howto:flim-fret_calculation_for_single_exponential_donors
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howto:flim-fret_calculation_for_single_exponential_donors [2020/04/22 07:02] – buschmann | howto:flim-fret_calculation_for_single_exponential_donors [2020/05/05 15:24] – [Step-by-Step Tutorial] ruckelshausen | ||
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- | Determine the donor only lifetime using the FLIM script | + | Determine the donor only lifetime using the " |
* Start [[products: | * Start [[products: | ||
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**Response: | **Response: | ||
- | {{ flim-fret_calculation_for_single_exponential_donors_Image_1.png }} | + | {{ :howto:flim-fret-1expd_samples_ws.png |}} |
* Highlight the file '' | * Highlight the file '' | ||
- | {{ flim-fret_calculation_for_single_exponential_donors_Image_2.png }} | + | {{ :howto:flim-fret-1expd_gret_gfp_and_mrfp.png |}} |
* Select the " | * Select the " | ||
- | {{ flim-fret_calculation_for_single_exponential_donors_Image_3.png }} | + | {{ :howto:flim-fret-1expd_analysis_imaging.png?500 |}} |
**Note:** The drop down menu can be opened and closed by clicking on the grey button on the left side of the header of the drop down menu {{grey_button.png}} | **Note:** The drop down menu can be opened and closed by clicking on the grey button on the left side of the header of the drop down menu {{grey_button.png}} | ||
- | * Start the " | + | * Start the " |
{{ flim-fret_calculation_for_single_exponential_donors_Image_4.png }} | {{ flim-fret_calculation_for_single_exponential_donors_Image_4.png }} | ||
- | **Response: | + | **Response: |
- | {{ flim-fret_calculation_for_single_exponential_donors_Image_5.png?600 }} | + | {{ :howto:flim-fret-1expd_analysis_lifetime-fret.png |}} |
The window contains five different regions: | The window contains five different regions: | ||
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{{ flim-fret_calculation_for_single_exponential_donors_Image_6.png }} | {{ flim-fret_calculation_for_single_exponential_donors_Image_6.png }} | ||
- | * In the image parameter panel, open the " | ||
- | * Activate "Use Threshold" | ||
- | * To set the threshold one can also use the bars in the intensity histogram. | ||
- | {{ flim-fret_calculation_for_single_exponential_donors_Image_7.png }} | ||
- | **Response: | + | * On top left, under Region of Interest, check "Use ROI" box and click on "ROI from Threshold" |
- | {{ flim-fret_calculation_for_single_exponential_donors_Image_8.png }} | + | |
+ | {{ : | ||
+ | |||
+ | |||
+ | **Response: | ||
+ | A large window with three sections will appear, where you can set a threshold for an image analysis visually. | ||
+ | {{ : | ||
+ | |||
+ | **Note: | ||
+ | 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 threshold to 500 counts. | ||
+ | * Click " | ||
+ | |||
+ | **Response: | ||
+ | {{ :howto:flim-fret-1expd_roi_from_thresh_2_v2.png?400 |}} | ||
* In the lifetime fitting panel, keep the default Fitting model " | * In the lifetime fitting panel, keep the default Fitting model " | ||
{{ flim-fret_calculation_for_single_exponential_donors_Image_9.png }} | {{ flim-fret_calculation_for_single_exponential_donors_Image_9.png }} | ||
+ | * Choose the corresponding ROI as Decay (in this case "ROI 0"). | ||
* Click " | * Click " | ||
- | **Response: | ||
- | {{ flim-fret_calculation_for_single_exponential_donors_Image_10.png? | ||
- | | + | **Response: |
+ | {{ : | ||
- | **Note:** In order to reduce statistical fluctuations, as many parameters | + | |
- | {{ flim-fret_calculation_for_single_exponential_donors_Image_11.png }} | + | **Note:** In order to reduce statistical fluctuations, |
+ | In this model, it is assumed that the long lifetime is caused by unbound donor molecules, and therefore this lifetime is labeled τ< | ||
+ | {{ : | ||
* Press " | * Press " | ||
| | ||
**Note:** Depending on the screen resolution, one may have to use the scroll bar to access this button. | **Note:** Depending on the screen resolution, one may have to use the scroll bar to access this button. | ||
- | + | {{ :howto:flim-fret-1expd_calcfret_bearb.png?400 |}} | |
- | {{ flim-fret_calculation_for_single_exponential_donors_Image_12.png?600px }} | + | |
**Response: | **Response: | ||
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* From the results, a "FRET Image" is calculated and plotted in the FLIM image area. The FLIM image is hidden behind in another tab. | * From the results, a "FRET Image" is calculated and plotted in the FLIM image area. The FLIM image is hidden behind in another tab. | ||
* On the right, the FRET efficiency histogram is plotted from the pixel values. | * On the right, the FRET efficiency histogram is plotted from the pixel values. | ||
- | * This graph shows a single peak, which is in contrast to the lifetime variations in the FLIM image. The explanation can be found in the Binding histogram below showingtwo | + | * This graph shows a single peak, which is in contrast to the lifetime variations in the FLIM image. The explanation can be found in the Binding histogram below showing two distinct peaks. |
* On the lower right, the Binding histogram is plotted. The Binding histogram plots the amplitude fraction of the donor lifetime in presence of FRET. As the amplitude of a donor dye is not changed by its proximity to a FRET acceptor, the binding ratio has a direct effect on the average lifetime. | * On the lower right, the Binding histogram is plotted. The Binding histogram plots the amplitude fraction of the donor lifetime in presence of FRET. As the amplitude of a donor dye is not changed by its proximity to a FRET acceptor, the binding ratio has a direct effect on the average lifetime. | ||
- | {{ flim-fret_calculation_for_single_exponential_donors_Image_13.png?600 }} | + | {{ :howto:flim-fret-1expd_calcfret_response.png |}} |
* To plot an image of the binding, simply change the parameter in the color scale to " | * To plot an image of the binding, simply change the parameter in the color scale to " | ||
- | {{ flim-fret_calculation_for_single_exponential_donors_Image_14.png }} | + | {{ :howto:flim-fret-1expd_bindingimage_bearb.png?400 |}} |
* Press "Save Result" | * Press "Save Result" | ||
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**Response: | **Response: | ||
- | {{ flim-fret_calculation_for_single_exponential_donors_Image_16.png }} | + | {{ :howto:flim-fret-1expd_pqres.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_single_exponential_donors.txt · Last modified: 2020/05/06 09:38 by ruckelshausen