Calibrating a Microscope

To properly calibrate your reticle with a stage micrometer, align the zero line (beginning) of the stage micrometer with the zero line (beginning) of the reticle. Now, carefully scan over until you see the lines line up again. You can then use a simple ratio to determine the value that each line represents in your reticle.


In the example above, the eyepiece micrometer (reticle image) is on the top and the stage micrometer image is on the bottom. The stage micrometer is 1 mm long with 100 divisions so each division of the stage micrometer is one one-hundredth of a mm (0.01mm or 10 um). Hint, you move the decimal point over three places to the right to change mm to micrometers.


The eyepiece micrometer is divided into 100 units. We don't need to know the actual distance between marks on it.


When the zero marks are lined up, scan across and look for a convenient point where the lines converge again. If you look at the 30 mark on the reticle, you will see pretty close alignment with the stage micrometer. How many divisions? Did you say 20? You are right! And, if each line is 10um wide, what will 20 lines equal? Answer: 200um.


Now it is just a simple math ratio. 30 divisions of the reticle (eyepiece micrometer) equal 200 micrometers. So what does one division on the reticle equal? Let's see, 30 is to 200 as one is to X. Remember how to do a ratio? Two fractions, 30 over 200 equals 1 over X. Cross multiply, you get 30X=200um, solve for X by dividing both sides by 30 and X equals 6.7 um. Notice that they line up again at 60 but alignment is off by one at 90. If we use 90 and 61 (610um) we get 6.8um. The wider the interval, the more accurate your results should be.


Remember, this distance between reticle lines is only good for that particular objective lens and it may not come out to be a nice round number. When you switch to a different objective, you must recalibrate.


Quiz time: Our stage micrometer has a line 1mm long with 100 divisions. That means that each division is one one-hundredth of a mm (.01mm or 10um). When looking at it with the reticle, you notice that the lines converge at 8 and again at 16. We will choose 16. At the 16 mark on the reticle, we notice 60 lines on the stage micrometer. What does each mark on the reticle represent?


If you got 37.5 um, give yourself an A!

For further information you may want to view this link about eyepiece reticle calibration.