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howto:flim_fret_calculation_for_multi_exponential_donors [2014/06/18 10:40] – adminhowto:flim_fret_calculation_for_multi_exponential_donors [2023/11/21 10:33] (current) – [FLIM FRET Calculation for Multi Exponential Donors] lan
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 ~~NOTOC~~  ~~NOTOC~~ 
  
-====== FLIM FRET Calculation for Multi Exponential Donors ======+====== FLIM-FRET Calculation for Multi Exponential Donors ======
  
  
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 ===== Step-by-Step Tutorial ===== ===== Step-by-Step Tutorial =====
  
- +**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 a 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 tailfit, a 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.
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 **Response:**\\ **Response:**\\
 The “Lifetime FRET Image” script is applied to the file ''CENP-labelled _cells_for_FRET.ptu''. Thereby, a new Window opens: The “Lifetime FRET Image” 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_26.png?600 }}+{{ :howto:flim-fret-multiexpd_lt-fret.png |}}
  
 The window contains five different regions:  The window contains five different regions: 
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 **Lower left:** Binding histogram. As the FRET efficiency image has not been calculated yet, this histogram is also still empty. As this diagram can only be calculated if the FRET donor dye has single exponential decay kinetics, changing the fitting model results in an exchange of this graph to a FRET distance histogram.\\ **Lower left:** Binding histogram. As the FRET efficiency image has not been calculated yet, this histogram is also still empty. As this diagram can only be calculated if the FRET donor dye has single exponential decay kinetics, changing the fitting model results in an exchange of this graph to a FRET distance histogram.\\
 **Lower center/left:** TCSPC histogram of all photons in the image in green (= dataset 0) and the TCSPC histogram of a single pixel in grey (= dataset 1). In red an estimation of the instrument response function (=IRF) is shown. The IRF reconstruction is deducted from the rising edge of the images TCSPC histogram. On the left of this graph, the fitting model and parameter controls are displayed. **Lower center/left:** TCSPC histogram of all photons in the image in green (= dataset 0) and the TCSPC histogram of a single pixel in grey (= dataset 1). In red an estimation of the instrument response function (=IRF) is shown. The IRF reconstruction is deducted from the rising edge of the images TCSPC histogram. On the left of this graph, the fitting model and parameter controls are displayed.
- 
  
   * Select only channel 2 as active channel.   * Select only channel 2 as active channel.
   * As the photon number/pixel is quite low in this image, select a Pixel Binning of 4 points.   * As the photon number/pixel is quite low in this image, select a Pixel Binning of 4 points.
   * Press "Calculate FastFLIM" to update the image.   * Press "Calculate FastFLIM" to update the image.
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_27.png }}+{{ :howto:flim-fret-multiexpd_calcfastflim.png?400 |}}
  
 **Response:**\\ **Response:**\\
 The pixel binning is applied to the image and the image is recalculated, using only photons from detector channel 2. The pixel binning is applied to the image and the image is recalculated, using only photons from detector channel 2.
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_28.png }}+{{ :howto:flim-fret-multiexpd_calcfastflim_response.png?400 |}}
  
   * Set the intensity scale from 0 counts – 200 counts and the lifetime scale from 0 to 5 ns.   * Set the intensity scale from 0 counts – 200 counts and the lifetime scale from 0 to 5 ns.
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_29.png }}+{{ :howto:flim-fret-multiexpd_fastflim_enhancedcontrast.png?400 |}} 
 + 
 +  *  On top left, under Region of Interest, check "Use ROI" box and click on "ROI from Threshold". 
 +{{ :howto:flim-fret-1expd_roiactive.png?300 |}}
  
-  * In the image parameter panel, open the "Threshold" drop down menu. 
-  * Activate "Use Threshold" and enter a threshold of 100. 
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_30.png }} 
  
 **Response:**\\ **Response:**\\
-In the image, only pixels with higher photon counts are highlighted.+A large window with three sections will appear, where you can set a threshold for an image analysis visually. 
 +{{ :howto:flim-fret-multiexpd_thesh_1.png |}}
  
 **Note:**\\ **Note:**\\
-One can also use the bar in the intensity histogram to set the threshold.+Left to right: Preview FLIM Image, Intensity Histogram and Lifetime Histogram. For any ROI you can set each of these parameters individually.  
 +There are two ways to define the new threshold. You can use the edit-box at the lower side to type the threshold and press enter to check the result or use cursor keys or mouse wheel to increase/decrease the value. Another option is using the blue vertical bars on the sides of the intensity or lifetime histograms; click and drag the bars to set the threshold. 
 + 
 +  * In this example set the lower intensity threshold to 100 counts. 
 +{{ :howto:flim-fret-multiexpd_thesh_2_bearb.png?400 |}} 
 +  * Click "OK" 
 + 
 +**Response:** In the image, only pixels with higher photon counts are highlighted. (eventually adapt the intensity maximum to see the highlighted pixels more clearly). 
 +{{ :howto:flim-fret-multiexpd_thesh_response.png?400 |}}
  
 {{ flim-fret-calculation_for_multi-exponential_donors_Image_31.png }} {{ flim-fret-calculation_for_multi-exponential_donors_Image_31.png }}
  
-  * For the analysis, only the spots within the nuclei of the cells are of importance. Therefore exclude the bright dots outside the nucleus of the cell on the lower right by placing the mouse cursor over the image, activate the context menu with a right mouse click, select "Rectangle ROI", press continuously "CRTL" and draw circles around the spots outside the nucleus.+  * For the analysis, only the spots within the nuclei of the cells are of importance. Therefore exclude the bright dots outside the nucleus of the cell on the lower right by placing the mouse cursor over the image, activate the context menu with a right mouse click, select "Rectangle ROI", press continuously "CRTL" and draw rectangles around the spots outside the nucleus.
   * **Response:** The marked regions are excluded from analysis.   * **Response:** The marked regions are excluded from analysis.
 {{ flim-fret-calculation_for_multi-exponential_donors_Image_32.png }} {{ flim-fret-calculation_for_multi-exponential_donors_Image_32.png }}
  
   * In the lifetime fitting pannel, set the Fitting model to "Multi-Exp. Donor".   * In the lifetime fitting pannel, set the Fitting model to "Multi-Exp. Donor".
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_33.png }}+{{ :howto:flim-fret-multiexpd_multiexpfit.png?400 |}}
  
   * Click on IRF "Import"   * Click on IRF "Import"
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_34.png }}+{{ :howto:flim-fret-multiexpd_irfimport.png?400 |}}
  
   * In the window appearing, select the file ''CENP-labelled cell_for_FRET_IRF_Det2.ptu'' as before in the FLIM script and click "OK".    * In the window appearing, select the file ''CENP-labelled cell_for_FRET_IRF_Det2.ptu'' as before in the FLIM script and click "OK". 
 {{ flim-fret-calculation_for_multi-exponential_donors_Image_35.png }} {{ flim-fret-calculation_for_multi-exponential_donors_Image_35.png }}
  
-  * Activate the imported IRF by choosing it in the IRF option.+  * Activate the imported IRF by choosing it in the IRF option (if not shown automatically).
 {{ flim-fret-calculation_for_multi-exponential_donors_Image_36.png }} {{ flim-fret-calculation_for_multi-exponential_donors_Image_36.png }}
  
 **Response:**The imported IRF is highlighted in red. **Response:**The imported IRF is highlighted in red.
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_37.png?600 }}+{{ :howto:flim-fret-multiexpd_irfimport_result.png |}}
  
   * As model parameters, select n="2".   * As model parameters, select n="2".
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 If a mixture of complexes performing FRET and not performing FRET is present, the decay can of course be more complex, as the Cerulean alone already shows a bi-exponential decay behavior. However, the limited number of photons per pixel does not allow to use a more complex model. If a mixture of complexes performing FRET and not performing FRET is present, the decay can of course be more complex, as the Cerulean alone already shows a bi-exponential decay behavior. However, the limited number of photons per pixel does not allow to use a more complex model.
  
-  * In the parameter panel, enter "2.675" ns as τ<sub>D</sub>.+  * In the parameter panel, enter "2.66" ns as τ<sub>D</sub>.
  
 **Note:**\\ **Note:**\\
-Whether you need to type "2.675" or "2,675" depends on the country specific settings of Windows program. For this tutorial, a computer with German specific settings was used, therefore, the decimal digits are separated with "," instead of ".". +Whether you need to type "2.66" or "2,66" depends on the country specific settings of Windows program. For this tutorial, a computer with German specific settings was used, therefore, the decimal digits are separated with "," instead of ".". 
  
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_38.png }}+{{ :howto:flim-fret-multiexpd_taud.png?400 |}}
  
   * Click "Initial Fit".   * Click "Initial Fit".
-**Response:**The fit is performed to the overall decay. +**Response:**The fit is performed to the ROI decay. 
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_39.png?600 }}+{{ :howto:flim-fret-multiexpd_initialfit_response.png |}}
  
   * In the Fitting panel, set the parameters Bkgr<sub>Dec</sub>, Shift<sub>IRF</sub> and Bkgr<sub>IRF</sub> constant by removing the mark. Set the background of the decay to 0.   * In the Fitting panel, set the parameters Bkgr<sub>Dec</sub>, Shift<sub>IRF</sub> and Bkgr<sub>IRF</sub> constant by removing the mark. Set the background of the decay to 0.
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 In order to reduce statistical fluctuations, as many parameters as possible need to be set constant. In a single pixel, the background counts of the pixel are negligible, therefore it is valid to set this to 0 in this case. Don't press "Initial Fit again". In order to reduce statistical fluctuations, as many parameters as possible need to be set constant. In a single pixel, the background counts of the pixel are negligible, therefore it is valid to set this to 0 in this case. Don't press "Initial Fit again".
  
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_40.png }}+{{ :howto:flim-fret-multiexpd_fitting_bgconst.png?400 |}}
  
   * Press "Calculate FRET" in the upper panel on the left.   * Press "Calculate FRET" in the upper panel on the left.
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 Depending on the screen resolution, one may have to use the scroll-bar in order to access this button. Depending on the screen resolution, one may have to use the scroll-bar in order to access this button.
  
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_41.png?600px }}+{{ :howto:flim-fret-multiexpd_calcfret_bearb.png?400 |}}
  
 **Response:**\\ **Response:**\\
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     * On the lower right, the FRET distance histogram is plotted. As we didn't enter any value of the Förster distance R0, the distance is plotted in terms of "R0".      * On the lower right, the FRET distance histogram is plotted. As we didn't enter any value of the Förster distance R0, the distance is plotted in terms of "R0". 
  
-**Note:**\\ +{{ :howto:flim-fret-multiexpd_calcfret_response.png |}}
-To transfer this in a nanometer scale, the real Förster distance must be known. The result is only meaningful, if the donor to acceptor ratio is 1:1 on the molecular level for each complex. This is not the case in our biological example with a more complex stoichiometry, therefore the graph is meaningless in this specific case.+
  
-Calculating the real Förster distance requires some effort and distinct knowledge about the sample. A few aids are available on the lab. These are not connected to PicoQuant and PicoQuant is not responsible neither for the content nor any possible errors; still these links may give an idea of what is required to calculate Förster distances. +  * Adjust the intensity scale to 0 counts – 200 counts in order to better compare the lifetimes in the Donor-Only and the FRET-cells.
  
-  * [[http://www.pymolwiki.org/index.php/Forster_distance_calculator]] +{{ :howto:flim-fret-multiexpd_calcfret_enhancedcontrast.png?400 |}}
  
-  * [[http://www.osmanbilsel.net/software/forster-distance-calculator]]+**Note:**\\ 
 +The FRET efficiencies calculated also show values below 0. Although physically impossible, mathematically this reflects the variation in the fit.
  
-  * [[http://en.wikipedia.org/wiki/F%C3%B6rster_resonance_energy_transfer]]+  * Set the maximum for the distance in the distance histogram to 2,5 R0. 
 +{{ :howto:flim-fret-multiexpd_maxdist.png?400 |}}
  
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_42.png?600 }}+**Note:**\\ 
 +To transfer the distance into a nanometer scale, the real Förster distance must be known. The result is only meaningful, if the donor to acceptor ratio is 1:1 on the molecular level for each complex. This is not the case in our biological example with a more complex stoichiometry, therefore the graph is meaningless in this specific case.
  
-  * In order to improve the FRET contrast, adjust the intensity scale to 0 counts – 200 counts.+Calculating the real Förster distance requires some effort and distinct knowledge about the sample. A few aids are available on the lab. These are not connected to PicoQuant and PicoQuant is not responsible neither for the content nor any possible errors; still these links may give an idea of what is required to calculate Förster distances. 
  
-**Note:**\\ +  * [[http://www.pymolwiki.org/index.php/Forster_distance_calculator]] 
-The FRET efficiencies calculated also show values below 0. Although physically impossible, mathematically this reflects the variation in the fit.+
  
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_43.png }}+  * [[http://www.osmanbilsel.net/software/forster-distance-calculator]]
  
 +  * [[http://en.wikipedia.org/wiki/F%C3%B6rster_resonance_energy_transfer]]
 +\\
   * Press "Save Result" to save the current analysis.   * Press "Save Result" to save the current analysis.
 {{ flim-fret-calculation_for_multi-exponential_donors_Image_44.png }} {{ flim-fret-calculation_for_multi-exponential_donors_Image_44.png }}
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 **Response:**\\ **Response:**\\
 A result file is saved and linked to the raw data file. A result file is saved and linked to the raw data file.
-{{ flim-fret-calculation_for_multi-exponential_donors_Image_45.png }}+{{ :howto:flim-fret-multiexpd_pqres_2.png?400 |}}
  
   * The image analysis is now finished. There are several possibilities to continue:   * The image analysis is now finished. There are several possibilities to continue:
howto/flim_fret_calculation_for_multi_exponential_donors.1403088001.txt.gz · Last modified: 2014/06/18 10:40 by admin