>> 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.
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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.
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