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howto:roi_selection_for_lifetime_fitting_using_the_flim_script [2020/05/12 15:33] – ↷ Page moved from writingroom:roi_selection_for_lifetime_fitting_using_the_flim_script to howto:roi_selection_for_lifetime_fitting_using_the_flim_script ruckelshausenhowto:roi_selection_for_lifetime_fitting_using_the_flim_script [2020/05/12 15:46] – ruckelshausen
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-{{tag>howto tutorial FLIM analysis SPT MT LSM_upgrade}}+{{tag>howto tutorial FLIM analysis SPT SymPhoTime MT LSM_upgrade}}
  
  
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   * Start the FLIM script by clicking on "Start".\\   * Start the FLIM script by clicking on "Start".\\
    
-{{ :writingroom:startflim.png?200 |}}+{{ howto:startflim.png?200 |}}
  
  
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   * Tick the checkbox of the detection "Data Channel" (i.e. detector), you want to use - in this case detector channel 4 - and press "Calculate FastFLIM".\\   * Tick the checkbox of the detection "Data Channel" (i.e. detector), you want to use - in this case detector channel 4 - and press "Calculate FastFLIM".\\
  
-{{ :writingroom:detectionchannel.png?400 |}}+{{ howto:detectionchannel.png?400 |}}
  
 **Response:​**\\ **Response:​**\\
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   * In the FLIM preview image, ROIs can be selected using the context menu (Right Click).\\    * In the FLIM preview image, ROIs can be selected using the context menu (Right Click).\\ 
  
-{{ :writingroom:selectmagicwand_w.png?300 |}}+{{ howto:selectmagicwand_w.png?300 |}}
    
 **Note:**\\ **Note:**\\
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   * In this step, as an example, we mark an ROI with similar lifetime range using the "Magic Wand" tool. The sensitivity of the tool to lifetime differences can be adjusted in a box below the intensity and color scale options.\\   * In this step, as an example, we mark an ROI with similar lifetime range using the "Magic Wand" tool. The sensitivity of the tool to lifetime differences can be adjusted in a box below the intensity and color scale options.\\
  
-{{ :writingroom:magicwand_w_thresh.png?300 |}}   +{{ howto:magicwand_w_thresh.png?300 |}}   
  
   * Increase the threshold to 25.\\   * Increase the threshold to 25.\\
  
-{{ :writingroom:inner_magicwand25_3.png |}}+{{ howto:inner_magicwand25_3.png |}}
    
 **Response:**\\ **Response:**\\
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   * In order to select an additional ROI, click "New" ROI.\\   * In order to select an additional ROI, click "New" ROI.\\
  
-{{ :writingroom:addroi.png?400 |}}+{{ howto:addroi.png?400 |}}
  
    
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   * For the next ROI, first we use the same tool, "Magic Wand" but adapt its threshold to 20.    * For the next ROI, first we use the same tool, "Magic Wand" but adapt its threshold to 20. 
-{{ :writingroom:tubul_magicwand20_3.png |}}+{{ howto:tubul_magicwand20_3.png |}}
    
   * Repeat the last two steps in order to select the cell nuclei as the last ROI.\\   * Repeat the last two steps in order to select the cell nuclei as the last ROI.\\
  
-{{ :writingroom:nuclei_magicwand30_3.png |}}+{{ howto:nuclei_magicwand30_3.png |}}
    
 **Response:**\\ **Response:**\\
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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:inner_monoexpo_3.png |}}+{{ howto:inner_monoexpo_3.png |}}
    
 ** Response:**\\  ** Response:**\\ 
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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:inner_biexpo_3.png |}}+{{ howto:inner_biexpo_3.png |}}
  
  
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 The fit quality increases. But if it still doesn't look sufficient. One sees a trend in residuals and not a random distribution around 0. Increase number of "Model Parameters" to three.\\ The fit quality increases. But if it still doesn't look sufficient. One sees a trend in residuals and not a random distribution around 0. Increase number of "Model Parameters" to three.\\
  
-{{ :writingroom:inner_3expo_3.png |}}+{{ howto:inner_3expo_3.png |}}
  
   * Here the fitting model shows a satisfying result. A 3-exponential fitting model should be used for ROI 0.\\   * Here the fitting model shows a satisfying result. A 3-exponential fitting model should be used for ROI 0.\\
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   * Try the last two steps with "ROI 1".\\   * Try the last two steps with "ROI 1".\\
  
-{{ :writingroom:tubul_3expo_3.png |}}+{{ howto:tubul_3expo_3.png |}}
  
  
   * Find the best fit for "ROI 2" in a same manner. It is better always to start from monoexponential and try to find the fitting model with a lower number of components.\\   * Find the best fit for "ROI 2" in a same manner. It is better always to start from monoexponential and try to find the fitting model with a lower number of components.\\
-{{ :writingroom:nuclei_3expo_3.png |}}+{{ howto:nuclei_3expo_3.png |}}
    
   * Three components exponential decay seems to be suitable for all three ROIs. If all ROIs can be fitted with the same model, you can also click on "Fit All" to get all curves fitted with the same model.\\   * Three components exponential decay seems to be suitable for all three ROIs. If all ROIs can be fitted with the same model, you can also click on "Fit All" to get all curves fitted with the same model.\\
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-{{ :writingroom:showall.png |}}+{{ howto:showall.png |}}
  
   * Uncheck the first two datasets on top of the parameter table and press "Fit All" again.   * Uncheck the first two datasets on top of the parameter table and press "Fit All" again.
  
-{{ :writingroom:deselectdataset1_3.png |}}+{{ howto:deselectdataset1_3.png |}}
  
  
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   * Tick the "tau_Av_Int" checkbox for Y-Axes and zoom in to be able to see the lifetime differences better.\\         * Tick the "tau_Av_Int" checkbox for Y-Axes and zoom in to be able to see the lifetime differences better.\\      
-{{ :writingroom:parameterplot_3rois_3.png |}}+{{ howto:parameterplot_3rois_3.png |}}
  
  
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-{{ :writingroom:SaveResultButton.png?300 |}}+{{ howto:saveresultbutton.png?300 |}}