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The calibration of your microscope is an essential part of working with cells if you want to record their measurements; which is often. It’s important because accuracy is important if you want to effectively monitor and compare the sizes of cells, or examine any differences between cells.
If you are new to microscopy and are just learning to use a microscope, let this article take you through the calibration process and give you guidance on how to calibrate a microscope with an eyepiece graticule and stage micrometer, starting with the basics.
If through reading this article you are unsure about any terms that are used, be sure to use our microscope glossary of terms as a guide.
Microscope Calibration: Definition
Using a microscope that’s calibrated means that the same results will be produced on the exact same sample under the same conditions if you were to use an entirely different microscope that was also calibrated. The reason to calibrate is to get the most accurate measurement of your sample.
Even if both of the microscopes used on the same sample were exactly the same but were not calibrated, a variation in magnification factors can lead to a mixture of results, one of which may not be accurate and can lead to incorrect data being recorded and used later.
Companies such as laboratories or hospitals may hire specialists to do their calibration for them but it’s still wise for the supervisor to know how calibration works, so they can ensure the specialists are doing a good job.
As students and hobbyists, you’ll need to know how to calibrate your own microscope to ensure the information you obtain is correct.
Calibration is performed using an eyepiece graticule (or reticle) and a stage micrometer. The stage micrometer is a slide that is marked with a known measurement, usually 1000µm (micrometers) or 1mm (millimetre) with each major division on the scale representing 100µm.
The eyepiece graticule is also a marked scale with divisions which is embedded within the eyepiece so that the scale mostly covers your field of view.
The point of this exercise is to align the two scales in order to ascertain a measurement which can be applied to the divisions of the eyepiece graticule and therefore to the sample you are observing.
The Method of Calibrating a Microscope
As an example: we will call the major divisions on the eyepiece graticule “ocular units” (ou). If you are looking at a cell at 10x magnification it may measure 2ou but then when you switch to the 40x magnification it may measure 8ou.
How many µm does this equate to? Well, we have to assign a µm value to the divisions of the eyepiece graticule scale so that we can calculate the measurement of the cell at any given magnification.
With that being said, note that each objective lens you will be using will need to be calibrated individually.
Set up the eyepiece graticule and stage micrometer
To start with, load your eyepiece graticule into the eyepiece of your microscope and set the stage micrometer in place so that it is centered under the objective just like any other slide.
Align the eyepiece graticule and stage micrometer
You may want to begin with your 10x objective lens first.
Line up the major divisions of the eyepiece graticule and stage micrometer so that the first major bar on the left of the eyepiece graticule lines up with the first bar on the left of the stage micrometer denoted as 0µm.
Using your course and fine focus dials, try to line up as many of the major bars as closely as possible just as shown in the image above.
Calculate the value of the ocular units in micrometers
As we know in this example, the stage micrometre is 1000µm in length and since it is divided into 10, each major division is worth 100µm.
We now need to work out the entire length of the eyepiece graticule, in the image above, the eyepiece graticule reached 6.9 on the stage micrometer and so the whole length of the eyepiece graticule (10ou) is 690µm.
If we divide both sides by 10, we can deduce that 1ou is equal to 69µm.
Stage Micrometer = 1000µm
10ou = 690µm
10/10:690/10 = 1ou:69µm
Going back to our example in which the cell under a 10x objective measured 2 ocular units; this means that the cell measures (2 x 69µm) 138µm!
Repeat the process for your other objective lenses
Now that you have a value for an ocular unit under 10x magnification, the same calibration must be performed for your every objective lens you are going to use. Let’s do the 100x next.
The stage micrometer is still 1000µm but let us say that this time the entire length of the stage micrometer is surpassing your field of view, and the whole eyepiece graticule reaches just 0.7 units on the stage micrometer because of the higher magnification.
If we multiply 0.7 by 100µm we know that 10ou is 70µm and divided by 10 is 7, meaning that 1ou equates to 7µm in this case.
Stage Micrometer = 1000µm
10ou = 70µm
10/10:70/10 = 1ou:7µm
In this scenario you would be looking at a much smaller cell than the one in the example given. This new cell reaches 5 ocular units on the eyepiece graticule.
If the new cell measure 5 ocular units we need multiply 7µm by 5 which gives us the measurement of 35µm.
I hope you found this tutorial useful and a simplified explanation of calibration. It does help to know the logic behind calibrating a microscope before learning how to do it, I think it just makes more sense to us that way and is easier to get to grips with the concept.
If you are still having trouble, or have some questions relating to microscope calibration, please feel free to comment below and I will do my best to get back to you.