I made some cell nuclei images in a confocal microscope. The analyst sent me the pictures, but they did not come with any scale. I know that to do the set scale in ImageJ we need to mesure a know distance in the picture (as the scale that was introduced during analysis), but as I do not have it, how can I do to set the scale by ImageJ, to be able to make nuclei measurements ? Also, when I open the imagej in the upper left corner some values appear in microns (212x212) followed by another value (1024x1024) without measuring unit.
Setting the scale on ImageJ
https://www.youtube.com/watch?v=8_yuEemH7Qs
ImageJ Tutorial: How to add scale bar
https://www.youtube.com/watch?v=rYt4CVv4zFo
Adding scale bars to images using ImageJ
http://www.swarthmore.edu/NatSci/nkaplin1/scalebar.htm
Dear Lisiane,
If your images are already in TIFF format, I wouldn't think that there is a real way to set a scale. However, if the analyst still has the main file coming from the confocal (which he should), the settings should be in the meta-data, usually, the scale can be added using the offline software that was used to do your imaging. For ZEISS and NIKON, I konw that you can get them for free on the net.
Concerning the numbers you see, they are probably related to the resolution of your image.
Unless the microscope has a variable zoom on the camera, each objective will have a microns per pixel value or an pixels per micron value that you should be able to ask the staff in the confocal facility to give you. Without having a calibration number, you cannot calibrate the image. You may also be able to get the numbers you need from the microscope makers web site or sales rep. It's unlikely that either of those numbers are in real microns if you can see the nuclei well enough to make accurate measurements, although a 100X objective typically has a 250 micro field of view and the image from the camera should be smaller than that. So it's possible that the 212x212 is the number of microns accross the image and 1024x1024 is the image array size which would make your calibration factor 0.207 microns per pixel.
https://imagej.net/Annotating_Images
https://www.swarthmore.edu/NatSci/nkaplin1/scalebar.htm
Hi Lisiane. I just had a similar problem to solve. Basically, I had jp2 macroscopy images taken with a Nikon camera, but in our lab the old camera and the software was replaced. When reopening the jp2 files with the new software all calibration data was lost. I solved it by installing NIS-Elements Viewer with which I could see all the details of my images, including um/pixel data. It is good to know, that even compressed jp2 files can contain quite some information.
You can follow any of these:
1. https://www.youtube.com/watch?v=tq9aH8fWY6c
2. https://www.youtube.com/watch?v=6dGAFawBDMU
3. https://www.youtube.com/watch?v=3bynnPQQxmg
I am having a same problem, I have clicked few images using a camera adapter (imager) attached to a stereozoom microscope . the images are in .jpg format. But it lacks in measurement scale. Is their any option to calibrate the software with the microscope magnification, such that every time we click a picture in desired magnification, we can add appropriate scale bar ?
by default those measurements are in pixel.The analyst who captured have to calibrate the microscope.Then only you will get the image with scale bar
Dear Thibault Bouderlique I ave used Nikon microscope wit a camera of the same Nikon. I also ave files in ND2 formate and Tiff. but do not ave scale can I add scale in ND2 file if yes will you recomend me how. Your elp will be appreciated.
Dear researcher,
I have the same problem. However, the photos were taken using a mobile phone and the pixels for all of the images are 1080*809. What should I do to set scale my ImageJ and size the particles?
Thank you
Regards
Hi Foroogh Khodadadi ,
You need an object of known size to be able to set the scale in ImageJ. This could be a ruler or any other object in the image you know the exact size of. Note that if the camera was not fixed (e.g. on a camera stand, the lens looking perpendicular at the scene you want to capture), then the distance and angle of the pictures will vary and it will be difficult to precisely determine the size of your particles (unless you set the scale for each individual image).
Once you have an object of known size, follow these steps to set the scale in ImageJ: http://microscopy.berkeley.edu/courses/dib/sections/04IPIII/IJsetscale.html
All the best,
Eszter
You wont need a calibration image if you find the resolution in the image info. go to image > show info. scroll to the end and you will find the resolution saying how many pixels per microns or inch. use this info to set the scale under set scale.
If you have a stage micrometer then take an image of the stage micrometer under the same magnification. Since you know how much the distance between two lines in the stage micrometer, use this distance for calibrating. Open image J > open stage micrometer (image) > Using the Line Tool draw a line between the two lines of the image of stage micrometer> Analyse >Set Scale > It will open a box in which distance in Pixel will be automatically filled, now fill the distance (known) between two lines, add units and OK. Then open any image, the line tool will give you the actual length in microns or another unit that you have used.
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