Nov 17, 2023
Analysis of immofluorescence images in ImageJ
- 1UCL
Protocol Citation: rachel.bates Bates 2023. Analysis of immofluorescence images in ImageJ. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9py4zg3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 30, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 85694
Keywords: ASAPCRN
Funders Acknowledgements:
ASAP
Abstract
Analysis at a single cell level of images taken on confocal
Import image into ImageJ as a TIFF file.
Set scale for Image by going to Analyze --> set scale. Set distance in pixels and unit of length to appropriate values for objective images were collected with.
Convert image to 8 bit going to Image --> type --> 8 bit.
Open up ROI manager using Analyser --> tools --> ROI manager.
Use freehand selection tool to carefully draw around the outline of each cell. For each cell outline add to ROI manager by selecting ADD.
Set up measurement parameters in Analyse --> set measurements --> select AREA, MIN & MAC GREY AREA, INTEGRATED DENSITY, MEAN GREY VALUE AND LIMIT TO THRESHOLD.
If a multichannel image convert to composite.
Go to IMAGE --> ADJUST --> THRESHOLD. From drop down menu select intermodes.
Use a consistent threshold for all images,set to threshold and in ROI manager select MEASURE.
Calculate a background fluorescence of each image aquired by drawing 3 ROI not including a cell. Take a measurement of background using the same threshold settings.
Calculate corrected total cell fluorescence using = integrated density measurement - (cell area * mean average background fluorescence).