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microscope

Fact file: Compound microscope (light)

Function: Uses visible light to illuminate a thin section of sample
Maximum magnification: Approximately x2000
School light microscopes that do not use oil immersion have a magnification range of x40-400
Best for:
Looking at some living things (for example, a single cell layer)
Looking at cells and tissues (preparation steps are less critical than for electron microscopy)
Getting an overview of a sample
Disadvantages:
Low resolution compared to electron microscope

Light microscopy
ALLAN MITCHELL
“There are two types of basic light microscope configuration. There is the compound microscope, which
is the microscope that shines light through a slice of a sample, and then there’s the stereomicroscope,
which looks at the surface of the sample.
The key advantages of light microscopy is you can look at living material. You can see processes that
may be occurring dynamically whereas in the electron microscope, because we have to do so much
preparation to get the sample in there, the sample is essentially dead – we get a moment in time.
When people do light microscopy, they start to run out of resolution at round about 2000 times
magnification, so a lot of assumptions are made about what they’re actually seeing when it comes to
fine detail. There are various specialised light microscopes coming on the market now which can
resolve this, but essentially, for most microscopy work, anything smaller than 200 nanometres is
invisible to a light microscope.”
Acknowledgements:
Ningbo Optical Microscopes Company
Rosa Henderson, Landcare Research
Leon Perrie, Te Papa
Jenni Stanley, Auckland University, Leigh Marine Laboratories
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microscope

Fact file: Confocal laser scanning fluorescence microscope

Function: Lets you look at thin ‘slices’ in a sample while keeping sample intact; lets you look
specifically at parts of a cell (such as individual proteins) by labelling them with fluorescence
Maximum magnification: Approximately x2000
Best for:
Looking at living cells
Understanding relationships between cells
Highlighting individual components of cells
Disadvantages:
Low resolution compared to electron microscope
See only fluorescent objects; no other structures visible
Fluorescence can cause artefacts

How confocal microscopy works

ALLAN MITCHELL
“In a conventional microscope, we have the light shining right through the sample, which means all the
information inside the thickness of that sample is superimposed on top of itself. However, with the
confocal microscope, we are able to select the particular levels within that section and just focus on
those. The information above that level and below that level is discarded from the image.”
REBECCA CAMPBELL
“So I explain this to my students as, if the tissue were a stack of pancakes, we can image each
individual pancake without having to take them apart and so then we can put them back together to
generate a 3D image of the pancake stack or we can look at the individual pancakes at the different
focal planes.”

Confocal microscopy of neurons

REBECCA CAMPBELL
“Confocal microscopy is very well suited for dealing with living tissue. We can look at larger sections of
that tissue so that we can appreciate the relationships between the cells.
It allows us to visualise whole neurons and their relationships with the other neurons. So although we
can’t see some of the ultrastructural features that we might need to go to the electron microscope for,
we can see the bigger picture of the GnRH neuron, its whole length and the inputs that are talking to
the GnRH neuron. If we went to the electron microscope to look at that, we would be dealing with
many serial thin sections, and it would be needle in a haystack kind of stuff.
So one of the things that we do is we fill GnRH neurons with low molecular weight molecules, and this
has allowed us to see the entire cytoplasmic contents of these cells, and there isn’t another technique
that’s known that allows us to do that. When we take that tissue then to the confocal microscope, we
can put together all of the different parts of this neuron to appreciate it as a whole cell.”
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microscope

Fact file: Scanning electron microscope (SEM)

Function: Lets you look at the surface of objects at high resolution.
Maximum magnification: Approximately x500 000
Best for:
Looking at surfaces of objects
Looking at objects in 3D
Disadvantages:
Resolution often not as high as the transmission electron microscope
Can’t be used to look at living things (samples need to be dried and coated in metal before visualising)
Costly to run

Why we use SEM

LIZ GIRVAN
“The SEM probably gives you the best depth of field out of any microscope, so you end up with
something that looks really, really three dimensional, just as it would in real life.
Some of the limitations of the SEM are the high vacuum we work at. That means that we can’t have a
sample that has any moisture in it in the SEM, unless it’s been treated carefully.
Probably the resolution is a limitation. Some people might want to look at things like viruses, which are
very, very tiny. With the current technology of SEMs, we can’t quite get down to that resolution.”
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microscope

Fact file: Transmission electron microscope (TEM)

Function: Lets you look at a very thin cross-section of an object (such as a cell)
Maximum magnification: Approximately 5 000 000x
Best for:
Looking at internal structure of objects
Looking at objects at very high resolution
Looking at relationships between structures at high resolution
Disadvantages:
Can’t be used to look at living things (samples need to be prepared extensively before visualising)
Costly to run

How TEM works

ALLAN MITCHELL
“The layout of the TEM is very similar to the compound light microscope. We have an illumination
source at the top which provides us our illumination. In our case, it’s electrons that come from an
electron generator which forms a small cloud of electrons about 50 micrometres in diameter – that’s 50
thousandths of a millimetre.
Below the electron gun, we have a series of lenses called the condenser lenses, and they converge that
50 micrometre spot of electrons down to a 1 micrometre spot which strikes a sample at this level. The
sample is inside the microscope at the end of this rod. Electrons pass through the sample and some
are deflected and some pass right through, and that forms our image, and they are focused by the
objective lens which is underneath the sample itself.”

Why use TEM?

ALLAN MITCHELL
“The transmission electron microscope’s particular strengths are its resolution – it can achieve far
higher resolution than light microscopy – and it’s used for looking at internal features in comparison
with the scanning microscope, which looks at surface features. So if you want to look at things that are
going on within your sample at high resolution, then the transmission electron microscope is the tool.
We’ve looked at everything from chocolate samples through to seaweed samples. We look at viruses.
We look at the way brain cells talk to each other. We look at particular organelles and their role within
the cells. We look at nanoparticle drug delivery systems. All these samples can be looked at inside the
transmission electron microscope as long as the electron beam can pass through.
The limitations of the transmission electron microscope, really, from a biological perspective, the
sample has to be dead, so you have to have quite an elaborate processing procedure to stabilise the
cell so it will survive inside the microscope and be able to be cut very, very thin”
Acknowledgements:
Abi Loughnan, University of Otago
Emily Wang and Eng Wui Tan, University of Otago