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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