howto:flim_fret_calculation_for_multi_exponential_donors
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howto:flim_fret_calculation_for_multi_exponential_donors [2014/05/23 07:43] – admin | howto: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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{{tag> | {{tag> | ||
- | ~~TOC~~ | + | ~~NOTOC~~ |
- | ====== FLIM FRET Calculation for Multi Exponential Donors ====== | + | ====== FLIM-FRET Calculation for Multi Exponential Donors ====== |
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This tutorial shows step-by-step, | This tutorial shows step-by-step, | ||
- | **Note:**\\ | + | {{ youtube> |
+ | |||
+ | ===== Background information ===== | ||
FLIM-FRET is a robust method to determine the FRET efficiency of a suited donor acceptor pair. | FLIM-FRET is a robust method to determine the FRET efficiency of a suited donor acceptor pair. | ||
If the FRET-donor molecule has a more exponential decay kinetic, the amplitude weighted average lifetime has to be used to calculate the average FRET efficiency. | If the FRET-donor molecule has a more exponential decay kinetic, the amplitude weighted average lifetime has to be used to calculate the average FRET efficiency. | ||
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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 '' | The FLIM script is applied to the file '' | ||
- | {{ 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 " | * Press " | ||
**Response: | **Response: | ||
The image and the graphs are recalculated, | The image and the graphs are recalculated, | ||
- | {{ flim-fret-calculation_for_multi-exponential_donors_Image_7.png?600 }} | + | {{ : |
- | * To enhance the lifetime and intensity contrast, set in the image the intensity from 1 count | + | * 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: | For more information about the different ROI selection tools, check the tutorial [[howto: | ||
- | * 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 " | + | * 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 " |
**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? | {{ flim-fret-calculation_for_multi-exponential_donors_Image_11.png? | ||
+ | |||
* 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 | + | The software offers the possibility to fit the data using a n-exponential tailfit, a n-exponential |
For explanation on the fitting model and the used equations, click on the " | For explanation on the fitting model and the used equations, click on the " | ||
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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 " | * At the IRF, click on the " | ||
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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=" | * As model parameters, select n=" | ||
+ | {{ : | ||
**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" | 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" | ||
- | |||
- | {{ flim-fret-calculation_for_multi-exponential_donors_Image_19.png }} | ||
* Click on " | * Click on " | ||
**Response: | **Response: | ||
- | {{ 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< | + | * The software calculates the amplitude weighted average lifetime τ< |
**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 ('' | A result file ('' | ||
- | {{ 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 '' | The “Lifetime FRET Image” script is applied to the file '' | ||
- | {{ 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/ | **Lower center/ | ||
- | |||
* Select only channel 2 as active channel. | * Select only channel 2 as active channel. | ||
* As the photon number/ | * As the photon number/ | ||
* Press " | * Press " | ||
- | {{ 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, | The pixel binning is applied to the image and the image is recalculated, | ||
- | {{ 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? |
+ | |||
+ | * On top left, under Region of Interest, check "Use ROI" box and click on "ROI from Threshold" | ||
+ | {{ : | ||
- | * In the image parameter panel, open the " | ||
- | * Activate "Use Threshold" | ||
- | {{ flim-fret-calculation_for_multi-exponential_donors_Image_30.png }} | ||
**Response: | **Response: | ||
- | In the image, only pixels | + | A large window |
+ | {{ : | ||
**Note:**\\ | **Note:**\\ | ||
- | One can also use the bar in the intensity | + | 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/ | ||
+ | |||
+ | * In this example set the lower intensity threshold to 100 counts. | ||
+ | {{ : | ||
+ | * Click " | ||
+ | |||
+ | **Response: | ||
+ | {{ : | ||
{{ 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 " | + | * 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 " |
* **Response: | * **Response: | ||
{{ 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 " | * In the lifetime fitting pannel, set the Fitting model to " | ||
- | {{ flim-fret-calculation_for_multi-exponential_donors_Image_33.png }} | + | {{ :howto:flim-fret-multiexpd_multiexpfit.png?400 |}} |
* Click on IRF " | * Click on IRF " | ||
- | {{ flim-fret-calculation_for_multi-exponential_donors_Image_34.png }} | + | {{ :howto:flim-fret-multiexpd_irfimport.png?400 |}} |
* In the window appearing, select the file '' | * In the window appearing, select the file '' | ||
{{ 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 |
{{ flim-fret-calculation_for_multi-exponential_donors_Image_36.png }} | {{ flim-fret-calculation_for_multi-exponential_donors_Image_36.png }} | ||
**Response: | **Response: | ||
- | {{ flim-fret-calculation_for_multi-exponential_donors_Image_37.png?600 }} | + | {{ :howto:flim-fret-multiexpd_irfimport_result.png |}} |
* As model parameters, select n=" | * As model parameters, select n=" | ||
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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< | + | * In the parameter panel, enter "2.66" ns as τ< |
**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 "," | + | 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 "," |
- | {{ flim-fret-calculation_for_multi-exponential_donors_Image_38.png }} | + | {{ :howto:flim-fret-multiexpd_taud.png?400 |}} |
* Click " | * Click " | ||
- | **Response: | + | **Response: |
- | {{ 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< | * In the Fitting panel, set the parameters Bkgr< | ||
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In order to reduce statistical fluctuations, | In order to reduce statistical fluctuations, | ||
- | {{ flim-fret-calculation_for_multi-exponential_donors_Image_40.png }} | + | {{ :howto:flim-fret-multiexpd_fitting_bgconst.png?400 |}} |
* Press " | * Press " | ||
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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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* This graph shows 2 peaks, which is expected, as in the image one can see two cells with donor and acceptor construct and two cells without. For the cells with the FRET donor only, a FRET efficiency around 0 is expected and also shows up in the histogram. | * This graph shows 2 peaks, which is expected, as in the image one can see two cells with donor and acceptor construct and two cells without. For the cells with the FRET donor only, a FRET efficiency around 0 is expected and also shows up in the histogram. | ||
* 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 " | * 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 " | ||
+ | |||
+ | {{ : | ||
+ | |||
+ | * Adjust the intensity scale to 0 counts – 200 counts in order to better compare the lifetimes in the Donor-Only and the FRET-cells. | ||
+ | |||
+ | {{ : | ||
**Note:**\\ | **Note:**\\ | ||
- | To transfer | + | The FRET efficiencies calculated also show values below 0. Although physically impossible, mathematically |
+ | |||
+ | * Set the maximum for the distance in the distance histogram to 2,5 R0. | ||
+ | {{ : | ||
+ | |||
+ | **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, | ||
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. | 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. | ||
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* [[http:// | * [[http:// | ||
- | + | \\ | |
- | {{ flim-fret-calculation_for_multi-exponential_donors_Image_42.png? | + | |
- | + | ||
- | * In order to improve the FRET contrast, adjust the intensity scale to 0 counts – 200 counts. | + | |
- | + | ||
- | **Note:**\\ | + | |
- | 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 }} | + | |
* Press "Save Result" | * Press "Save Result" | ||
{{ 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: | ||
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* To analyze many images with the same FRET pair, it is possible to save the starting values for the fits by clicking on "Save Defaults" | * To analyze many images with the same FRET pair, it is possible to save the starting values for the fits by clicking on "Save Defaults" | ||
* If you don't want to do anything more, just close the result window. | * If you don't want to do anything more, just close the result window. | ||
- |
howto/flim_fret_calculation_for_multi_exponential_donors.txt · Last modified: 2023/11/21 10:33 by lan