This is a really good and practical question — one many researchers run into when analyzing histology images.

Here’s the situation:

1. Using magnification alone

If the only information you have is “40×” or “20×” magnification, that by itself is not reliable for calibration.

The actual pixel-to-micron ratio depends on the camera sensor, binning, image resolution, and any intermediate optics. Two labs using “40×” objectives can produce images with very different pixel sizes.

So: magnification information is not sufficient to set a measurement scale in ImageJ.

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2. Alternative approaches

If you lack scale bar and metadata, you can still try some workarounds:

Tissue landmarks: If the histology section includes structures of known size (e.g., red blood cells ≈ 6–8 µm in diameter, nuclei of adipocytes or other cells), you can use these as internal “biological rulers.” This gives an approximate calibration but not as accurate as a true scale bar.

Request metadata: If possible, contact whoever acquired the images (core facility, collaborator, technician). Microscope software usually records pixel size in the image file header — it may just need the right viewer to extract.

Reacquire a reference image: If the microscope setup is still available, take a picture of a stage micrometer (calibration slide with 10 µm divisions) at the same magnification and camera settings. Use this in ImageJ to set your pixel-to-micron ratio, then apply it to the experimental images.

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3. Best practice in ImageJ

Open your calibration/reference image (stage micrometer or internal landmark).

Use Analyze → Set Scale in ImageJ to define pixels = microns.

Save this calibration and apply it consistently to your adipose images.

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✅ Summary:

You cannot reliably measure adipocyte size using only the nominal magnification. For accurate measurements in ImageJ, you need either (a) a scale bar or stage micrometer image acquired under the same conditions, (b) access to the original image metadata with pixel size, or (c) as a rough approximation, use known biological structures (e.g., RBCs) for internal calibration.

Thank you so much for your information 🙏🙏🙏🙏🙏