Under the Microscope

>>Hello and welcome to
the penguin prof channel. Today’s video is part
of my lab skills series. And we’re going to talk about
microscopes and microscopy. As always, if you find
these videos helpful, I would ask that
you take a moment and click those buttons below. It makes a big difference. Thanks. You want to stay tuned. We’re going to be talking
about all things microscope. Including the parts of
a compound microscope. How to figure out magnification
and depth of field. And we’re going to
talk about refraction. How to make a wet mount. And how to find stuff. And how to not kill your
eyes while doing all of that. The type of microscope that you
are going to use, of course, depends on what you’re
looking at. I just threw this in here
for a sense of scale. If you want to see a little
bit more on size and scale and even some microscope
history, I have a couple of videos. And I will, of course, put
those links down there. These are compound microscopes. Depending on upon what kind of scope you have it’ll
look something like this. You may have one eyepiece. We call those “monocular.” If you’ve got two,
those are binocular. There are actually
trinocular scopes. That is for your third eye. No. It’s for a camera. But they all basically
look something like this. So, you’ve got an eyepiece with
an ocular lens and that’s fixed. And usually those are
10x but it varies. And then you’ll have a
“nosepiece” as it’s called. And you’ll have revolving
objective lenses. These are the lenses close to
the object you are looking at. And the magnification on those
will be printed right there on the lens. Then you have the
stage, of course. And the stage you may
have to move the specimen on the stage with your hands. Hopefully, you have what are
called “coaxial” stage controls. These are little knobs usually
underneath or next to the stage. And that allows you
to move the slide in an x-y direction
very, very smoothly. So, hopefully, you have that. And then you have your focus. And we have two knobs. We’ve got a coarse knob
and then a fine focus knob. Usually the coarse
one is bigger. And we’ll talk about those. And then down here you’ll
have a light source. And then usually a variety of
things including an iris diagram that works just like the
iris diaphragm in a camera or in your eye actually. As well as a condenser lens. And all of these help to
control the light as it passes through your specimen. Now, when you use the
microscope, of course, I have to say please always use
both hands when you carry it. Even when you move it around on
the table — on your lab bench. Go ahead and pick it up. Don’t drag it across the table. It makes that horrible
bouncing sound and that’s probably
the condenser getting out of alignment. When you sit down to use it and
you plug it in, the first thing that you want to do is
maximize the distance between the objective
lens and the stage. Now, some scopes what you’ll do
is you will move the nosepiece up and down with
respect to a fixed stage. And other microscopes you
actually move the stage up and down against
a fixed nosepiece. But either way you want
to increase that distance between those two when
you’re getting started. You want to turn the
reinvolving nosepiece so that the lowest
power objective — we call that the
“scanning lens” — it has the smallest number
on it and it is the shortest. You want to click that
little guy into place. Put your slide on the stage. And if you have a tension arm,
you want to pull that back. And then there’s a little
frame in there as you can see. So, this little frame
helps to hold the slide. And then the tension arm
keeps it from moving around. Those are really slick to have. And that way you can move
the slide back and forth with those coaxial controls
that are right here. So, now you can use those
co-axial controls to very, very smoothly move the
slide and adjust it so that the light is shining
directly through this center. If you don’t have
those controls, your scope probably
looks like this and you have these little clips. And you have to use your hands
to move the slide around. It’s not as smooth,
not as slick. But, you know, it is what it is. The next thing you want to do is
use that coarse adjustment knob to bring the specimen
toward the lens. Again, you’re either moving the
nosepiece or moving the stage but either way you’re bringing
them together and you want to bring your specimen
into focus. And then you can use the
fine focus adjustment to fine tune that focus. You want to center the specimen and then you can
increase the magnification and now you only want to
use those fine focus knobs. So, the coarse adjustment is
for scanning lens work only. Now, compound microscopes
have two lenses. That’s the name. And the magnification that
you see is the product of the two lenses
that you’re using. So, for example, if you have
an ocular lens that’s 10x and you happen to be using
the 40x objective lens, you multiply those
numbers together to get your total magnification
which would be 400x. You will probably have to make
a wet mount as it’s called. And what you’re going to
do is use a drop of liquid. The liquid you use
depends on your specimen. So, you would be using
tap water for plant cells, saline for animal cells. If you don’t have saline around,
contact lens solution will work. Get your specimen. If you need to cut it,
especially if it’s a plant, you want to make that
slice as thin as possible. And then just place the
specimen in the fluid. Next, you’re going
to grab a cover slip which is a little square piece
of plastic and add a stain if you’re told to do so. But most people add the stain after the cover slip
is already in place. I’ll show you that. Then you’re going to set
one edge of the cover slip at about a 45-agree angle
and then slowly let it drop. And this technique will
reduce air bubbles. Now, these are air bubbles. Many students spend many
hours drawing air bubbles because they look alive
and they’re very pretty. Okay. Guys, they’re air
bubbles so just move on. Like I said, often
you add the stain after the coverslip is in place. So, to do that, you just add a
drop of the stain at the edge where the coverslip
and the slide meet. And then you place an
absorbent material. You can use a paper towel or
a Kimwipe at the opposite end and it will wick the fluid and draw the stain
across the specimen. It’s a really nice way of
evenly distributing the stain across the slide. Finding stuff under a
microscope slide is a lot harder than it sounds because it seems
that the slide is very small. But, in fact, when
you magnify it, you can spend all day
looking for stuff. It takes a lot of practice. I tell my students
to approach this like a search and
rescue mission. Okay. So, when you are
doing a search and rescue, you start by flying high. And what you see is a
very, very large area. What we called the
“field of view.” You don’t see very
much detail, though. So, if you spot a possible
target, then what they do is fly over it and then come
down in altitude. What they’re doing
is getting closer so you can see it
in more detail. So, the field view — that
is that area that you can see and the magnification,
which is the detail, they’re inversely related. Okay. So, just so you could
look at something pretty, here have a very, very
large field of view but this is very
low magnification. We don’t see very much detail. As we fly in and we’re looking
for a target, now the field of view is decreasing but
we’re seeing more detail. And, finally, there’s
your target. Here’s Flops. This is grace bay in the
Turks and Caicos islands. I just wanted to give you
something fun to target on. High magnification. But a very, very
small field of view. Under the microscope, under
a lower magnification, you see more of the
slide but less detail. As you bump up the
magnification, you’re going to see more and more detail. So, the idea is you
want to start with that scanning
objective and look for the stuff that’s
interesting. And then center it. And then increase
your magnification. And that’s really
a good strategy. An interesting note
is that because of refraction things
are inverted. And as you move the slide
around in your x-y direction, it can be a little confusing. If you cut out a letter e from
a magazine or something — we make our students
do this sometimes — and you put it under the
microscope like this, when you look through the scope,
that’s what it looks like. So, it’s a little
strange, you know. And it takes a little
while to get used to. The other thing that
takes a while is to realize you’re working in the 3-d even though the slide
seems very, very flat to us. There is a narrow depth of
field where your object will be in focus and the
depth of the depth of field changes
with magnification. So, sometimes you’ll get a
slide with these crossed threads to kind of explore this. And the idea is that as
magnification goes up, the depth of field goes down. That is to say, the higher
the magnification is, the less of the slide in a
vertical plane will be in focus. And that’s a really
important idea. So, as you increase
your magnification — here we’re looking at paramecia. Under low magnification, we
have a high depth of field. So, lots of the slide
is in focus. And as you increase
magnification, you see more and more detail but your
depth of field goes down. And that’s how we actually get
these beautiful images where, as you can see, the — the
background is — is blurred. So, you need to keep
that in mind as well. You will most likely have to be measuring things
under the microscope. You need to know how
big things really are. Now, some microscopes have
ocular scales like this one. So, when you look through that
eyepiece, there’s a little scale in there and you
— you can compare that at different magnifications
against a micrometer that you put on the stage. This is obviously the
best way to go about it. But a lot of student
scopes don’t have those. All you need is a small
clear plastic metric ruler. And what you’re going to do
is measure the field of view under the scanning
objective lens. And then based on that you will
be able to calculate the field of view for all your
other lenses. So I want to show
you how to do that. So, first of all, just so you
know what I’m talking about, under your scanning
objective — let’s say 4x. If you look at a ruler, maybe you see something
that look likes this. But as you bump up
the magnification — so, here’s your field of view in
yellow using the 10x objective. And, finally, here’s your field
of view under the 45x objective. So, obviously, you
can’t measure that. In other words, you can’t
use this metric ruler to calculate the field of
view of the blue sphere. But you can use math to do that. All you need to remember is
the magnification and field of view are inversely related. So, if you double
the magnification, then you reduce the
field of view by half. So, pretty straight forward. Let’s pretend the field of view under my 4x scanning
lens is 5 millimeters. If I had an 8x lens, if I
doubled the magnification, the field of view would be half. 2.5 millimeters. So, that’s it. Now, what you need to do is
calculate the field of view for all the lenses you have. So, if my 4x field of
view is 5 millimeters, can you calculate what the field of view would be
for 10x and 45x? Can you do it? Can you try it? Can you press pause
and give it a go? I know you didn’t press pause. Of course, I’m going
to tell you. So, here all you’re doing is
comparing how many times more magnified each lens is compared
to the one that you counted. So, in other words, 10x is 2.5
times more magnified than 4x. So, then all I want to do is
take the field of view under 4x. And I say, well, what’s
2.5 times less than that? So, do you see what I mean? It’s pretty straight forward. Now, I did convert
millimeters into microns because that’s a more
biologically relevant unit. But that’s basically
all you have to do. And this will give you a
really quick and easy way to estimate the size of things if you do not have
an ocular scale. So, here I am under my 45x. Now, remember, my total
magnification would be 450 if my ocular lens is 10. So, now I know that diameter is. It’s 440 microns. And if I’m looking at my
cheek cells and I estimate that the length of one
of my cheek cells is about a fifth maybe of
that total diameter, then I get 88 microns
as a length. And that’s pretty close. Last thing I want
to talk about is how to save your eyes while
spending all this time looking in the microscope. Number one, don’t
behind yourself. I find my students tend
to use way too much light. Especially under the scanning and low power lenses you
don’t need that much light. As you increase magnification,
that’s when you need to increase the amount
of light coming through. So, make sure you adjust that
and you’re not always looking under maximum illumination. The other thing you want to watch is what’s
called “eye relief.” And this is something you
have to experiment with. And that is the distance
between your eyeball and that ocular lens. So, the higher the magnification that you’re using the shorter
the eye relief should be. So, as you decrease
magnification, try and pull your eyes
away from the ocular lens. You know, like I said,
you got to experiment. You should shoot for
somewhere around six, six and a half millimeters
distance. If you wear glasses,
you’re going to need to experiment a little bit more. But this is something that really makes a big
difference it you’re spending a lot of time in front
of a microscope. Here’s another thing. Your focal point. Your eyes automatically adjust
as objects move toward and away from you and we call
this “accommodation.” Most people approach microscopy
with a near point accommodation. So, in other words, you
kind of focus your eyes as if you were reading but actually it’s far
point accommodation. So, you know, this is — it
sounds kind of weird I know — try to look at the specimen
and not into the specimen. It sounds a little touchy-feely
but experiment with it. And you’re going to find that you can help to
reduce eye strain. My last suggestion. Take breaks and rest your
eyes every 20 to 30 minutes as you build up that
microscopy stamina. As always, I hope
that was helpful. Thank you so much for visiting
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