So in this tip, I'm going to show you how to measure the
point spread function of your microscope. And so to see
how your microscope diffracts the light, you first need an object
that is smaller than the resolution of your microscope. And so that
object is given by these beads that have a size of, in this case,
100 nanometers. So this is an example of green fluorescent
beads. These ones here are very popular, these TetraSpeck beads,
because they fluoresce in multiple colors. But today, we're going to use
these yellow-green fluorescent beads. You can see here that you
buy a bottle with quite a few milliliters, however to get a good
spread of -- a good density of beads on the coverslip. All we need
is a dilution of 100,000 fold out of this one. And that means that,
so basically, we'll take 1 microliter of beads and dilute that
100,000 fold. And you can use -- people use various different
strategies to make these samples. What I like to do is dilute it
with ethanol, since the ethanol evaporates very rapidly. And that
gives us a very nice easy to make sample. So first dilute
the beads 1,000 fold. And then go on and dilute it another 100 fold.
Now since the beads are fluorescent, they also photobleach.
And that's why I'll put them away now so that they are stored
in the dark and don't photobleach. So here's our sample of beads diluted
at 100,000 fold. Now, to prepare the coverslip, you could in principle put them
straight down on the coverslip itself. But these coverslips are
relatively dirty. And so, what I tend to do is wash them in ethanol
and then flame them. So I'll switch on the bunsen burner, and when you do
this, be careful because ethanol is highly flammable. And you don't
want to set the whole thing on fire. So this is a relatively
crude way of cleaning the coverslip, but since these beads
are highly fluorescent, it is actually good enough.
So at this point, we can pipet roughly 4 microliters on one of these
18x18 mm coverslips. And that will spread then very quickly
over the coverslip and fill the whole surface area. And then
to both speed up the drying of the ethanol, but also to bake
the polystyrene beads onto the coverslip itself, what I like to do
is to put the coverslips, bead side up, onto a heating block. And that
seems to like melt the bottom of these polystyrene beads onto the
glass. You don't have to do this, the beads will also stick without it.
But this makes them wiggle less, and under certain circumstances that can be a good
idea. So now, an alternative strategy is to pipet the 4 microliters
onto the coverslip and use this little centrifuge, in which you can spin
the coverslip. And the advantage of this is that the solution then
spreads very nicely and dries in one go. So I'll put a coverslip into the
centrifuge, pipet on the beads solution, and spin it.
And then, so the coverslip is now dry. But I'll also heat that up
for a few seconds. Then what remains to be done is mounting
the coverslip onto a slide. I like to wipe the slide clean with a
Kimwipe. Now there are different types of mounting media you can
use. One of the simplest is 100% glycerol. I know this because it's
highly viscous. The refractive index is somewhat elevated over
that of water, and because of its viscosity and lack of oxygen,
it also happens to be a pretty good anti-fade reagent,
in its own right. So we now take the coverslip, bead side up,
turn it upside down so that the beads are facing the glycerol,
put it down, the glycerol spreads out. One important thing is
labeling the slides, so that you later still know what it is.
So these are 100 nm green beads. And we're done preparing
our beads slide.
Okay, so we have here at the fluorescent microscope. We have our
bead slide. We are using a 63X 1.4 NA oil objective. This
particular objective actually has a phase ring in it. So, we'll give it a
tiny drop of oil, of the appropriate immersion oil. And then put the
slide on it, coverslip side down. So it's advantageous to look at
the oil and to judge roughly where you are, where the objective is
with respect to the coverslip. Because these beads are tiny,
diffraction limited, and they're also not super bright, so they're actually
hard to find. And so being close is going to help you dramatically.
So then, we can now observe them. Okay, and I see my
tiny little beads. And this literally looks like stars at night. I see the
big dipper there. What you might already be seeing, what becomes
a little bit more obvious when we zoom in onto this image,
and then again go live. You see that as I slowly go through focus,
so here we see a single bead. And you see that from one side,
there is a bit of a line in this direction, and when I go through
focus, there's a bit of a line in that direction. And that is
a sign of astigmatism. So there's a tiny bit of astigmatism in this
optical system here. Now to collect the whole point spread function,
we're going to use the software to do that for us. So we're starting
out now here in the middle, and we tell the software to take a
z-stack. We want that z-stack to go from 5 microns below the
point of focus to 5 microns on top of the point of focus, and
steps of 0.2 microns. So when I now acquire, you see the
z-motor slowly stepping up and taking an image at every next z.
You now see that bead image forming as we go.
We now have the whole data set here, and we can quickly
manually go through focus. What we can do is to get the
cross-section of this image. The ImageJ command, image, stacks,
orthogonal views. We now get two extra images that display
this sideways view. And at the moment, we don't see anything there.
But if we put the cursors now near a spot, and then go through
focus here, we now see the side view of our point spread function.
And so here, you see that typical shape with the airy discs, so the same
you can see here on this side, where we see the airy discs in
cross-section. Now there's also auto scaling going on. And that makes
this all a little bit harder to see. And I'll now take off that auto scaling,
so you now can see that most of the light is actually contained in this
small central area here. We have the cross-section of one of these
z-slices. So z goes here, and this is x. And you now do see that there's
a bit of an asymmetry in this. So there's some energy going off
in this direction. But you also do see that this diffraction pattern
is you know, pretty nice. So here we are at a Nikon inverted
fluorescent microscope. This has a turret worked in here, and so
there are dichroic cubes sitting in here that we can bring in place.
We have an epi light source, and so we'll use it here to image
the beads. And then when I now open the shutter, we have blue light coming out.
I send it to the eyepiece. We can cover up the sample with like a
black lid or something dark, so the overhead light doesn't make
it into the camera. And then that way we can have some light on
while filming. I'm now manually going through focus, we have
the software on auto stretching. And you can now live see these
rings. And what is also highly apparent here is that when I go in
z in one direction, we have no rings, whereas if I go in the other
direction, we see beautiful rings forming there. And this is a
hallmark of spherical aberration. So now going in one direction,
and we see beautiful airy discs, although a little bit asymmetric.
Then when I go the other direction, I don't see them at all.
It's just fuzziness. So now, we will do the same as on that other
system. So we're going to do a z-stack, and again we'll go from -5
to +5, with 0.2 micron steps. Let's go. There we go. So we see
the point spread function as we speak. So we're going from the
glass to the beads and then we go through. Okay, now with
this set we can look at the orthogonal view. So what you
now can see very, very clearly, for instance, let's say in this side view
here, is how asymmetric the point spread function is. So it's nicely
defined here at this end, but it's kind of a blur here.
And that's really a hallmark of spherical aberration.