This tutorial shows step-by-step, how the FLIM script of SymPhoTime 64 (V2.5) can be used to perform a pixel-by-pixel lifetime fit of a FLIM image and how to extract and read the results. In detail, the FLIM script is started using an image of mouse kidney tissue in the “Tutorials” workspace,https://figshare.com/s/4957fcfa684daef86c23, a suited fitting model is chosen and a pixel-by-pixel fit is performed.
The files of the sample workspace are displayed in the workspace panel on the left side of the main window.
Kidney_Cells_FLIM.ptuby a single mouse click.
The drop-down menu can be opened and closed by clicking on the grey button on the left side (small set of arrows) of the header of the drop down menu or by clicking onto the “Analysis” header:
The window contains five different sections:
1: Setting panel
Imaging analysis options can be defined in this section.
2: Fast FLIM image
The FLIM preview image displayed in a false color scale. The brightness encodes the intensity, while the color encodes the average “Fast FLIM” lifetime, i.e. the mean arrival times of the photons after the laser pulse. When not defined otherwise, intensity and color scale stretch from minimum to maximum. As the mean arrival time of the background photons in the areas where no fluorescence is detected, is randomly spread over the TCSPC window, the mean photon arrival time of the dark background is usually very long (with an average up to 1/2 of the TCSPC window), which makes the color scale loaded by default sometimes unsuited for displaying the lifetime contrast in the actual sample. In this case, the scale can be adapted.
3: Fluorescence Lifetime Histogram
Here the frequency of photon counts corresponding to the individual mean lifetimes is plotted. The settings of the plot can be adapted using the controls on the right of the plot. The histogram is photon weighted.
4: Decay Fitting Parameters
Here the lifetime fitting model can be selected. Fitting is then applied to the lifetime graph(s) on the lower center (TCSPC histogram).
5: TCSPC Decay window
By default, this set shows the TCSPC histogram of all photons in the image in green (= dataset 0) and the TCSPC histogram of a single pixel in grey (= dataset 1). In red an estimation of the instrument response function (=IRF) is shown. The IRF reconstruction is deducted from the rising edge of the TCSPC histogram. Check the SymPhoTime online help for more details.
FLIM Preview image will get updated. Optimize contrast by adapting “Min” and “Max” values.
A large window with three sections will appear, where you can set a threshold for an image analysis visually.
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/decrease the value. Another option is using the blue vertical bars on the sides of the intensity or lifetime histograms; click and drag the bars to set the threshold.
<CTRL>button, we remove the structures appeared on the left side of the image.
FLIM preview image of
ROI 0, as well as its Lifetime Histogram will get updated. A corresponding decays with gray color appear in TCSPC window under “Overall Decay”.
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 software offers the possibility to fit the data using a n-exponential tailfit, n-exponential reconvolution or rapid reconvolution fit. A tailfit can be used when the fitted lifetimes are significantly longer than the instrument response function. A reconvolution fit is usually preferable, because the complete decay is fitted, while for a tailfit, the start of the fitting range is usually a bit arbitrary. A rapidReconvolution fit is needed, if the count rates, with which the image was acquired are significantly above the Pile Up limit. It is also needed, if the pulse frequency is too high for the fluorescence decay to decrease to the background level before the end of the decay window (often, this is the case for 2-Photon-Excitation data acquired with a TiSa-laser with 80MHz repetition rate). Check out the rapidFLIM-analysis tutorial for this case.
For explanations about the fitting model and the used equations, click on the “Help” button next to the selected model. This opens a help window containing the fitting equation and the explanation of the different parameters.
ROI 0and click “Initial Fit” (marked in orange).
A single exponential reconvolution fit is performed. In the TCSPC window, the fit is displayed as a black line. Below, the residuals (= difference between raw data and fit values) are displayed.
Usually, a decent fit is characterized by the following criteria:
The fitted curve overlays well with the decay curve. In the residual window, the values spread randomly around 0.
The χ²-value approaches 1.
The calculated fitting values are reasonable.
The fitting model with least parameters is preferred.
The fit quality increases. But if it still doesn't look sufficient, increase number of “Model Parameters” to three.
The fourth component appears with a negative amplitude, which is physically not possible. Also, the fit quality has apparently not increased. That means a three component exponential fits the best in this case. So we get back to n=3.
For pixel-by-pixel fits, we are interested about how the lifetime parameters are distributed in the image. ROI fitting for this analysis is the first step to determine the number of fitting parameters here to be used in the pixel-by-pixel fits.
The aim of this procedure is to minimize the number of variable parameters, as every variable parameter also adds noise to the fit. It depends on the sample, to which extend this can be done. In order to increase the fitting quality however, we assume that
A 3-exponential reconvolution fit is performed on each pixel of “ROI 1”. The result is:
I, I, I correspond to τ, τ, τ, respectively in the TCSPC fitting panel. It is also possible to plot the amplitudes instead of the intensities or to switch off individual components. You can adapt the intensities of each color as preferred by modifying the displayed false color scale.
A result file “FLIM.pqres” is stored under the raw data file