Calibrating Your Microscope

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 and 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. So what is microometry? It is the measuring of linear distance such
as width, length 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 dominant eye
reticle. Using your dominant eye only, while keeping
the other eye closed, focus the eyepeice crosshairs by rotating the uppermost eyelens of the dominant
eye eyepience until the crosshairs are in focus. 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
non-dominant eye reticle. While closing your dominant eye, turn the
diopter adjustment ring at the base of the non-dominant eye eyepeice until the sample
is in sharp 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 calculation 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 little closer 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: Total scale length/# of divisions=Length
of each division 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 is 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. The first row 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 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. Chuck: okay, I think we are ready to start
taking some questsions. If you have any questions, please go ahead
and type them into the questions box. We will answer them as they come in. Looks like we have a question from Alex, “Which
stage micrometer do you recommend and where can one be purchased?” Nicole: Sure, there are several different
kinds, and you can find a list on our website. I recommend getting in contact with one of
our sales representatives from McCrone Microscopes and Accessories because they can help you
determine which stage micrometer would be best for the type of microscope you have and
the application you use it for. There are different types, including transmitted
and reflected light and SEM micrometers. We sell them to anyone that will need them
for a manufacturinglab, forensic lab or a cleanroom. Chuck: yeah, they are used in a lot of different
environments for different applications. So, they have alot of them listed on the website
and we can help you out with that. Looks like we have a question from Louie,
“Should the stage microcometer be recertified, and if so how often do you recommend doing
this?” Nicole: Yes, they definitely can be. They do not expire, and it is up the user
and the company’s policy as to whether they need to be recertified. recertification can be handled through McCrone
Microscope and Accessories, so we will also be able to help you with that when you need
it. Chuck: Okay, I think that does it for the
questions. Thanks again to everyone out there who tuned
into this webinar. We hope to see you soon at another McCrone
Group webinar. Thanks.

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