Time-Resolved Fluorescence Wiki

User Tools

Site Tools

Trace:
howto:lifetime-fitting_using_the_rapid_reconvolution_algorithm

Differences

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

Link to this comparison view

Both sides previous revisionPrevious revision
Next revision		Previous revision
Last revisionBoth sides next revision
writingroom:lifetime-fitting_using_the_rapid_reconvolution_algorithm [2020/05/13 07:24] – ruckelshausenhowto:lifetime-fitting_using_the_rapid_reconvolution_algorithm [2021/06/12 05:05] – [Introduction] rezvani
Line 11: Line 11:
 This tutorial shows step-by-step, how to use the Multi-Exponential Reconvolution fitting model with correction for rapidFLIM. This new approach allows better fitting for data acquired with rapidFLIM capable hardware with count-rates way beyond classical pile up limit. This tutorial shows step-by-step, how to use the Multi-Exponential Reconvolution fitting model with correction for rapidFLIM. This new approach allows better fitting for data acquired with rapidFLIM capable hardware with count-rates way beyond classical pile up limit.
    
-For the principle of rapidFLIM - recording, check the technical note available here: [[https://www.picoquant.com/images/uploads/page/files/16651/2016_soh_appnote_rapidflim.pdf|Application Note: rapidFLIM]]+For the principle of rapidFLIM - recording, check the application note available here: 
 +[[https://www.picoquant.com/dl_technotes/AppNote_rapidFLIM_HiRes.pdf|Application Note: rapidFLIM]].
  
-Here in detail the FLIM analysis of SymPhoTime 64 is used to perform a lifetime fit and how to extract and read the results. A mixture of Dragon Green and Nile Red fluorescent microspheres were imaged with more than three frames per second. + 
 +Here in detail the FLIM analysis of SymPhoTime 64 is used to perform a lifetime fit and how to extract and read the results. A mixture of Dragon Green and Nile Red fluorescent microspheres were imaged with more than three frames per second. Tutorials workspace can be downloaded from [[https://figshare.com/s/4957fcfa684daef86c23]].
  
 ===== Step-by-Step Tutorial ===== ===== Step-by-Step Tutorial =====
Line 40: Line 42:
   * Start the FLIM analysis by clicking on "Start".   * Start the FLIM analysis by clicking on "Start".
  
-{{ :writingroom:flimgui.png?200 |}}+{{ howto:flimgui.png?200 |}}
  
  
Line 46: Line 48:
 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 |}}
  
  
Line 66: Line 68:
  
  
-{{ :writingroom:contrastadaptation2.png?600 |}}+{{ howto:contrastadaptation2.png?600 |}}
  
  
Line 72: Line 74:
   * 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 |}}
  
  
Line 85: Line 87:
   *  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 |}}
  
  
Line 91: Line 93:
 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:**\\
Line 100: Line 102:
   *  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 |}}
  
  
Line 120: Line 122:
  
  
-{{ :writingroom:roi0.png |}}+{{ howto:roi0.png |}}
  
  
Line 126: Line 128:
  
  
-{{ :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 |}}
  
  
Line 139: Line 141:
 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 |}}
  
  
Line 161: Line 163:
    
  
-{{ :writingroom:rapidfitmodel_rois.png?300 |}}+{{ howto:rapidfitmodel_rois.png?300 |}}
  
  
Line 167: Line 169:
   * 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 |}}
  
  
Line 179: Line 181:
   * 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 |}}
  
  
Line 192: Line 194:
  
  
-{{ :writingroom:monoexpo_roi1.png |}}+{{ howto:monoexpo_roi1.png |}}
  
  
Line 199: Line 201:
  
  
-{{ :writingroom:biexpo_roi1.png |}} +{{ howto:biexpo_roi1.png |}} 
  
  
Line 207: Line 209:
  
  
-{{ :writingroom:3expo_roi1.png |}}+{{ howto:3expo_roi1.png |}}
  
  
Line 222: Line 224:
   * 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".
  
  
-{{ :writingroom:flimfitroi0.png |}}+{{ howto:flimfitroi0.png |}}
  
  
Line 234: Line 236:
  
  
-{{ :writingroom:saveresult.png?400 |}}+{{ howto:saveresult.png?400 |}}
  
  
Line 245: Line 247:
  
  
-{{ :writingroom:flimfitroi1.png |}}+{{ howto:flimfitroi1.png |}}
  
  
howto/lifetime-fitting_using_the_rapid_reconvolution_algorithm.txt · Last modified: 2023/02/17 12:40 by admin