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Microscope scale calibration compares an image readout with a known specimen length. This documented practical uses an eyepiece graticule, stage micrometer and Köhler illumination. Focus the graticule; place the reference on the stage with the objective-appropriate cover glass. Focus both scales with the chosen microscope objective, then rotate and translate them into parallel alignment. Read widely separated coincidences using consistent line edges. Record the optical configuration and recalibrate for each objective.^[ref]

The stage micrometer is the reference; preserve the optical configuration when substituting the specimen. Line width, focus, edge selection and optical aberrations limit accuracy. Additional recommended checks are realignment and another known interval: agreement tests repeatability and scale consistency, not absolute reference accuracy.^[ref]

Here is an original toy calculation, not the source's calibration example or measured data. Suppose a reference span $L=100\mu \mathrm{m}$ coincides with $N=40$ eyepiece intervals. Then the calibration factor and the length corresponding to $n=18$ specimen intervals areThis works because both readouts use the same image-to-specimen scale: $\ell /L=n/N$. One must count intervals, not tick marks; forty intervals have forty-one bounding ticks. In an ideal linear scale model, if identifying the calibration endpoints introduces a total readout error of $\pm 1$ interval, the inferred factor ranges from $100/41$ to $100/39\mu \mathrm{m}$ per interval. Holding $n=18$ exact gives $43.90\le \ell /\mu \mathrm{m}\le 46.15$. This isolates one contribution to measurement uncertainty; it is not a complete uncertainty budget or a measured confidence interval.

For a recorded real image, image a stage micrometer at the same capture settings to calibrate length per detector pixel. Resizing changes length per pixel; update the scale bar accordingly. Eye-viewing angular magnification alone supplies no digital image scale.^[ref]

An optical microscopist and author of material on optical microscopes.^[ref]