This tutorial shows step-by-step, how to fit the lifetime of a measured sample. As an example, a single exponential reconvolution fit is used to determine the lifetime of ATTO655 diluted in water.
Select a file and start the script
Note: The “Samples” workspace is delivered with the SymPhoTime 64 and on the CD-ROM and contains example data to show the function of the SymPhoTime data analysis. If you haven't installed it on your computer, copy it from the DVD onto a local drive before going through this tutorial.
ATTO655_diff_FLCS-pattern.ptuby a single mouse click.
Note: The window contains two different regions:
Note: The software offers the possibility to fit the data using a n-exponential tailfit or a n-exponential reconvolution fit. A tailfit can be used when the fitted lifetimes are significantly longer than the instrument response function. Still 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.
For explanation 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.
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.
Usually the fitting model with least parameters is selected.
⇒ In this example, the fit is already sufficient.
Note: The .dat file contains the TCSPC curve, the estimated instrument response function (IRF) and the fitted curve.
ATTO655_diff_FLCS-pattern.ptuwith one mouse click each and start the TCSPC fitting script.
Response: The TCSPC fitting window is opened and the TCSPC histograms of both files are loaded. The TCSPC histogram from the file “ATTO655_diff_FLCS-pattern.ptu is marked in green, indicating that it is the active file. Under decay data all files are listed, the active file is always highlighted in green.
Note: The file
Cy5_diff_IRF+FLCS-pattern.ptu contains a lifetime measurement of the dye Cy5 in water. In the TCSPC histogram it can be clearly seen that the lifetime of this dye is significantly shorter than the lifetime of ATTO655.
Response: Both data sets are fitted with a single exponential tailfit model. The values of the last dataset are displayed in the fitting table.
Response: The parameter plot is shown. The parameters to be displayed are plotted as points in a graph, with the first point on the left belonging to the first data set, etc.
Note: The parameter plot is in this case not very illuminating, as only 2 datasets are present. It's full potential can be generated, if the same dye is measured several times, because in this case it graphically shows the deviation of the fitting values. It also calculates an average for each fitting value over the data sets. In our example, it is of course meaningless, as two different dyes were fitted.
Response: A window opens and asks for a file name and a folder to store the data, e.g. as
.pqres), which in this case is storedalong with both corresponding raw data files (