I searched about how to measure thickness tunica media of rat artery in http://www.bmj.sk/2011/11206-03.pdf, I found that many journals use an ocular micrometer. I am asking if it is possible to measure from a photo we take from microscope and transfer it to Image J and analyze measure thickness?
Take a photo of a ruler on the microscope. Open it in ImageJ. Use the line tool and draw a line over the ruler (the line should be straight and you should know the distance or length of that line).
Go to analyze/set scale...put the length of the line you draw in the space "known distance" and set the length unit.
Note: You should take the photo of the ruler with the same parameters that you use with your tissue (objective, etc)
Take a photo of a ruler on the microscope. Open it in ImageJ. Use the line tool and draw a line over the ruler (the line should be straight and you should know the distance or length of that line).
Go to analyze/set scale...put the length of the line you draw in the space "known distance" and set the length unit.
Note: You should take the photo of the ruler with the same parameters that you use with your tissue (objective, etc)
To complete the post above, if you have any object with known dimensions -- a bead, ruler, micrometer slide, etc. Photograph it with the settings you use for your unknowns -- binning, objective, etc. Then use the Line tool to draw the known distance and Analyze>Set Scale... to establish the pixel-distance relationship and check Global to use this scale in your other images. Next, in your unknown/sample images, taken with the same mag, and use the line tool to draw a segment you want to measure and hit Analyze>Measure. Check Analyze>Set Measurements... if you want to adjust what is measured.
Finally, you can add a scale bar using Analyze>Tools>Scale Bar...
Fiji has plugins to do this in an automated way as outlined here: http://occm.otago.ac.nz/resources/Quantifying_Fibre_Thickness.pdf
The steps involved can be summarized as:
* Calibrate the pixel size
* Segment the image (e.g. by threshold)
* Generate thickness map using BoneJ plugin.
That is exactly the same problem as measuring the intima media thickness of the carotid artery (see our publications). You need to detect the boundaries of the tunica by edge detection. Then you need to discretize the contour and finaly use snakes or actoive contours to deform the contour in order to fit to the actual borders of the tunica. There are differnt snake or active contour implementations.
You can use also Image-Pro Plus/Premier software's. Are commercial but are very easy to use and you can record easily a macro's to do what every you want. For the spatial calibration you need only of a scale bar in the image.
The problem could be that the thickness is not uniform and it is different at different measuring spots. So how to accurately measure something that is not uniform in thickness? Same is true for glomerular basement membrane.
So most of you were talking about spacial calibration in ImageJ. What after?
When the thickness is not uniform, then what is usually done is to measure the thickness in many different equidistant locations (take several measurements). Then estimate the mean of the thickness by averaging all different measurements. You may also find a median, maximum and minimum thickness.
I agree... Just make sure to Take several measurements to do a strong statistical probe...
You should also be able to look up the pixel size for your objective and microscope combination. You can then enter the pixel size value in imageJ and the measure tool will use this value to calculate the thickness of the tissue. This is a more accurate option than setting the distance with an image of a ruler. When you calibrate using an image of a ruler at high mag where do you place the beginning and end of line- at the start of the hash mark, middle, or end? These differences will create large variations at the scale of a micrometer. The relative differences between measurements will be valid this way, but your measurements won't be very accurate.
how to calibrate micrometer in Image J when we don't have micrometer tool?
You need to know the spatial image resolution. When images are acquired they do have a resolution which is measures in pixels/mm. Then you can measure any distance in pixels. Now if you want to calibrate then you need to select some objects that you know there exact dimensions. Then digitize them and measure them in pixels. You can then find the size of the images and estimate their resolution (pixels/mm).
Maybe you may have a look at some of our publications in research gate where we segment the intima-media complex of the carotid artery and measure distances.
You don't need a micrometer tool, per se, though it's obviously ideal.
You could use anything with a known size, for example some latex microspheres or a fiber with a known diameter. Your calibration may be less accurate, but it's better then nothing.
You would need to obtain this information for your objective/camera combination from the manufacturer/support company, assuming you bought a turn-key system.
It's not something you can calculate without a calibration tool.
I use my toolset with cycle to calculate distance between lumen and external perimeter to calculate thickness of blood vessels. You can try to use the same algorithm.
I attached video of my algorithm in the supplementary resources of this publication
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