Calibration
Microscope pixel size calculator (µm per pixel)
Find how many micrometres one pixel covers in your image. Three ways: from the camera and objective, from a measured distance such as a stage micrometer, or for a level of a whole-slide image pyramid. That number is what makes a scale bar, a length or an area measurement correct.
How pixel size at the sample is calculated
pixel size (µm/px) = camera pixel (µm) × binning ÷ (objective × camera adapter)
The camera's own pixel (for example 6.5 µm on many sCMOS cameras, 3.45 µm on common CMOS sensors) is projected onto the specimen through the objective and any camera adapter. Binning 2 × 2 merges four pixels into one, doubling the pixel size. The microscope's eyepiece plays no part.
Worked example: 6.5 µm pixels, 60× objective, 1× adapter, no binning: 6.5 ÷ 60 = 0.108 µm per pixel, or 9.2 pixels per µm. Compare that with the Nyquist pixel size for your objective to see whether the camera samples the optics properly.
Calibrating with a stage micrometer
The formula assumes the nominal magnifications are exact; they are usually within a few percent. For measurements you will publish, calibrate each objective once: image a stage micrometer (a slide with a ruled scale, often 1 mm in 100 divisions of 10 µm), draw a line along as many divisions as fit, and divide the real length by the line's length in pixels.
Worked example: 100 µm of the micrometer spans 400 pixels: 100 ÷ 400 = 0.25 µm per pixel. In Fiji, Analyze › Set Scale does the same; in SlideScope, View › Set Pixel Size… stores it for that image when the file does not carry one.
Microns per pixel (MPP) in whole-slide images
Slide scanners record the base-level pixel size in the file (Aperio SVS writes "MPP", other formats an equivalent tag). By convention a 40× scan is about 0.25 µm per pixel and a 20× scan about 0.5 µm per pixel; the exact value depends on the scanner (the OpenSlide CMU-1 test slide, an Aperio scan at 20×, records 0.499 µm/px). Each pyramid level multiplies the MPP by its downsample, so level 2 of a 40× slide with downsample 16 is 4 µm per pixel, equivalent to about 2.5×.
Read it in Python with openslide.OpenSlide(path).properties["openslide.mpp-x"] (see reading SVS files in Python). Desktop viewers, including SlideScope, read it automatically and draw the scale bar from it.
Questions
How do you calculate microns per pixel?
Divide the camera pixel size by the total magnification between specimen and camera (objective × camera adapter), and multiply by the binning. Or image a stage micrometer and divide a known length by its length in pixels.
What is MPP in pathology?
Microns per pixel: the width of one pixel at the specimen in a whole-slide image. About 0.25 µm/px is called 40× and 0.5 µm/px 20×, by convention.
Why does my image show the wrong scale bar?
The file is missing its pixel size or carries a wrong one (some TIFF exports store 1 pixel per inch). Set the pixel size by calibration; in Fiji use Analyze › Set Scale, in SlideScope View › Set Pixel Size….
Does the eyepiece change the pixel size?
No. The camera sees the image formed by the objective (and adapter), not through the eyepiece.
Is this calculator free?
Yes. It runs in your browser and sends nothing anywhere. SlideScope, the desktop microscopy app made by the same team, is a paid app with a free preview.
Sources
Results are theoretical or arithmetic; check them against your instrument's documentation before relying on them for clinical or regulated work.
Measure on your real images
SlideScope opens 34 microscope and slide-scanner formats, CZI, ND2, SVS and LIF among them, with their own calibration, so lengths, areas, cell counts and scale bars come out in µm without setting anything up. Open a few of your own images with the free preview. Already decided? Subscribe now.