howto:flim-fret_calculation_for_single_exponential_donors
Differences
This shows you the differences between two versions of the page.
Both sides previous revisionPrevious revisionNext revision | Previous revision | ||
howto:flim-fret_calculation_for_single_exponential_donors [2020/04/22 07:02] – buschmann | howto:flim-fret_calculation_for_single_exponential_donors [2020/05/06 09:38] (current) – [Step-by-Step Tutorial] ruckelshausen | ||
---|---|---|---|
Line 26: | Line 26: | ||
===== 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 " |
* Start [[products: | * Start [[products: | ||
Line 38: | Line 39: | ||
**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: | ||
Line 65: | Line 67: | ||
{{ 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 intensity threshold to 500 counts. | ||
+ | {{ : | ||
+ | * Click " | ||
+ | |||
+ | **Response: | ||
+ | {{ :howto:flim-fret-1expd_roi_from_thresh_2_v3.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 }} | + | * Choose the corresponding ROI as Decay (in this case "ROI 0"). |
+ | {{ :howto:flim-fret-1expd_fittingmodel.png? | ||
+ | * Click " | ||
+ | |||
+ | **Response: | ||
+ | {{ : | ||
- | * Click " | ||
- | **Response: | ||
- | {{ flim-fret_calculation_for_single_exponential_donors_Image_10.png? | ||
* In the Fitting panel, set the parameters τ< | * In the Fitting panel, set the parameters τ< | ||
- | **Note:** In order to reduce statistical fluctuations, | + | **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< | |
- | {{ flim-fret_calculation_for_single_exponential_donors_Image_11.png }} | + | {{ : |
* 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: | ||
Line 97: | Line 112: | ||
* 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" | ||
Line 108: | Line 123: | ||
**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