howto:flim_fret_calculation_for_multi_exponential_donors
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howto:flim_fret_calculation_for_multi_exponential_donors [2014/06/18 10:39] – admin | howto:flim_fret_calculation_for_multi_exponential_donors [2020/05/12 12:39] – [Calculate the FRET efficiency image using the FLIM-FRET script] ruckelshausen | ||
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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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===== 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 script ==== | ||
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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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* 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 " | ||
- | **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, | + | |
- | Calculating | + | * Adjust |
- | * [[http:// | + | {{ :howto: |
- | | + | **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/ | + | * Set the maximum for the distance in the distance histogram to 2,5 R0. |
+ | {{ : | ||
- | {{ 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, | ||
- | * In order to improve | + | Calculating |
- | **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 }} | + | * [[http:// |
+ | * [[http:// | ||
+ | \\ | ||
* 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: |
howto/flim_fret_calculation_for_multi_exponential_donors.txt · Last modified: 2023/11/21 10:33 by lan