Loading TCSPC Data

Note that it might be useful to add a "space" or "underscore" character at the end of the Name field to separate original file name from image number (this is not necessary for AlliGator to read the images correctly).

Don't forget to note the relevant acquisition parameters in the Original Metadata window. You will typically need the value of the following parameters:

• • SP_TAC_TC: Time-to-amplitude bin size (in s)

  • SP_TAC_TD: Time-to-amplitude window duration (in s)

It is recommended to save the Original Metadata (as a .csv file) for the record.

• Converting a .ptu file into a folder of gate images

As for the .sdt files, the first step consists in converting the file into a series of "gate images".

An ImageJ plugin developed by Eugene Kathrukha does just that, as explained on the corresponding Github repository: https://github.com/ekatrukha/PTU_Reader.

A simple dialog pops up when using the PTU Reader plugin. Unless you need the intensity and lifetime average stacks (which did not seem to make much sense when I tried this option with a test file), the following options are sufficient for exporting the gate images:

Once the gate image stack has been loaded, proceed as for the .sdt file. Specifically, save the image stack as an image sequence (File>>Save as.>>Image Sequence...): the default parameters will usually be fine.

The metadata present in .ptu file is available in a window named "Log", formatted (except for a few lines) as a series of lines with syntax:

Parameter_Name: Parameter_Value

which it is a good idea to save (as a .txt file) for future reference.

Warning: close the log file before you open a new .ptu file, otherwise the metadata of that file will be appended to the existing log window.

The interpretation of these various parameters can be found in the Matlab repository created by PicoQuant at https://github.com/PicoQuant/PicoQuant-Time-Tagged-File-Format-Demos/blob/master/PTU/Matlab/Read_PTU.m

The following parameters are useful in AlliGator:

• MeasDesc_Resolution: bin size (gate step in AlliGator)

• MeasDesc_GlobalResolution: measurement window (= laser repetition rate?)

Notes on .ptu Files

1) .ptu FLI dataset files are generally comprised of photon timestamps rather than histogrammed nanotimes for each pixel. This results in rather large files (which take some time to decode) with no obvious way to convert the data into gate (or rather, binned) images. To open these files, the user need to specify the desired number of bins (G) into which to histogram the photon nanotimes (also called microtimes by other authors) with the Settings>>Fluorescence Decay>># Gates parameter.

The resulting bin size b (or Gate Duration and Gate Step) is defined as b = T/G, where T, the laser period is read from the .ptu file, where its inverse is saved as "Sync Rate (cps)" (this parameters, as well as others, can be looked up in the Dataset Information Window).

If b is not chosen to be a multiple of the intrinsic hardware resolution, binning artifacts can occur, which appear as regular 'spikes" or "gaps" in decay histograms. The Intrinsic hardware resolution is given by the "Resolution (s)" parameter in the .ptu file and can be looked up in the Dataset Information Window.

2) .ptu files save detection channel information. At this stage (v 0.25), all photons are read and used to form the gate images. If it is useful for you to distinguish between detection channel, please let the author know by posting in the AlliGator Feature Suggestions forum.

3) As mentioned in point 1, loading and reading .ptu files can be time- and resource-consuming. It is recommended to save .ptu datasets binned appropriately as HDF5 FLI dataset to obtain files with reduced size and thus, faster to load subsequently.