Source Image

The Source Image is located on the left hand side of AlliGator's main window.

Some of the associated tools are highlighted in the snapshot shown above. Their description can be found in the following sections.

The Source Image displays different data depending on the position of the Time Gate Slider, on the position of the Time Point Slider (if it is shown) and the Displayed Image selector.

1. Time Gate Slider

Note: The Time Gate Slider is not shown when Sum of All Gates is selected in the Displayed Image pull-down list.

Located above the image area, the Time Gate Slider allows browsing the individual images comprising the time gated dataset. The numeric indicator to the right shows the number of gates in the dataset. Moving the slider to the left will show early gates, while late gates can be seen by moving the slider to the right.

When moving the slider, the displayed image's contrast might be inappropriate (overly dark or bright image). In this case, switch to the Image Histogram panel and make sure of the following:

- The Use Image Histogram for Contrast checkbox is checked.

- The Min and Max cursors are visible and encompass most of the histogram bins, as shown below:

Alternatively, uncheck the Use Image Histogram for Contrast checkbox. This will automatically adjust the contrast of each gate.

2. Time Point Slider

Note: The Time Point Slider is only visible when a Dataset Series is opened.

Located to the right of the image area, this slider allows navigating through the different time points in a time series. Move the slider to the bottom to access the early time points, or to the top to access the late time points. The name of the current dataset folder is indicated in the text box at the bottom of the panel (in the example shown above, the folder name is M1H00_19). If a corresponding .set file is present, the time stamp information for this dataset will be displayed next to its name (915 s in the example shown above). 

While the slider is moved, the image is not updated. Image update only occurs when the slider is released, in order to reduce CPU load.

The selected time point is indicated in the box in the lower right (19 in the example above).

3. Displayed Image Selector

The Displayed Image selector allows switching from a mode where individual gates are shown (Single Gate) or only their sum (Sum of All Gates). The latter mode is the default mode. If a White Light Image or a ROI Mask Image have been loaded, they can also be displayed using the selector.

4. Image Color Ramp

The color ramp located to the right of the source image indicates the color scale as well as the Min and Max values set in the Image Histogram.

The Min value (shown at the bottom of the scale) and Max value (shown at the top of the scale) define the range of pixel values which are mapped to the color scale. Pixels with value outside this range will be displayed with the Low or High colors shown at the bottom and top of the color scale, respectively.

The color scale can be changed using the Image Source Palette context menu. For more information on how to use and change color palettes, see Context Menu section below and the Image Color Palette page of this manual.

5. Refresh & Phasor Highlight Tools

The cluster of three objects on the top-left corner of the image are used to:

6. Overlay Mode

The Overlay Mode selector allows choosing between 3 different modes:

ROI, Phasor Ratio Color Map and Phasor Color Map, described next:

• • ROI: ROIs defined in the Phasor Plot are highlighted in the Source Image.

  • Phasor Ratio Map: pixels whose phasor is located in the vicinity of the segment connecting the two phasor references defined in the Phasor Plot are highlighted in the Source Image with a color reflecting their phasor ratio. See the Phasor Ratio Map page of the manual for details.

• Phasor Color Map: pixels are colored according to their location in the Phasor Plot with respect to multiple vertices defined in the Phasor Map Color Picker window. See the Phasor Color Map page of the manual for details.

7. Use ROI as Search Area

The button with a red cross in it (Use ROI as Search Area) is used to toggle a special ROI definition mode, described next.

When selected (green background), the ROI defined by the user is not the one used to compute the Fluorescence Decay and Phasor Data point of the Phasor Graph, but instead is used as a Search Area to define this ROI. The final ROI is a disk centered on the brightest pixel (in the sum image) within that search area, and ROI Diameter defined in the Source Image panel of the Settings window. If there are more than one pixel with the same maximum brightness, the first found from the top-left corner of the image is used.

Note that each time the Search Area ROI is moved, the button toggles back to its off position.

8. Image Tool Palette

The cluster of tools shown on the lower left side of the image are used to zoom, grab or move the image, or to draw regions of interest (ROIs).

Special combinations:

• • Pressing and holding down the Alt key while drawing a rectangle ROI will zoom on the circumscribed area (and keep a 1:1 aspect ratio)

  • Holding the Shift key while resizing or dragging a selected ROI will modify the definition of that ROI.

9. Save Image

The Save Image checkbox at the bottom right corner of the Source Image is used to automatically save the displayed image (including overlay) each time the overlay is updated. The file is saved in the folder containing the current dataset folder, and the file name used is identical to that of the dataset folder, with an extension corresponding to the Saved image File Format option selected in the Miscellaneous panel of the Settings window.

If the displayed image is the sum of all gates, the file name with be prefixed by "Sum of ". If a file with an identical name already exists, it will be overwritten.

This option is convenient for time series analysis, since it allows saving all source images in a time series (with their overlay and the palette chosen for display) in the same folder. The same function can be performed using the Save Displayed Image with Overlay context menu item (see below).

10. Context Menu

The Source Image context menu is shown below:

• Copy Data: copies the whole Source Image object bitmap image to the Clipboard for reuse in other software (or in the Notebook).

• Copy Image Only (with Overlay): copies the visible part of the image (with overlay) to the Clipboard for reuse in other software (or in the Notebook).

• Save Image Data as TIFF saves the data displayed in the Source Image as a 8- or 16-bit image, following the selected options offered to the user at the beginning of the process. The resulting file can be used for external analysis. Note that if the Sum of All Gates is shown, that sum is saved, while if a Single Gate is shown, that single gate is saved. To save a complete dataset, check the File>>Save>>Dataset submenu.

• Save Image with Overlay saves the same image as copied by Copy Image Only (with Overlay)

• Save Overlay saves the current image overlay as an 8-bit image in which any non-black pixel is assigned the value 255, resulting in an effective mask image.

• Palette: this submenu is described in detail in the Image Color Palette manual page.

• ROIs: this submenu is described in the Using ROIs manual page.

• Zoom to Fit: stretches the image so that it fits in the 400 x 400 pixel image display are. If the source image is not square, this will result in artificial deformations.

• Zoom to Fit (Keep Aspect Ratio): as above, but ensuring that the longest dimension of the source image is stretched to 400 pixels.