2DFRETratiometrics is an ImageJ macro for image analysis, that covers the processing of raw image data sets into ratiometric measurements, capable of illustrating relative differences in the protein activation states within a single cell. This tool was spcifically developed to analyse ratiometric FRET signal on images acquired in widefield inverted microscopes or laser point scanning confocal systems. This tool follows the main steps of the ratiometric FRET analysis pipeline, including multiple options of background subtraction, uneven illumination correction, object segmentation and the ratiometric measurements of FRET.
Mouse Schwann cells FRET result:
In this macro, the FRET ratiometric pipeline is followed by correcting image illumination and subtracting image background. Cells are segmented either by automatic threshold or by user selection, and finally, the ratio between fret and donor channels is made. Some user-friendly dialogs are available to perform multiple options during the workflow execution. Background subtraction can optionally be made either using a background image or by subtracting mean intensity background values. Segmentation step can be performed from the first channel or by using a third channel, in the case it is available. The input is divided into two modes: single file or batch files, allowing processing a single opened image or a full folder of images. The output result combines the 2D_FRET_ratiometrics RGB image with royal LUT to illustrate the FRET ratio; the 2D_FRET_ratiometrics results table for each image; the 2D_FRET_ratiometrics results table for all images in batch mode; the structure/cell mask and the ROI outline for quality control.
Note that while 2DFRETratiometrics is easy to use and semi-automatized, it only works efficiently with images with individual cells.
- File in .tif or .tiff formats, 2D with two or three channels
- Divided into two modes:
- single image already opened
- batch mode, to process a folder of images.
The macro can output 5 types of files:
- The 2D_FRET_ratiometrics RGB: an RGB image with royal LUT to illustrate the FRET ratio
- The 2D_FRET_ratiometrics output results for each image: .xls file with mean grey values of structure/cell FRET
- The 2D_FRET_ratiometrics output results for all images in batch mode: .xls file with mean grey values
- The structure/cell mask: The mask applied to the original image, for quality control.
- The Roi outline of the structure/cell.
- Run on ImageJ/Fiji v1.53
- Install plugin Polynomial Shadig Corrector, by OptiNav (https://www.optinav.info/Polynomial_Shading_Corrector.htm)
- Download the "2D_FRET_ratiometrics.ijm" macro file somewhere on your computer (You can put it in Fiji "macros" folder for example)
- Start Fiji.
- In Fiji, run the macro: Plugins > Macros > Run…, and then select the “2D_FRET_ratiometrics.ijm” file.
This macro should NOT be redistributed without the author's permission. Explicit acknowledgement to the ALM facility should be done in case of published articles (approved in C.E. 7/17/2017):
"The authors acknowledge the support of i3S Scientific Platform Advanced Light Microscopy, member of the national infrastructure PPBI-Portuguese Platform of BioImaging (supported by POCI-01-0145-FEDER-022122)."
Date: October/2020 Author: Mafalda Sousa, [email protected] Advanced Light Microscopy, I3S PPBI-Portuguese Platform of BioImaging