I am taking photos of cells in culture using a FLoid™ Cell Imaging Station. I would like to add a scale bar on ImageJ. I know I need to go to Analyse->set scale, and input the information regarding pixel size in microns. I know the image resolution is 1296 x 964 pixels. But I am unsure of how many microns are per pixel. What am I missing?
you should ask your imaging facility it depend upon the resolution of the camera and the magnification you use. i pick un an example on google
Most such turn-key imaging systems have embedded scale info: the number of pixels per micron for each objective. Try using the software that came with the system and look for a Scale bar feature that you can place. Then export the image and open it in ImageJ. Use the IJ line tool to place a line over the scale bar then use Analyze>Set scale... to set the scale. The number of pixels should be prepopulated and you can enter the micron length of the bar into the known distance.
Alternately, use a micrometer slide or a hemocytometer, anything really with known line spacing. Image with each objective and then generate a table of pixel/micron values.
You should use "Scale Bar" tool under Analyze->Tools tab. If you change the numbers in "Set Scale" it would artificially change the micron->pixel scale of the original image. Most of the imaging softwares save metadata with image files, meaning from Image->Properties tab you should see how many microns one pixel correspond to directly, without any need to enter that info into "Set Scale".
If the info is there (which probably is):
To add a scale bar, simply go to Analyze->Tools->Scale Bar and specify the width you want in microns (50, 100 etc) and height in pixels. Important Note: If you do this for a single channel your Image Type is probably using pseudocolor LUT, and set to 8, 16 or 32 bit. You should make it "RGB Color" from Image->Type before adding your scale bar in white. Otherwise white scale bar will be in the color of your channel too.
@Muhammed Fethullah Simsek, I usually do what you mention when the info is there, however the info isn't in the metadata in this case.
Thank you Peter Sobolewski. I saved an image with a scale bar embedded by the software, measured it on ImageJ and added that data on "set scale bar" as you suggested. I am now able to add the scale bar on ImageJ.
The microscope software only burns the scale if the image is saved as png and not when it is saved as tif, so being able to add a scale on a tif image is great. Thanks! :)
Glad it worked.
I would recommend taking an image of anything with each of the objectives and at each bin setting using your system. Then embed the Scale bar using the FLiod software as you did and measure the length in ImageJ. Then just make a table of the conversion factor between pixels and microns for each objective and binning for future reference.
Thanks Peter Sobolewski. I wrote a macro now that adds the scale bar for my measurements. So now I can select input/output directory, and select which magnification I used and voilà it adds the scale bar :)
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