How to calibrate a microscope for measurement

Before you can accurately measure an object under your microscope, you first need to calibrate it.

To properly calibrate your microscope, you’ll need an eyepiece reticle and a stage micrometer.. Then you can learn how to use eyepiece micrometers and reticles.

Why do you need to calibrate your microscope? 

When you try to measure something, you need something to give you a sense of scale. You need a frame of reference.

In everyday life, you’d use a ruler, or a tape measure, or an object of known size.

Unfortunately, microscopes don’t have any convenient measuring tools you can line up next to a tiny cell or bacterium. 

Even if you know the approximate size of what you’re looking at, each microscope is a little different magnifications can be slightly off, and optical pathways can differ.

If you want accurate measurements, you need to give your microscope a frame of reference ahead of time by calibrating it.

Eyepiece reticles and stage micrometers

The two handy tools you need to calibrate your microscope are the eyepiece reticule and a stage micrometer.

An eyepiece reticle is a transparent disc with an unmarked measurement scale that slots into your microscope eyepiece. It acts a bit like a crosshair – overlaying a scale onto your image.

The reticle scale is unmarked because you’ll be using it at different magnifications, so it can’t have fixed values.

A stage micrometer is simply a glass slide with a measurement scale on it. It’s essentially a transparent ruler that you put under your microscope.

However, unlike the reticle, the micrometer has known dimensions. The marked scale is inscribed with precisely measured intervals so you can use it as a frame of reference for calibration.

The interval width will depend on your specific micrometer, so be sure to check the specifications.

Step-by-step guide to calibration

Calibrating your microscope is straightforward, but it does involve a little maths.

 Fortunately, you only need to do it once for each objective.

1. Install your eyepiece reticle. This procedure will depend on your microscope, but usually it involves popping out one eyepiece, unscrewing the bottom and inserting the reticle.

2. Place your stage micrometer in position under your microscope, and swap to the lowest magnification objective.

3. Look down the eyepiece at the stage micrometer. Align the 2 scales. Some people prefer to have them side by side, others prefer to overlap them. The important thing is to line up the ‘0’ values. 

4. Find a point where the big interval lines on each scale are aligned, just like at the ‘0’ point.

5. Take note of the number of intervals on the stage micrometer scale at that point and multiply them by their interval width (usually written on the micrometer). This gives you the total length of that span. For example, the number of stage intervals (20) x interval width (0.01mm) = total length (0.2mm).

6. Take that total length and divide it by the number of intervals on the eyepiece scale at that point. This will give you the real length of a single interval of the eyepiece reticle scale at this magnification. For example, length (0.2mm) divided by the number of reticle scale intervals (15) equals the reticle interval length (0.013mm). Write that number down – you’ll be using it to perform your future measurements using the eyepiece reticle.

7. Repeat this process for each different objective lens.

8. Switch out the micrometer for your sample slides and use your freshly calibrated eyepiece reticle to measure your specimens.

You won’t need to recalibrate unless you switch microscopes or swap any of your microscope components.  Learn more about Microscope eyepiece reticles and micrometers