howto:lifetime-fitting_using_the_rapid_reconvolution_algorithm
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writingroom:lifetime-fitting_using_the_rapid_reconvolution_algorithm [2020/05/12 16:16] – ruckelshausen | howto:lifetime-fitting_using_the_rapid_reconvolution_algorithm [2023/02/17 12:40] (current) – admin | ||
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This tutorial shows step-by-step, | This tutorial shows step-by-step, | ||
- | For the principle of rapidFLIM - recording, check the technical | + | For the principle of rapidFLIM - recording, check the application |
+ | [[https:// | ||
- | Here in detail the FLIM analysis of SymPhoTime 64 is used to perform a lifetime fit and how to extract and read the results. A mixture of Dragon Green and Nile Red fluorescent microspheres were imaged with more than three frames per second. | + | Here in detail the FLIM analysis of SymPhoTime 64 is used to perform a lifetime fit and how to extract and read the results. A mixture of Dragon Green and Nile Red fluorescent microspheres were imaged with more than three frames per second. Tutorials workspace can be downloaded from [[https:// |
===== Step-by-Step Tutorial ===== | ===== Step-by-Step Tutorial ===== | ||
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* Start the FLIM analysis by clicking on " | * Start the FLIM analysis by clicking on " | ||
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The FLIM analysis is applied to the data file. Thereby, a new Window opens: | The FLIM analysis is applied to the data file. Thereby, a new Window opens: | ||
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* Set Proper Detection Channel, in this case 3, and press " | * Set Proper Detection Channel, in this case 3, and press " | ||
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* On top left, under Region of Interest, check "Use ROI" box and click on "ROI from Threshold" | * On top left, under Region of Interest, check "Use ROI" box and click on "ROI from Threshold" | ||
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A large window with three sections will appear, where you can set a threshold for an image analysis visually. | A large window with three sections will appear, where you can set a threshold for an image analysis visually. | ||
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**Note:**\\ | **Note:**\\ | ||
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* First adapt the contrast by setting the Number of Events [Cnts] in brightest pixel and Fast Lifetime color code. This will help you to select your ROI or threshold easier. | * First adapt the contrast by setting the Number of Events [Cnts] in brightest pixel and Fast Lifetime color code. This will help you to select your ROI or threshold easier. | ||
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*As a next step try to distinguish compartments of your specimen in FLIM image by setting the intensity or lifetime thresholds. For instance select the area with higher intensity by dragging the indicator bars. | *As a next step try to distinguish compartments of your specimen in FLIM image by setting the intensity or lifetime thresholds. For instance select the area with higher intensity by dragging the indicator bars. | ||
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* Or try different lifetime range: | * Or try different lifetime range: | ||
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* As an example here an area with longer lifetime is selected. | * As an example here an area with longer lifetime is selected. | ||
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Corresponding decays with gray color will appear under Overall Decay curve. | Corresponding decays with gray color will appear under Overall Decay curve. | ||
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* Start with ROI 0 and only one component (monoexponential) fit. Press " | * Start with ROI 0 and only one component (monoexponential) fit. Press " | ||
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* If the fitted curve is not overlaid not so well with the decay curve, like in this case, increase the "Model Parameters" | * If the fitted curve is not overlaid not so well with the decay curve, like in this case, increase the "Model Parameters" | ||
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* One the single pixel level, the background can be neglected, and the parameter " | * One the single pixel level, the background can be neglected, and the parameter " | ||
* Set constraints for A1 and A2, so they cannot become negative. | * Set constraints for A1 and A2, so they cannot become negative. | ||
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* and press "FLIM fit". | * and press "FLIM fit". | ||
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howto/lifetime-fitting_using_the_rapid_reconvolution_algorithm.txt · Last modified: 2023/02/17 12:40 by admin