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writingroom:lifetime-fitting_using_the_rapid_reconvolution_algorithm [2020/05/13 07:24] – ruckelshausenwritingroom:lifetime-fitting_using_the_rapid_reconvolution_algorithm [2020/05/13 07:50] – ↷ Links adapted because of a move operation ruckelshausen
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   * Start the FLIM analysis by clicking on "Start".   * Start the FLIM analysis by clicking on "Start".
  
-{{ :writingroom:flimgui.png?200 |}}+{{ howto:flimgui.png?200 |}}
  
  
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 The FLIM analysis is applied to the data file. Thereby, a new Window opens: The FLIM analysis is applied to the data file. Thereby, a new Window opens:
  
-{{ :writingroom:sptwindows_rapidflim.png |}}+{{ howto:sptwindows_rapidflim.png |}}
  
  
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   * Set Proper Detection Channel, in this case 3, and press "Calculate FastFLIM".   * Set Proper Detection Channel, in this case 3, and press "Calculate FastFLIM".
  
-{{ :writingroom:ch3_fastflim.png?500 |}}+{{ howto:ch3_fastflim.png?500 |}}
  
  
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   *  On top left, under Region of Interest, check "Use ROI" box and click on "ROI from Threshold".   *  On top left, under Region of Interest, check "Use ROI" box and click on "ROI from Threshold".
  
-{{ :writingroom:roiactive.png?300 |}}+{{ howto:roiactive.png?300 |}}
  
  
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 A large window with three sections will appear, where you can set a threshold for an image analysis visually. A large window with three sections will appear, where you can set a threshold for an image analysis visually.
  
-{{ :writingroom:selectthreshold.png |}}+{{ howto:selectthreshold.png |}}
  
 **Note:**\\ **Note:**\\
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   *  First adapt the contrast by setting the Number of Events [Cnts] in brightest pixel and Fast Lifetime color code. This will help you to select your ROI or threshold easier.   *  First adapt the contrast by setting the Number of Events [Cnts] in brightest pixel and Fast Lifetime color code. This will help you to select your ROI or threshold easier.
  
-{{ :writingroom:adaptcontrast_selectroi.png?500 |}}+{{ howto:adaptcontrast_selectroi.png?500 |}}
  
  
   *As a next step try to distinguish compartments of your specimen in FLIM image by setting the intensity or lifetime thresholds. For instance select the area with higher intensity by dragging the indicator bars.   *As a next step try to distinguish compartments of your specimen in FLIM image by setting the intensity or lifetime thresholds. For instance select the area with higher intensity by dragging the indicator bars.
  
-{{ :writingroom:select_high_intensity.png?500 |}} +{{ howto:select_high_intensity.png?500 |}} 
  
    
   * Or try different lifetime range:   * Or try different lifetime range:
  
-{{ :writingroom:select_by_lifetime.png |}}+{{ howto:select_by_lifetime.png |}}
  
  
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-{{ :writingroom:startroi1.png |}}+{{ howto:startroi1.png |}}
  
  
   * As an example here an area with longer lifetime is selected.     * As an example here an area with longer lifetime is selected.  
  
-{{ :writingroom:roi1_fromhistogram.png |}}+{{ howto:roi1_fromhistogram.png |}}
  
  
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 Corresponding decays with gray color will appear under Overall Decay curve. Corresponding decays with gray color will appear under Overall Decay curve.
  
-{{ :writingroom:roi_decays.png |}}+{{ howto:roi_decays.png |}}
  
  
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   * Start with ROI 0 and only one component (monoexponential) fit. Press "Initial Fit" (marked in orange).   * Start with ROI 0 and only one component (monoexponential) fit. Press "Initial Fit" (marked in orange).
  
-{{ :writingroom:monoexpo_roi0.png |}}+{{ howto:monoexpo_roi0.png |}}
  
  
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   * If the fitted curve is not overlaid not so well with the decay curve, like in this case, increase the "Model Parameters" to n = 2. Again, click on "Initial fit".   * If the fitted curve is not overlaid not so well with the decay curve, like in this case, increase the "Model Parameters" to n = 2. Again, click on "Initial fit".
  
-{{ :writingroom:biexpo_roi0.png |}}+{{ howto:biexpo_roi0.png |}}
  
  
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   * One the single pixel level, the background can be neglected, and the parameter "Backgroudn Decay" can be fixed to 0 in this example.   * One the single pixel level, the background can be neglected, and the parameter "Backgroudn Decay" can be fixed to 0 in this example.
   * Set constraints for A1 and A2, so they cannot become negative.   * Set constraints for A1 and A2, so they cannot become negative.
-{{ :writingroom:limitamplitude_beads_2.png?600 |}}+{{ howto:limitamplitude_beads_2.png?600 |}}
  
   * and press "FLIM fit".   * and press "FLIM fit".
  
  
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howto/lifetime-fitting_using_the_rapid_reconvolution_algorithm.txt · Last modified: 2023/02/17 12:40 by admin