If you have been collecting data without doing this I would trash all of it as the procedure done to collect it hasn't controlled for anything.Īlso as for where to go to ask this stuff. Whatever image you are overlaying on you must also split the channels of that as well and use the channel that contains the wavelength you are measuring (ie: Alexaflour 488 would be in the GFP channel). You should use these settings:Īfter all of this then you apply the overlay. Right now this option is available if you are measuring the time trace data (using a button GetAverage) without updating the Stack Window. Now I assume you are measuring human cancer cells. an influence on the result: which is good if you plan to measure intensities in the projection image (although quantitative analysis of projections can be. The check box 'Recenter for measuring mean' is to tell the plugin whether to use the re-centering option when you measure the mean intensity trace. Also look for odd shaped cells or micro-nuclei, which should also be deleted. Any outlines containing more than one cell should be deleted. However it is not perfect so once ROI pops up and the cells have been outlined by the particle analysis you must go through and ensure it is measuring singular cells. This way you can measure their individual intensities. You also need to use the "Watershed" function after the threshold so it will outline individual cells allowing you to avoid outlining a group of cells globed together. tif formatīefore threshold you must split the channels, especially if you are looking for florescence from a certain wavelength. Save channels separately not as an RGB stack if you are using.If your microscope cannot save files as RAW format you must use.ImageJ automatically converts images to 8-bit.The are some things you need to be aware of when measuring intensity in imageJ: Right click in the Results window and click Summarize.Īre we correctly measuring mean intensity data using the above steps?. In ROI Manager: press “measure.” (a Results window with individual data points will pop up).Then, Image > Overlay > From ROI Manager. Analyze > Analyze particles > Show > Bare Outlines.Threshold the image (Image > Adjust > Threshold) to outline all the regions and click Apply.while it does seem correct, my question is -> are we actually measuring intensity correctly using the following steps or are we erroneously measuring something else and believing that that value is the intensity? we came up with the below steps to measure the intensity. We have to measure the intensity of the fluorescence in certain regions of images using imagej.
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