The tool measures in FLIM-FRET images, for each cell, the total volume of the cell and the volume occupied by values in a given range. It displays the positions of the values in the range in a result image.

The tool uses the 3D Objects Counter plugin [1].

Getting started

To install the tool save the file FLIM-FRET_Volume_Tool.ijm into the folder macros/toolsets of your FIJI installation. You must have the 3D Objects Counter (already included in FIJI) installed.

Select the "FLIM-FRET_Volume_Tool" toolset from the >> button of the ImageJ launcher.

• the first button opens the help page
• the m-button measures the volumes in the current image
• the b-button runs the measurements in batch-mode on the images in a folder

Method

The tool determines a background value by looking for maxima around minima in the image. The image is thresholded with the found background value. A binary watershed is applied and the resulting mask is eroded. Then the 3D Objects Counter is used to create an indexed mask of the cells. The indexed mask is used to measure each cell individually.

Options

Right-click on the m-button to open the options-diaog.

• lower threshold - the lower threshold of the range of values that will be measured
• upper threshold - the upper threshold of the range of values that will be measured
• create control image - if selected an image, showing the positions of the values in the range, is created

Literature

[1] S. Bolte & F. P. Cordelières, A guided tour into subcellular colocalization analysis in light microscopy, Journal of Microscopy, Volume 224, Issue 3: 213-232