**Presenter:** Nicole Groshon, Cleanroom Microscopist, McCrone Associates

Every microscope and every objective is slightly different. In this webinar, Nicole discusses how to properly calibrate your microscope’s eyepiece reticle to a stage micrometer so that you can obtain true, correct particle measurements. 15 minutes.

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## Transcript

**Introduction**

**Charles Zona (CZ):** Okay, I think we are ready to get started. My name is Charles Zona and I would like to welcome everyone to today’s McCrone Group webinar. Our presenter is Nicole Groshon. Nicole is a cleanroom microscopist with McCrone Associates, and she is going to talk to us about how to calibrate your microscope using the microscope’s eyepiece reticle using a stage micrometer so that you can obtain true, correct particle measurements.

This webinar is being recorded and will be available on The McCrone Group website under the Webinars tab.

And now I will hand the program over to Nicole.

**Nicole Groshon (NG): **Thank you for the introduction, Charles. Good afternoon everyone. Thank you for taking the time out of your day to join us. If you have any questions, there will be a chance to ask them at the end. So let’s get started.

Micrometry is the measuring of linear distance (width, length, etc.) of microscopic samples. Before we can accurately report particle dimensions, we need to calibrate our microscope.

There are three major components of calibration. First, you will want to focus your eyepiece reticle to your eyesight. The benefit in doing so is that your eyes may differ in acuity, so focusing your eyepieces separately from one another will prevent squinting, eye strain, tension, and even headaches.

Second, you will want to find true magnification. Calculating true magnification for each click stop will give perspective to the images you take. Saying an image was taken at click stop 4 does not mean much to the viewer since click stops vary from microscope to microscope, but saying the image was taken at 60X is important.

Lastly, you will want to calibrate your click stops to a certified stage micrometer. Doing so will allow you to measure samples accurately.

Here is an image of a stage micrometer we use at McCrone. We do sell them at McCrone Microscopes & Accessories, if you are interested in purchasing one. My contact information will be available at the end of the presentation.

At McCrone Associates, we do check our calibration annually. If the measurements are not within 5% of our original calculations, it is an indication that there might be an issue with the microscope’s alignment.

Today, my calculations will be using the click stops from the Nikon SMZ1270; however, the calculations I will be sharing with you in the following slides can be used for any stereo- or polarized light microscope.

So now we will jump into greater detail on the three steps of calibration that I discussed—the first one being that we need to focus our eyepiece reticle.

You will want to find a sample to focus on, so something with fine features, such as small typed font on a piece of paper, would be a perfect example to use.

Second, you will want to determine your dominant eye, and place the reticle on that side. Eyepieces are usually removable and interchangeable with one another. If you have this option on your microscope, the reticle—or the scale bar—should be placed on the side of the user’s dominant eye.

So I have a quick exercise we can do right now to help determine your dominant eye, in case you aren’t sure which one it is.

Extend one arm out and hold the thumb of that hand in an upright position. Keeping both eyes open and focused on a distant object, such as a clock, superimpose your thumb on that object. Alternately close one eye at a time. The eye that keeps your thumb directly in front of the object while the other eye is closed is your dominant eye.

Here, I have an image of how the eyepieces are interchangeable with one another.

The third step is to focus your non-dominant eye eyepiece. Start with focusing the eyepiece that does not have the scale bar in it. While keeping your dominant eye closed, use coarse and fine focus knobs to adjust your view until the fine details of the sample you are viewing come into sharp focus.

The fourth and final step is to focus your dominant eye reticle. Using your dominant eye only, while keeping the other eye closed, focus the eyepiece crosshairs by rotating the uppermost eye lens of the dominant eyepiece until the crosshairs are in focus.

The second component to calibrating your microscope is finding true magnification for each of your click stops. There’s a three-step calculation used to determine this.

First, you will want to find the eyepiece magnification. Sometimes it will have it printed on the piece itself, other times you will need to look in the manual to find this information.

Next, you multiply this number by the coaxial illumination magnification. On the Nikon, along with many other ‘scopes, it is printed right on the front. the Nikon, for example, is a 1.5X magnification.

Next, you multiply by the objective magnification, which can be found printed directly on them. It’s difficult to tell from the angle in this image, however, there are two objectives here—one is in the back. The one that we’ll be using for our calculations today is a 1X; the one in the back is a 1.5X. So you’ll need to recalculate if you switch out your objectives.

The final step to the calibration is multiplying by the knob magnification and/or click stops, found printed on the side knob, here.

By multiplying these magnifications, you find your true microscope magnification.

So now let’s set up a chart to keep our calculations organized. There are seven click stops for the Nikon, so there will be seven different calculations.

For this Nikon, the eyepiece magnification is 10, and that will remain constant for all of the calculations.

The coaxial illumination magnification is 1.5, and as I mentioned earlier, it is printed on the front of the ‘scope.

The objective magnification will be 1 for these calculations. If you change objectives, you need to recalculate.

The knob magnifications, or click stops, are found on the knob here, and I have listed them below.

When you multiply these number across, you find the true magnification for the microscope. It is important to keep these numbers handy for the chart that I’ll be showing you in the following slides.

Now I will give a brief overview for the five steps for calibrating your objectives. We will go into greater detail for these steps in the following slides.

First, you will want to calculate the distance of each stage micrometer division.

Second, line up the micrometer with your eyepiece reticle scale bar.

Third, count divisions and calculate size for that magnification.

Fourth, increase magnification and repeat calculations for each click stop.

And last, you will want to create a sizing chart to keep at your workstation.

Now we well take a look at each of the five steps.

First, you will want to calculate the distance of each stage micrometer division. Observe the unit of measure of the certified stage micrometer from the units on the micrometer itself or on the Certificate of Analysis.

Calculate the distance of each division using this formula:

Sometimes, the stage micrometer will have the distance between each division noted right on it. If you’d like a thorough understanding of where these numbers come from, or would like to convert from millimeters to micrometers, knowing this formula will come in handy.

Here, I have an image of what a typical micrometer scale will look like.

We can run through the example using the formula above. Total scale length, which is 1 mm—or 1,000 µm—divided by the number of divisions, which is 100, equals length of each division. So for the stage micrometer shown here, every division equals 10 µm.

Moving on to the second step in calibrating your objectives. Line up the micrometer with your eyepiece reticle scale.Place the stage micrometer on the stage of the microscope and bring the scale into focus. Position the stage micrometer so that its zero overlaps with the zero on the eyepiece reticle scale, as I’ve shown here.

Onto the third step of calibrating your objectives. Count divisions and calculate size of a single division for that magnification. Count the number of stage micrometer divisions that match up with the largest visible number of the eyepiece reticle divisions and enter in the following equation:

The number of stage micrometer divisions—in the example I have here (see image in step 2 above), it’s 100; divided by the number of eyepiece reticle divisions—and this example is dead-on at 45; times (multiplied by) the number of micons (micrometers) per stage micrometer division—in step 1, we calculated this to be 10 µm; equals one eyepiece reticle division—for this example, one eyepiece reticle division is 22µm at 45X.

Let’s do another practice problem using the equation from step 3. For this example, I took an image of the scale bars at click stop 6, magnification 90X. So to recap on the formula I just shared with you: The number of stage micrometer divisions, which in this example is also 100; divided by the number of eyepiece reticle divisions, in this case, it’s 91, times the number of microns per stage micrometer division, and this will still be 10, equals one eyepiece reticle division. So for this specific example, one division equals 11 µm at 90X.

On to the fourth step of calibrating your objectives. Increase magnification and repeat calculations for each click stop. Every time you increase magnification, you will need to realign the zeros from both scales and count the divisions. Continue this process until you have calculated the size of one division at each magnification.

Here I have some examples of what the different click stops and magnifications would look like. It’s best to start with your lowest magnification and work your way up.

On to the fifth, and final, step of calibrating your objectives: create a sizing chart for your work station. Instead of making calculations every time you measure a particle, create a cheat sheet to expedite your observation process.

Now grab your click stop and magnification numbers from the previous calculations, and start to fill them into a chart in Excel (this will be the header). The first row (following the header) will be the length on one eyepiece reticle division at that click stop. You can easily expand your chart by using simple multiplication. For example, two divisions at 105 µm gives you 210 µm; three divisions gives you 315 µm, and so on, and so forth.

This chart comes in handy, say, for example, if you were working at click stop 3, you can grab your chart and see that you are working at 45X. And if you find a particle that’s 7 divisions long, you know that it’s 154 µm.

Now that we’ve completed our chart, let’s practice using it. I have an image here of several particles at click stop 8, magnification 120X. Let’s try to measure this (darkest) particle.

It looks like it’s about 8 divisions across, so if you head over to your chart, 8 divisions at 120X equals 64 µm.

That’s a lot easier than pulling out a calculator every time you want to measure a single particle.

And that sums up how to calibrate your microscope to acurately measure particles.

I have some references and acknowledgments. I used some images and information from John Delly’s *Essentials of Polarized Light Microscopy and Ancillary Techniques*, and a special thank you to Leslie Bolin, our graphic artist, for helping with photography and editing.

That wraps up my presentation for today. Thank you for joining us. If you have any questions, we’ll take them now. Otherwise, my contact information is listed here.

**CZ:** It looks like that might do it for the questions. Thanks again for attending today’s webinar. Please visit McCrone.com for a schedule of our future webinars.

## Comments

Is it possible to measure visible particles inside the ampoules/vials without breaking them? Which microscope/instrument do you suggest for use?

Fabiana, thank you for your question. It is possible to estimate the size of particles before breaking the vials, but it really depends on the nature of the particulate. It’s easier to measure them if they are stuck to the side of the vial or float in the solution. If they tend to sink to the bottom, the curvature of the glass might distort the view, making the measurement less accurate. Measurements should be taken using a stereo microscope; a polarized light microscope would be too high of magnification to focus on particles moving in solution.