Loading a Folder of Gate Images

Data in time-gated or binned decay phasor analysis consists in folders of images, each folder comprising a series of N images.

Each series of N images represent N "gates". A gate is an image acquired with the image intensifier (or more generally detector) turned on at a specific time after the laser pulse (gate offset) and off after a constant duration (gate duration). Each gate is separated from the next by a constant step (gate separation).

(Gate Duration and Gate Separation parameters are specified in the Settings>>Fluorescence Decay panel or the Fluorescence Decay panel of AlliGator).

Although each gate takes some time to acquire, and a series of images takes about N times this amount, we will refer to such a series of images as a "time point".

AlliGator allows the analysis of individual time points, or a series of such time points (i.e. a time series).

In the latter case, each time point is a folder identified by a name specifying its order in the time series. In other words, a time series with P time points will look something like this on disk:

or, more generally: 

time series/time point 1/image 1

time series/time point 1/image 2

...

time series/time point 1/image N

time series/time point 2/image 1

time series/time point 2/image 2

...

time series/time point 2/image N

...

time series/time point P/image 1

time series/time point P/image 2

...

time series/time point P/image N

time series is the name of the folder (Mouse in the Figure above) in which all time point subfolders are located (M1H00_nn in the Figure above). These subfolders should be named using a common root name followed by an increasing number suffix.

For instance, folders named TimePoint_001, TimePoint_002.tif, ..., TimePoint_100.tif constitute a valid series of names, but TimePoint1 , TimePoint2, ..., TimePoint10,... etc. is also an appropriate naming convention (*).

The naming convention for images in each folder should follow a similar convention (**): root name followed by a numeric suffix.The software will assume that the files, ordered numerically (using their suffix) are also ordered temporally, i.e. correspond to successive gates, starting at offset 0 and incremented by a constant step equal to the specified Gate Separation parameter (see the Fluorescence Decay page of the manual). The Gate Separation parameter is currently not used and can therefore be omitted  (see the Fluorescence Decay page of the manual).

For instance, files named Image000.tif, Image001.tif, ..., Image100.tif constitute a valid series of names, but other naming conventions can be used. For instance,  Image1.tif, Image2.tif, ..., Image10.tif,... etc., is also an appropriate naming convention.

An example of image folder is shown below:

Supported file formats are: BMP, TIFF, JPEG, JPEG2000, PNG. The files can be 8 or 16 bits gray scale images.

1. Loading a single time point

To load a single time point (consisting of N gates), use the File>>Load >>Dataset>>Open Gate Image Folder (Ctrl+L) menu item. The path to the dataset folder will be displayed in the title bar.

The image displayed on the left side will represent a single gate, or the sum of all gates, depending on the status of the Sum checkbox.

If the Sum checkbox is unchecked, moving the top Time Gate Slider will cycle through the different time gates. This slider has no effect if the Sum checkbox is checked.

The image is displayed with the palette selected by the user (right-click >> Palette). Palette choice is discussed further in the Image Palette section of the manual. Adjusting contrast and similar image manipulations are discussed in the Image Histogram page of the manual.

Note that it is possible to exclude gates from the analysis (see the Settings>>Fluorescence Decay>>Basic Analysis window and the discussion in the Fluorescence Decay page of the manual). Changing these options will require reloading the data set to take effect.

2. Loading a time series

In order to load a time series (or any succession of data sets to be analyzed as a series), use the File >>Load>>Dataset Series>>Open Gate Image Folder Series (Ctrl+Shift + O) menu item. To also load the time of acquisition of each time point (based on the series of associated .set files), check the Use File Timestamp chekbox in the Time Trace panel of either the Settings or AlliGator windows, before you load the time series.

After the folder containing the time series has been selected, the first data set in the series will be loaded and displayed as described in the previous section.

In addition, a vertical slider (Time Point Slider) will be displayed on the right-hand side of the image,allowing to explore the time series. The name of the data set currently displayed will be indicated in the Current Folder text box below the image.

Note that to avoid slowing down the software, there is no update of the image as you move the vertical slider around: only the name of the Current Folder is updated. As soon as the slider is released, the corresponding data set is loaded and represented.

Notes:

(*) The acquisition software seems to name folders in a time series in the following manner:

Folder

Folder_1

Folder_2

etc.

This unfortunately is not compatible with the algorithm used to figure out the common root name of all folders as well as their order. Fortunately, the fix is simple and consists in renaming the folder corresponding to time point 0 (Folder in the example above) as Folder_0.

(**) The LaVision camera recently (2017) decided to output gate files with names of the kind: root_nameXXXXX_Delay=YYYps.tif, where XXXXX is the gate number and YYY is the gate delay with respect to the laser trigger. Versions of AlliGator above 0.9.13 support this unconventional name format.

(***) The same parameters and formula are used for the Save Image Data as TIFF right-click menu function of the Source Image and Phasor Plot Image.