Hello everyone,
I havae taken pictures of Golgi-Cox stained brains in a 100x magnification and am now trying to measure dendritic segments. However, the measurement doesn't seem to make any sense:
The image information gives me a resolution of 300 pixels per inch.
That's 300 pixels per 25400 µm.
This again means 0.018 pixels/micron.
When I use these data via analyze -> set scale however, it gives me impossible lengths of over 70000 microns for a small dendritic segments - whicht makes sense with the resolution given by the image.
But how do I get the appropriate measurement now?
Thanks,
Alexandra
Dear Alexandra Abromeit-Sundermann
The PPI value of an image depends on the resolution of the camera,
E.g. if if you have two cameras with different resolutions and you capture an image of the same object at the same magnification with each camera, then you would have captured the same real dimensions but each image will have different PPIs.
If the object is 100 micrometers in length then doubling the PPI will double the number of pixels across the 100 micrometers of the object and the actual length reflected by each pixel will decrease by half.
To solve the issue, you need to know the yield or field of view (dimensions) of the microscope camera. To do this, take an image of a small object with known dimensions (E.g. microscope ruler) with the camera you used at the same magnification, then you can identify the real dimensions reflected by each pixel.
Once that is done, then you can set the scale in ImageJ or let the software calculate the lengths with pixels and then convert them to micrometers.
I hope this addresses your question.
Good Luck
Hello,
300 dpi is the image information for the printer, not the size of your field of view seen by the detector of the microscope.
If the detector of the microscope is 900x900 pixels for example, if you print the image at 300 dpi, the image will have a size of 3x3 inch on the paper.
Normally images from the microscope should be saved as metadata, meaning that in addition of the images, there are information as text containing all image acquisition conditions such as resolution of your field of view, depending on your magnification. Microscope magnification with detector has to be calibrated. For example 1 pixel of the detector corresponds to, let say, 1 micron at a given magnification. If your detector is 1000x1000 pixels, then your image correspond to a field of view of 1000x1000 micron in your sample.
By giving the information scale coming from the microscope in ImageJ, if your structure is 200 pixels, so it has a size of 200 micron.
Dear Nicolas Barois and Amjad Hasan Bazzari
thanks four your replies.
Unfortunately, I only have the tiff images and no longer access to the microscope. Is there anything I can do?
Thanks,
Alex
Dear Alexandra,
In this case, you may contact the persons who have access to the microscope and ask them if they know the field of view at the used magnification or if they can determine it as it should not take more than few minutes using a microscope ruler (stage micrometer) or perhaps send you a raw data file captured with the same objective.
Other possible options:
look for the field of view online if you know the type and specifications of used hardware (E.g. microscope, camera and objective) but it might vary.
If there is anything captured in the images that you know the exact dimensions of then you can use it as a scale.
Best Wishes
Hello,
Your tiff images may be tagged (containing text information) but not see by ImageJ.
Use Bio-Formats plugin to open an image > Bio-Formats importer > Bio-Formats import options/ Metadata viewing / select display metadata.
Or like mentioned above, contact the person in charge of the microscope to know the calibration.
here is a screen-print of an image from my transmission electron microscope.
The tagged tiff file contains acquisition information. Opening the image in ImageJ by drag and drop will not give me information about resolution.
But opening the image with Bio-Formats displaying metadata, I know that 1 nm correspond to 3.589 pixels.
Hey Nicolas Barois
thanks for the suggestion, but I fear it also doesnt work (see attached pic).
I have the microscope and camera now:
Zeiss AXIO Imager M2 (objective 100x)
Zeiss Axiocam 506 => pixel size according to manual: 4,54 μm x 4,54 μm
I also uploaded a picture in question in the original question post... maybe you can try with it?
The Neurolucida software shows a value of 0.04µm/pixel ... is that a reasonable value?
Thanks!
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