected Regions (ROIs) ====
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   * From "Decay" drop-down list select ''ROI 0'' and click "Initial Fit" (marked in orange).\\   * From "Decay" drop-down list select ''ROI 0'' and click "Initial Fit" (marked in orange).\\
  
-{{ :writingroom:selectroi_initfit_multiframe_2.png?300 |}}+{{ howto:selectroi_initfit_multiframe_2.png?300 |}}
    
 ** Response:**\\  ** Response:**\\ 
 A single exponential reconvolution fit is performed. In the TCSPC window, the fit is displayed as a black line. Below, the residuals (= difference between raw data and fit values) are displayed.\\  A single exponential reconvolution fit is performed. In the TCSPC window, the fit is displayed as a black line. Below, the residuals (= difference between raw data and fit values) are displayed.\\ 
   * Fit values appear in the fitting table. Check the fitting curve, residuals and χ².\\   * Fit values appear in the fitting table. Check the fitting curve, residuals and χ².\\
-{{ :writingroom:monoexpo_roi0_10framebinning_2.png |}}+{{ howto:monoexpo_roi0_10framebinning_2.png |}}
    
 **Note:**\\ **Note:**\\
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   * In this example it is obvious that the applied single exponential reconvolution fit is not suited to fit the decay curve. Increase the "Model Parameters" to n = 2. Again, click on "Initial fit".\\   * In this example it is obvious that the applied single exponential reconvolution fit is not suited to fit the decay curve. Increase the "Model Parameters" to n = 2. Again, click on "Initial fit".\\
  
-{{ :writingroom:biexpo_roi0_10framebinning_2.png |}}+{{ howto:biexpo_roi0_10framebinning_2.png |}}
  
 ** Response:**\\ ** Response:**\\
 The fit quality increases. But if it still doesn't look sufficient, increase number of "Model Parameters" to three.\\ The fit quality increases. But if it still doesn't look sufficient, increase number of "Model Parameters" to three.\\
  
-{{ :writingroom:3expo_roi0_10framebinning_2.png |}}+{{ howto:3expo_roi0_10framebinning_2.png |}}
  
   * We apply fit now all ROIs using the fitting model and start parameters from the ROI0-fit by pressing "Fit All".   * We apply fit now all ROIs using the fitting model and start parameters from the ROI0-fit by pressing "Fit All".
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   * The fit results for all datasets can be seen (and exported) as a table using the "Show all"-button.\\   * The fit results for all datasets can be seen (and exported) as a table using the "Show all"-button.\\
  
-{{ :writingroom:showall_button.png?300 |}}+{{ howto:showall_button.png?300 |}}
  
   * Clicking on "Fit All" will fit all active data sets with the same model, using the start parameters of the active data set. If you do not want to fit the complete decay (dataset 1) and the decay of a single pixel (Dataset 2), press "Show All" button, uncheck the first two datasets on top of the parameter table and press "Fit All" again.\\    * Clicking on "Fit All" will fit all active data sets with the same model, using the start parameters of the active data set. If you do not want to fit the complete decay (dataset 1) and the decay of a single pixel (Dataset 2), press "Show All" button, uncheck the first two datasets on top of the parameter table and press "Fit All" again.\\ 
  
 **Response:**\\ **Response:**\\
-{{ :writingroom:showall_table_2.png |}}+{{ howto:showall_table_2.png |}}
  
   * On the right hand of the TCSPC window select “Parameter Plot” to get a graphical impression of the change of the fit values and compare for instance average lifetime of your ROIs.   * On the right hand of the TCSPC window select “Parameter Plot” to get a graphical impression of the change of the fit values and compare for instance average lifetime of your ROIs.
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   * Select the intensity averaged lifetime for the Y-Axis; tick the "tau_Av_In" checkbox and deselect the unwanted fitting parameters.   * Select the intensity averaged lifetime for the Y-Axis; tick the "tau_Av_In" checkbox and deselect the unwanted fitting parameters.
  
-{{ :writingroom:parameterplot_10framebinning_2.png |}}+{{ howto:parameterplot_10framebinning_2.png |}}
  
 **Response:**\\ **Response:**\\
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   * Click on "Export all frames” with the selection "Bitmap".    * Click on "Export all frames” with the selection "Bitmap". 
  
-{{ :writingroom:exportbitmap.png?400 |}}+{{ howto:exportbitmap.png?400 |}}
  
 **Note:**\\ **Note:**\\
howto/visualizing_dynamics_using_the_multiframe-flim_script.txt · Last modified: 2023/02/17 12:41 by admin