All ROIs Single-Pixel Analysis

The multi-ROIs single-pixel analysis scripts accomplish a series of steps that can speed up the single-pixel level analysis of a FLI datasets. A number of options are available and are defined in a Multi-ROIs, which provide some flexibility in the actual series of steps, settings or outputs of the analyses, as described below.

The general idea is, for NLSF analysis, to start from a list of pre-defined ROIs, convert them to single-pixel ROIs and perform NLSF for this new list of single-pixel ROIs. Optionally, a preliminary step of IRF extraction (NLSF), can be included at the beginning, and ASCII Files of some of the parameters saved as well as custom histograms build at the end of the analysis.

For phasor analysis, the steps are similar, with the difference tha each ROI is processed individually, converted to single-pixel ROIS, and a single phasor plot output for each ROI.

NLSF Analysis

The dialog window used to specify the NLSF analysis steps looks as follows:

_images/AlliGator-Multi-ROIs-NLSF-Analysis-Dialog.png

  • Step 1: The first parameter, IRF File is optional and not visible unless the top left Use IRF Dataset checkbox is checked off. The corresponding IRF file can be selected using the Browse File button on the right side of the box. It is used to extract single-pixel IRFs for the subsequent single-pixel NLSF analysis.

    If a single, common IRF is to be used instead, or if the single-pixel IRFs have already been defined and are still in memory, this checkbox should be left unchecked.

  • Step 2: When and if an IRF file has been selected, the next step consists in specifying all the options to be used to process these IRFs. This includes file loading options (Data Information panel of the Settings window), pixel rejection options (Source Image panel) and decay preprocessing options (Settings:Fluorescence Decay:Decay Pre-Processing panel). In particular, decays from the IRF file might need to be extrapolated, which would require defining the relevant parameters in the Decay Pre-Processing panel.

    To store these options, click on the Set IRF Parameters button to the right. The IRF Settings Saved LED next to the button lights up to indicate that the options have been saved. It is always possible to modify them later, but do not forget to press the Set IRF Parameters button to store the new options.

  • Step 3: The dataset to be analyzed is selected with the Browse File button to the right of the Dataset File box (alternatively drag and drop the file into the Dataset File box).

  • Step 4: When done, change the settings to those to be applied to the actual datasets and save them using the Set Dataset Parameters button to the right. The Dataset Settings LED turns on to indicate that the parameters to be used for the dataset have been stored.

  • Step 5: ROIs to be used in the analysis can be predefined before the analysis or loaded from a file. In that case, the file is specified in the ROI File box.

  • Step 6: To save some of the fitted (or derived) fit parameters as ASCII files and generate histograms, check the Save Results checkbox and define a Results Folder, as well as the list of Decay Fit Parameters to save, as well as the histogram bin size for each parameter in the list at the bottom. The parameter ASCII files will be named Dataset-File-Name_parameter.txt, where parameter is the name of the parameter. The histograms will be generated but not saved.

  • Step 7: To clear the Phasor Graph (in which all the individual ROI single-pixel phasor plots will be displayed) before analysis, check this box.

To run the script, click on the Run Script button. The data corresponding the computed Decay Fit Parameter Map will be saved automatically in a HDF5 file named Dataset-File-Name IRFs & Fit Data.hdf5, where Dataset-File-Name is the file name (without extension) of the analyzed dataset.

Phasor Analysis

The dialog window used to specify the phasor analysis steps looks as follows:

_images/AlliGator-Multi-ROIs-Phasor-Analysis-Dialog.png

  • Step 1: The first parameter, IRF File is optional and not visible unless the top left Use IRF Dataset checkbox is checked off. The corresponding IRF file can be selected using the Browse File button on the right side of the box. It is used to extract single-pixel IRFs, compute their phasor and create a phasor plot in the Phasor Graph.

    If the calibration to be used for the dataset analysis has already been defined and is stored, this setp can be ignored and the Use IRF Dataset checkbox should be unchecked.

  • Step 2: When and if an IRF file has been selected, the next step consists in specifying all the options to be used to process these IRFs. This includes file loading options (Data Information panel of the Settings window), pixel rejection options (Source Image panel), decay preprocessing options (Settings:Fluorescence Decay:Decay Pre-Processing panel) and calibration options (Phasor Calibration panel). In general, no calibration should be applied, and the calibration lifetime shold be set to the one associated with the IRF dataset (generally zero),

    To store these options, click on the Set IRF Parameters button to the right. The IRF Settings Saved LED next to the button lights up to indicate that the options have been saved. It is always possible to modify them later, but do not forget to press the Set IRF Parameters button to store the new options.

    The script, will load the file, compute the individual pixel phasors and use the resulting phasor plot as a phasor calibration map.

  • Step 3: The dataset to be analyzed is selected with the Browse File button to the right of the Dataset File box (alternatively drag and drop the file into the Dataset File box).

  • Step 4: When done, change the settings to those to be applied to the actual datasets and save them using the Set Dataset Parameters button to the right. The Dataset Settings LED turns on to indicate that the parameters to be used for the dataset have been stored.

  • Step 5: ROIs to be used in the analysis need to be loaded from a file, specified in the ROI File box.

  • Step 6: To save scatterplots of pairs of some of the computed (or derived) parameters as ASCII files, check the Save Results checkbox and define a Results Folder, as well as the list of Phasor Parameters Scatter Plots. The parameter ASCII files will be named Dataset-File-Name (ROI n, f = XXX MHz)_parameter 2 vs parametr 1.txt, where n is the ROU number, XXX is the phasor frequency and parameter 1 & parameter 2 are the names of the selected parameter. The phasor plots will not be saved, but this can be easily done at the end of the script using the Phasor Graph Save Phasor Plots (.phplot):Save All Phasor Plots menu option.

To run the script, click on the Run Script button.