I saw at least 20 videos on Image J and trust me this one genuinely helped me to understand finally. This is a complete video to learn how to measure the diameter. Very good effort. Anyone can understand if listen carefully. Every details are there. Thank you so much for uploading this one. I highly recommend this video. Worth watching.
Thank you so much for this informative video. I would like to ask does calibration need to be done even if I added a scale bar via Zen Black? I have images with 20X, 40X, and 63X. If I click global, will it apply to every image? How could I solve that problem? In addition, does the calibration is necessary for the intensity measurement? Thank you so much.
If your image has a scale bar through Zen Black then you should be able to calculate it's size without going through calibration. Instead, you can input that information in ImageJ and hit global to apply to all the images. You have to remember to change the calibration for each set of magnifications though and reset global accordingly. I would start by organizing the images into folders for each magnification e.g. all the 20x images together, all the 40x images together etc. Next, you would need to know what the scale bar provided by Zen Black is equal to in each case. I believe you should be able to find that information on the image bar itself. You would then open one of these images in Image J. Follow my calibration instructions and in this case measure the scale provided by ZenBlack and measure it in pixels, then set it to the measurement in uM shown in the image by ZenBlack. You can set it to Global and analyze ALL images with the same magnification taken by the same program. You would then repeat these steps for each set of images at different magnifications. To just measure intensity you do not need any calibration. Hope this helps.
So helpful video ! Thank you very much BUT Here in example you showed, the scales where so close to each other.... What if the image has different scale than the one used for calibration ? Could I copy the scale of calibrated one and use them for the other?
You video was very informative. I have one doubt, when I am setting same scale(100 um) for different images , the number of pixels per um is varying a bit. Why that is happening?. If I set scale of 100 um to different images one would show 0.5978/um and other would be 0.5981/um. I am using the same technique to measure and same edges I am focusing. Could you explain the reason behind this?
This amount of variation is normal in this calibration method due to it's partially manual nature. May be the line you drew was slightly inside the edge or outside the edge in your different measurements leading to the variability. If you want to be more precise and create a standard for yourself, you can take average of the calibrations you got from three separate images and set it manually in the scale as global calibration anytime you are examining an image with that magnification.
You have to take the stage micrometer image with the stage micrometer slide using the same microscope and imaging system that you are using for your own experimental images. You get an image of the micrometer using each of the objective lenses so you can calibrate your image analysis software for each magnification. I'll post a video explaining that process next week.
I saw at least 20 videos on Image J and trust me this one genuinely helped me to understand finally. This is a complete video to learn how to measure the diameter. Very good effort. Anyone can understand if listen carefully. Every details are there. Thank you so much for uploading this one. I highly recommend this video. Worth watching.
You're very welcome! Glad to hear it was useful.
Thank you so much for this informative video. I would like to ask does calibration need to be done even if I added a scale bar via Zen Black? I have images with 20X, 40X, and 63X. If I click global, will it apply to every image? How could I solve that problem? In addition, does the calibration is necessary for the intensity measurement? Thank you so much.
If your image has a scale bar through Zen Black then you should be able to calculate it's size without going through calibration. Instead, you can input that information in ImageJ and hit global to apply to all the images. You have to remember to change the calibration for each set of magnifications though and reset global accordingly. I would start by organizing the images into folders for each magnification e.g. all the 20x images together, all the 40x images together etc. Next, you would need to know what the scale bar provided by Zen Black is equal to in each case. I believe you should be able to find that information on the image bar itself. You would then open one of these images in Image J. Follow my calibration instructions and in this case measure the scale provided by ZenBlack and measure it in pixels, then set it to the measurement in uM shown in the image by ZenBlack. You can set it to Global and analyze ALL images with the same magnification taken by the same program. You would then repeat these steps for each set of images at different magnifications. To just measure intensity you do not need any calibration. Hope this helps.
@@biologybasicsThank you so much for this detailed answer. It helped so much. No amount of thanks is enough.
So helpful video ! Thank you very much BUT Here in example you showed, the scales where so close to each other.... What if the image has different scale than the one used for calibration ? Could I copy the scale of calibrated one and use them for the other?
THANK U SM URE A LIFESAVER
Thank you. Glad it helped
Thank you so muchh!! This is very helpful
it's really helpful , thank you
You video was very informative. I have one doubt, when I am setting same scale(100 um) for different images , the number of pixels per um is varying a bit. Why that is happening?. If I set scale of 100 um to different images one would show 0.5978/um and other would be 0.5981/um. I am using the same technique to measure and same edges I am focusing. Could you explain the reason behind this?
This amount of variation is normal in this calibration method due to it's partially manual nature. May be the line you drew was slightly inside the edge or outside the edge in your different measurements leading to the variability. If you want to be more precise and create a standard for yourself, you can take average of the calibrations you got from three separate images and set it manually in the scale as global calibration anytime you are examining an image with that magnification.
Hi how did you get the stage micrometer image?
You have to take the stage micrometer image with the stage micrometer slide using the same microscope and imaging system that you are using for your own experimental images. You get an image of the micrometer using each of the objective lenses so you can calibrate your image analysis software for each magnification. I'll post a video explaining that process next week.
my 20x lens barely capture the two borders of 1 mm......
It doesn't have to capture the entire micrometer image. Whatever is visible is enough. As long as there are 2 tick marks visible
Thank you so much
You're most welcome