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howto:flim_fret_calculation_for_multi_exponential_donors [2020/05/12 14:39]
ruckelshausen [Calculate the FRET efficiency image using the FLIM-FRET script]
howto:flim_fret_calculation_for_multi_exponential_donors [2020/05/12 14:44] (current)
ruckelshausen [Determine the donor only lifetime using the FLIM script]
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 **Note:** This Step-by-Step tutorial was written for SymPhoTime version 2.5. There might be slight differences compared to other software versions. For example the way of setting an intensity threshold has changed. In the video the former version is shown. **Note:** This Step-by-Step tutorial was written for SymPhoTime version 2.5. There might be slight differences compared to other software versions. For example the way of setting an intensity threshold has changed. In the video the former version is shown.
-==== Determine the donor only lifetime using the FLIM script ​====+==== Determine the donor only lifetime using the FLIM analysis ​====
  
  
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 **Response:​**\\ **Response:​**\\
 The FLIM script is applied to the file ''​CENP-labelled_cells_for_FRET.ptu''​. Thereby, a new Window opens: The FLIM script is applied to the file ''​CENP-labelled_cells_for_FRET.ptu''​. Thereby, a new Window opens:
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_5.png?600 }}+{{ :howto:flim-fret-multiexpd_analysis_flim.png |}}
  
 **Note:**\\ **Note:**\\
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   * As data channels, check only Channel 2, as in this channel the donor emission has been recorded.   * As data channels, check only Channel 2, as in this channel the donor emission has been recorded.
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_6.png }}+{{ :howto:flim-fret-multiexpd_ch2.png?400 |}}
  
   * Press "​Calculate Fast FLIM" to update the image.   * Press "​Calculate Fast FLIM" to update the image.
 **Response:​**\\ **Response:​**\\
 The image and the graphs are recalculated,​ using photons only from channel 2. The image and the graphs are recalculated,​ using photons only from channel 2.
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_7.png?600 }}+{{ :​writingroom:​flim-fret-multiexpd_fastflim.png |}}
  
-  * To enhance the lifetime and intensity contrast, set in the image the intensity from 1 count ​to 30 counts and the lifetime from 0 to 5 ns.+  * To enhance the lifetime and intensity contrast, set in the image the intensity from 1 count to 30 counts and the lifetime from 0 to 5 ns.
 **Response:​**\\ **Response:​**\\
 The image scales are adapted. The image scales are adapted.
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 Now four cells are visible, the two nuclei on the left have longer Fast lifetimes then the other two. These are the cells transfected only with the donor constructs. Now four cells are visible, the two nuclei on the left have longer Fast lifetimes then the other two. These are the cells transfected only with the donor constructs.
  
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_8.png }}+{{ :howto:flim-fret-multiexpd_enahncedcontrast.png?400 |}}
  
   * Keep the mouse pointer over the image and open the context menu by a right mouse click. Select the "Free ROI" tool.   * Keep the mouse pointer over the image and open the context menu by a right mouse click. Select the "Free ROI" tool.
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 For more information about the different ROI selection tools, check the tutorial [[howto:​roi_fitting_using_the_flim_script|ROI fitting using the FLIM script]]. For more information about the different ROI selection tools, check the tutorial [[howto:​roi_fitting_using_the_flim_script|ROI fitting using the FLIM script]].
  
-  * Draw ROI around one of the left cells by maintaining the left mouse button activated while circling the selected part of the image. Then release the left mouse button, press "​Shift"​ continuously and cycle the other left nucleus. This way, the donor only cells are marked as ROI 0.+  * Draw an ROI around one of the left cells by maintaining the left mouse button activated while circling the selected part of the image. Then release the left mouse button, press "​Shift"​ continuously and cycle the other left nucleus. This way, the donor only cells are marked as ROI 0.
  
 **Response:​**\\ **Response:​**\\
     * Only the selected area is now shown in color. ​     * Only the selected area is now shown in color. ​
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_10.png }}+{{ :howto:flim-fret-multiexpd_roi.png?400 |}}
  
     * In the TCSPC window, the photons from ROI 0 are added in grey.     * In the TCSPC window, the photons from ROI 0 are added in grey.
 {{ flim-fret-calculation_for_multi-exponential_donors_Image_11.png?​600 }} {{ flim-fret-calculation_for_multi-exponential_donors_Image_11.png?​600 }}
 +
  
     * In the decay form on the lower left, select n-exponential reconvolution as fitting model.     * In the decay form on the lower left, select n-exponential reconvolution as fitting model.
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_12.png }}+{{ :howto:flim-fret-multiexpd_reconvfit.png?400 |}}
  
 **Response:​**\\ **Response:​**\\
     * In the TCSPC window, the IRF is displayed in bright red, the data fitting limits are moved to the border of the TCSPC window.     * In the TCSPC window, the IRF is displayed in bright red, the data fitting limits are moved to the border of the TCSPC window.
     * The new fitting parameters "Shift IRF" and "Bkgr IRF" (=background IRF) appear in the fitting parameter table.     * The new fitting parameters "Shift IRF" and "Bkgr IRF" (=background IRF) appear in the fitting parameter table.
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_13.png?600 }}+{{ :howto:flim-fret-multiexpd_reconvfit_response.png |}}
  
 **Note:**\\ **Note:**\\
-The software offers the possibility to fit the data using a n-exponential tailfit ​or a n-exponential reconvolution fit. A tailfit can be used when the fitted lifetimes are significantly longer than the instrument response function. Still a reconvolution fit is usually preferable, especially for FRET determinations,​ because the complete decay is fitted, while for a tailfit, the start of the fitting range is usually a bit arbitrary, and the short component can be underestimated.+The software offers the possibility to fit the data using a n-exponential tailfita n-exponential ​reconvolution fit or a rapid reconvolution fit. A tailfit can be used when the fitted lifetimes are significantly longer than the instrument response function. Still a reconvolution fit is usually preferable, especially for FRET determinations,​ because the complete decay is fitted, while for a tailfit, the start of the fitting range is usually a bit arbitrary, and the short component can be underestimated. The rapid reconvolution ​ model is used for systems with rapid reconvolution electronics (e.g. hybrid PMA-detector + MultiHarp photon counting board), where the peak count rates have been significantly higher than the pile up limit, or in cases, where the repetition rate has been too high to fit the decay completely into the TCSPC-window,​ e.g. 2PE excitation with 80MHz and dyes with a lifetime above ~3ns.
 For explanation on the fitting model and the used equations, click on the "​Help"​ button next to the selected model. This opens a help window containing the fitting equation and the explanation of the different parameters. ​ For explanation on the fitting model and the used equations, click on the "​Help"​ button next to the selected model. This opens a help window containing the fitting equation and the explanation of the different parameters. ​
  
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 **Response:​**\\ **Response:​**\\
 The TCSPC curve of ROI 0 is highlighted in green. The TCSPC curve of ROI 0 is highlighted in green.
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_14.png?600 }}+{{ :howto:flim-fret-multiexpd_roi-graph.png |}}
  
   * At the IRF, click on the "​Import"​ button.   * At the IRF, click on the "​Import"​ button.
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 **Response:​**\\ **Response:​**\\
 The imported IRF is highlighted in red. The imported IRF is highlighted in red.
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_18.png?600 }}+{{ :howto:flim-fret-multiexpd_irf-graph.png |}}
  
   * As model parameters, select n="​2"​.   * As model parameters, select n="​2"​.
 +{{ :​howto:​flim-fret-multiexpd_n2_bearb.png?​400 |}}
  
 **Note:**\\ **Note:**\\
 In this case, the decay is double exponential. As it is not the scope of the tutorial to explain standard lifetime fitting (see the tutorial "ROI fitting using the FLIM script"​),​ we skip the steps of finding the correct fitting model here. In this case, the decay is double exponential. As it is not the scope of the tutorial to explain standard lifetime fitting (see the tutorial "ROI fitting using the FLIM script"​),​ we skip the steps of finding the correct fitting model here.
- 
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_19.png }} 
  
   * Click on "​Initial Fit".   * Click on "​Initial Fit".
 **Response:​**The fit is calculated. **Response:​**The fit is calculated.
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_20.png?600 }}+{{ :howto:flim-fret-multiexpd_initialfit.png |}}
  
-  * The software calculates the amplitude weighted average lifetime τ<​sub>​Av. Amp.</​sub>​of 2.675. This is the result of the average donor lifetime in absence of FRET.+  * The software calculates the amplitude weighted average lifetime τ<​sub>​Av. Amp.</​sub>​of 2.66. This is the result of the average donor lifetime in absence of FRET.
  
 **Note:**\\ **Note:**\\
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   * Save the result by clicking on "Save Result"​.   * Save the result by clicking on "Save Result"​.
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_21.png }}+ 
 +{{ :howto:flim-fret-multiexpd_saveresult_bearb.png?400 |}}
  
 **Response:​**\\ **Response:​**\\
 A result file (''​FLIM.pqres''​) is stored under the raw data file (''​CENP-labelled_cells_for_FRET.ptu''​). A result file (''​FLIM.pqres''​) is stored under the raw data file (''​CENP-labelled_cells_for_FRET.ptu''​).
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_22.png }}+{{ :howto:flim-fret-multiexpd_pqres.png?400 |}}
  
   * Now the first part is finished and the FLIM window should be closed.   * Now the first part is finished and the FLIM window should be closed.
howto/flim_fret_calculation_for_multi_exponential_donors.txt · Last modified: 2020/05/12 14:44 by ruckelshausen