In this tip, we’re gonna talk a little bit more
about spherical aberration,
and we’re structural talk specifically about
how to minimize or eliminate spherical aberration
in the laboratory under the imaging conditions
that you’re working on.
Just to review, spherical aberration,
as shown in this figure here,
is the uneven focus of light, axially,
from light that passes through the periphery of the lens
— that focuses closer to the lens —
or light that passes through the axis of the lens —
focusing further away from the lens.
Spherical aberration,
as I had mentioned in the previous lecture,
is one of those aberrations that’s extremely difficult,
in fact impossible, to completely correct for
in the manufacture of lenses
because the design criteria that we have to use
to make lenses don’t necessarily match the imaging conditions
that you need to use in the laboratory
to address the questions of interest.
Okay?
So, we have this paraxial focus
and peripheral focus,
and we need to bring those together.
So, there’s a number of ways to address that, okay?
So, one can be utilizing different refractive index immersion media.
So, basically, what you’re doing to correct for spherical aberration,
no matter how you do it,
is adjusting the optical path length
to best fit the design criteria of the lens.
And we can do that with immersion media,
and there are actually kits that you can buy
which have many, many different refractive index immersion media
that vary at the third decimal place —
say, 1.515, 1.516, 1.517, and so on.
And you can look in the specimen
at the depth that you need to address a question
— say you’re looking at the endoplasmic reticulum,
and you want to then focus to what looks like
it’s something that’s at the limit of resolution
close to that depth in the cell —
and then test different immersion media
at that focus depth
so that you get the best correction for spherical aberration.
And again, the best correction is found when you see,
focusing through, up and down,
you have the best symmetry of in and out of focus light, okay?
So, you can use this immersion media
to correct for spherical aberration
and oftentimes that works pretty well.
It’s a bit tedious and can be a little messy as well.
Another thing you can do is to go ahead
and measure your coverslips.
To get the design criteria of 170 microns thickness of coverslip,
you’ll use a micrometer to measure them
and then throw out ones that are too far outside the criteria
that you need to have the proper optical path length.
In addition to that, you’ll also want to go ahead
and, ideally, use a lens with a correction collar,
which is really the easiest way to correct for spherical aberration.
There are limits, though.
So, you have to understand
if you’re outside the range that you can correct for,
you’ll have to go to these other methods.
But a correction collar,
which was traditionally available for adjustment of coverslip thickness
in air and water immersion lenses,
is now also available in oil immersion lenses.
And that’s because oil
that you use for oil immersion lenses
actually has a temperature dependence
to its refractive index —
as it warms up, the refractive index decreases.
So, a correction collar
actually works by changing the angle
of the peripheral rays versus the axial rays
in the objective lens.
And you can see the objective lens cut away here.
The way a correction collar works
is we take the area
where you have the steepest angle,
right here in the front,
between two lens groups in the ray trace,
and we take all of the lenses behind that angle
— say, here back —
we put them in a brass tube,
we score a groove around that brass tube,
put a pin in it, attach a collar to that,
and then we fix the other lenses in another brass tube.
When we turn that collar, it moves all of those rear lenses
back and forth relative to the front lenses,
and those steep angles there then go like this,
so that you’re adjusting the peripheral rays
while the axial rays don’t get changed quite as much.
And that’s exactly how a correction collar works.
And then it will bring the paraxial focus,
which was further away,
back to the peripheral focus, shown here.
Okay? So, that’s basically spherical aberration.