Microscope scale bar length.

Three ways to get micrometres per pixel

Everything on this page comes down to one number: how many micrometres of the specimen one pixel covers. A scale bar is then just the bar length in micrometres divided by that number. The three routes differ in what you already know.

From the sensor and the objective

Divide the sensor’s edge by its pixel count to get the pixel pitch in micrometres, then divide by the total magnification. The total is the objective multiplied by whatever the adapter or relay lens adds. That last factor is the one people forget: Krebs measured it at about 2.12× for his setup, which turns a 10× objective into 21.2× recorded magnification. If you do not know the adapter factor, use one of the other two routes.

From a ruler across the frame

Photograph a stage micrometer with the same setup and read how many millimetres fill the frame width. Krebs’ formula is recorded magnification = sensor dimension ÷ millimetres recorded. In his article a 2× objective showed about 5.25 mm across a 22.2 mm sensor, so 22.2 ÷ 5.25 = 4.23× recorded, and dividing by the objective’s 2× gives his 2.11× relay factor. Micrometres per pixel here is simply the millimetres recorded times 1000, divided by the pixel count.

From a measured distance

Open the stage micrometer photo in an image editor, measure how many pixels a known distance spans, and divide. A typical stage micrometer, as Evident Scientific describes it, is a slide with a scale of 1 mm and 100 graduations, so each division is 10 µm. Measure across as many divisions as you can, because a pixel of error matters less over a long span.

Calibrate every objective

Evident’s guide says you need to calibrate your microscope camera for each objective individually, because the magnifications differ. A university lab protocol from Boston University does the same, creating a named calibration for each camera and objective and checking it by photographing a ruler. If you resize or crop the exported picture, say so in the exported width field, and the scale is corrected.

Worked example

Using Krebs’ published figures: Canon 350D, 22.2 mm edge, 3456 pixels, so the pixel pitch is 22.2 × 1000 ÷ 3456 = 6.42 µm. With a 10× objective and his 2.12× relay factor the recorded magnification is 21.2×, so one pixel covers 6.42 ÷ 21.2 = 0.303 µm and a 100 µm bar is 100 ÷ 0.303 = 330 pixels. The full picture is 3456 × 0.303 = 1047 µm wide, so a bar of 200 µm (about a fifth of the width) is 660 pixels. If you export the picture at half size, 1728 pixels wide, each pixel covers twice as much, 0.606 µm, and the 100 µm bar becomes 165 pixels. The magnifications are Krebs’ own measurements for his camera, not ones you should reuse for yours.