I took pictures from the same tissue stained with HE and IF (two slides, same piece of tissue, different stainings) using different microscopes (both Nikon) with the same objetive (10x). However, when I set the scale bar it comes in different sizes fot each image. Is there any way of correcting this?
Before you can make precise measurements with a measuring eyepiece, and/or camera, you must first calibrate with an object micrometer, each microscope separately. This calibration gives the micrometer value for the measuring eyepiece, and/or camera. The micrometer value indicates how large the distance between two graduations of the micrometer in the specimen is. An object micrometer consists of a conventional slide on which a mostly 1 mm long, consisting of 100 graduations scale is applied. The distance between 2 divisions is therefore exactly 10μm. The object micrometer must first be placed on the stage exactly like a specimen for the calibration process. The calibration then takes place after the focusing of the object micrometer through the eyepiece.
Due to the different magnification of the objectives, the calibration of the eyepiece micrometer must of course be done for each objectives!
Please compare the image attached showing the magnification of 6 different objectives and the resulting with of image in detail.
Best regards,
Guenter Grundmann
Before you can make precise measurements with a measuring eyepiece, and/or camera, you must first calibrate with an object micrometer, each microscope separately. This calibration gives the micrometer value for the measuring eyepiece, and/or camera. The micrometer value indicates how large the distance between two graduations of the micrometer in the specimen is. An object micrometer consists of a conventional slide on which a mostly 1 mm long, consisting of 100 graduations scale is applied. The distance between 2 divisions is therefore exactly 10μm. The object micrometer must first be placed on the stage exactly like a specimen for the calibration process. The calibration then takes place after the focusing of the object micrometer through the eyepiece.
Due to the different magnification of the objectives, the calibration of the eyepiece micrometer must of course be done for each objectives!
Please compare the image attached showing the magnification of 6 different objectives and the resulting with of image in detail.
Best regards,
Guenter Grundmann
Guenter Grundmann explained it as required to perform before capturing the images.
Now, as you have already took images and can't take them again. Then, I think you can do only one thing.
Your both Nikon cameras are having different magnifications (or optical zoom), that's why images are not matching.
As any image captured on microscope is a combination of objective magnification and camera magnification.
You already have objective magnification, you have to just multiply it with each camera magnification. This will be the only way to get the results correct.
Depends on what kind of microscopes you are using.
If you have one which doesn't have an automatic stage you're going to need to get a micrometer and sit there calibrating the objectives by hand, its easy but time consuming. Take an image of something where you know it's size like a micrometer, fluorescent bead or whatever. Measure the distance in pixels with a straight line in imageJ, actual distance/number of pixels = pixel width in um, nm whatever, that is your scale.
If one of them was say a C2 or an A1 of some sort with an automated stage go to objective configurations -> calibrate objective -> manual calibration and follow the instructions measuring the distance of some structure or ruler of a known size.
Once you know the scale it's easy to apply it to all your other images. The macro for command is:
run("Set Scale...", "distance=[distance in pixels] known=[known distance] unit=[units your known distance is in]");
so run("Set Scale...", "distance=1 known=10 unit=micron"); would set the scale such that every pixel is 10 microns.
To apply this to all the images you've already taken open imageJ and go Process -> batch -> Macro. Paste you're "run(Set Scale..." command into the textbox, locate the folder that contains the images with the incorrect scale for your input, pick an appropriate folder for output and click run. ImageJ will go through the input directory open all the images, apply the correct scale to them and save them in the output directory. Now when you open
Via using adequate software, which inserts correct bars of a specific size!
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