howto:flim_fret_calculation_for_multi_exponential_donors
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howto:flim_fret_calculation_for_multi_exponential_donors [2020/05/12 12:39] – [Calculate the FRET efficiency image using the FLIM-FRET script] ruckelshausen | howto:flim_fret_calculation_for_multi_exponential_donors [2020/05/12 12:44] – [Determine the donor only lifetime using the FLIM script] ruckelshausen | ||
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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 '' | 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. |
howto/flim_fret_calculation_for_multi_exponential_donors.txt · Last modified: 2023/11/21 10:33 by lan