Hello dears,
I am new in the field of FRET imaging. I am doing FRET imaging by Multiphoton microscope system Leica TCS SP5 MP OPO.
I use YC3.60 OTI mice. In this mouse CD8+ T cells express CFP and some YFP but after calcium signaling CFP decreases and YFP increases in CD8 T cells.
This change in the ratio of YFP to CFP shows Ca+2 signing.
By Metamorph software, we can calculate this image ratio and show it in another color such as red.
So cells with the high ratio of YFP/CFP become red.
Unfortunately, our Leica Metamorph is too old (Version 1.4) and I could not use it for this purpose.
I have access to IMARIS software and ImageJ.
I am wondering that IMARIS maybe have some option for this Image ratio calculation.
If I should use ImageJ, How could I export data from Leica microscope to ImageJ to be usable by ImageJ?
If there is any way to do it by IMARIS or ImageJ please show me how to do it.
Thank you very much
Masoud
In ImageJ, you need the BioFormats Importer to read lif files. The best way to get this plugin is to download FIJI, which has Bioformats and many other useful plugins preinstalled: http://fiji.sc/Fiji
If you have a PC, you can open your lif file using Bioformats by simply dragging and dropping the lif file to the ImageJ title bar. If you have a mac, you will need to go to Plugins->Bio-Formats->Bio-Formats Importer to open the file.
To measure the ratio, the easiest way would be to use Image Calculator (Process->Image Calculator). Then choose the YFP image as image1 and the CFP as image2 and choose divide as the operation. Also, be sure to check the box that says "32-bit (float) result" as this allows for decimals. Otherwise each pixel's value will be rounded to the nearest integer. Finally, choose the LUT you want, by going to Image->Look-Up tables. Phase or Physics will probably be the more helpful LUTs since they are colorimetric heat-maps.
I would also remove the background fluorescence for each channel before measuring the ratio.
By doing FRET in a way were you compare the intensities you should make sure to identify the crosstalk in your channels to get more reliable and exact data. Anyway I do support the awnsers of Smith and Campa.
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