Which microscope?

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In this interactive, students explore the strengths and weaknesses of seven types of microscope. They hear why individual scientists have chosen to use particular microscope types in their research.

Transcript

Welcome

When planning an experiment with microscopes, you need to think carefully about which microscope to use. It all depends on what you want to learn. What do you want to do?

  • Look at organisms, cells or tissues that are currently alive?
  • Look at the surface of a living thing?
  • Look at whole cells and how they connect in 3D?
  • Look at the surface of a sample at high resolution?
  • Look at a cross-section of a sample at high resolution?
  • Build up a 3D model from the results?
  • Avoid removing moisture from the sample?

This interactive takes you through some of the important differences between microscopes. Click on the micrograph images to find out more about each microscope or answer the questions to find out which microscope is best to use.

Fact file: Stereomicroscope (light)
Fact file: Compound microscope (light)
Fact file: Confocal laser scanning fluorescence microscope
Fact file: Scanning electron microscope (SEM)
Fact file: CryoSEM
Fact file: Electron tomography
Fact file: Transmission electron microscope (TEM)

Fact file: Stereomicroscope (light)

Function: Uses visible light to illuminate the surface of a sample

Maximum magnification: Approximately 2000x
School light microscopes that do not use oil immersion have a magnification range of 40-400x

Best for:
Looking at living things
Looking at things without disrupting them

Disadvantages: Usually lower resolution than the compound light microscope

Video: 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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Fact file: Compound microscope (light)

Function: Uses visible light to illuminate a thin section of sample

Maximum magnification: Approximately 2000x
School light microscopes that do not use oil immersion have a magnification range of 40-400x

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

Video: 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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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 2000x

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

Video: 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.

Video: 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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Fact file: Scanning electron microscope (SEM)

Function: Lets you look at the surface of objects at high resolution.

Maximum magnification: Approximately 500 000x

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

Video: How SEM works

LIZ GIRVAN
This is a scanning electron microscope or SEM. The action starts at the top of the column, which is this piece here, which is where the electron gun is. Inside here we have got different electromagnetic lenses, which focus that, all those electrons into a nice little beam. At the bottom down in here we’ve got a final lens, which is where the electrons are focused again and then pop out and hit the sample, which is sitting just below it.

That beam, instead of just coming straight down, it actually moves quite rapidly backwards and forwards across the sample. You get electrons being knocked off the surface of the sample and then picked up by a detector, and they’re called secondary electrons. They show you most of the information.

Video: 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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Fact file: CryoSEM

Function: Lets you look at the surface of objects that contain liquid (avoids sample preparation steps for conventional SEM)

Maximum magnification: Approximately 500 000x

Best for:
Looking at biological samples in a lifelike state
Looking at biological samples too delicate to survive preparation for conventional SEM
Looking at hydrated or wet surfaces of biological samples

Disadvantages:
Resolution often not as high as the transmission electron microscope
Can’t be used to look at living things (sample needs to be frozen before visualising)
Frozen water may hide important information
Costly to run

Video: CryoSEM versus standard SEM

LIZ GIRVAN
CryoSEM is quite different from conventional SEM. So in cyroSEM, we take the sample as it is from nature or from industry, and we simply freeze it and then view it frozen. So we don’t do any of the fixation steps, we don’t need to dry it – we just freeze it and look at it.

BRONWYN LOWE
Why would you use cryoSEM looking at plant materials? In order to prepare a sample to put in an SEM you have to treat it, you have to dry it, you have to chemically set it, and when you do that, you can introduce a lot of changes into your sample that are then artefacts of your preparation steps rather than what the plant really looks like. And because I really wanted to know what does the plant look like immediately it comes off the bush, all of those preparation techniques were not possible.

So cryoSEM gets around those problems because you take your sample and it gets dunked into liquid nitrogen so it’s snap frozen, and so you capture all the cells, all the arrangement of the cells, everything is just frozen instantly. And so that means that you don’t alter the sample much in putting it in the microscope.

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Fact file: Electron tomography

Function: Lets you build up a 3D model of a sample from TEM data

Maximum magnification: Approximately 5 000 000x

Best for:
Understanding relationships between objects in a sample at high resolution
Understanding three-dimensional structure
Looking at objects in very high resolution

Disadvantages:
Can’t be used to look at living things (samples need to be prepared extensively before visualising)

Video: How electron tomography works

ALLAN MITCHELL
The technique that allows us to make 3D models of our biological structures is called electron tomography. If you imagine that this book is the section that we use for electron tomography and it’s thicker than a normal section would be, we place this section inside the microscope and then, while illuminating with our electron beam, we start to tilt it. We take an image at 1 degree tilt, 2 degree tilt, 3 degree tilt, and in this way, the electron beam is building data that is through the thickness of the sample rather than data that is superimposed over the top of each other.

The information looks very, very confused. However, with special computer programs, we can take that confused information and put that image back into its three-dimensional space, and we end up with a three-dimensional model of what we were looking at.

Video: Electron tomography of the primary cilium

TONY POOLE
Tomography allows you to see more material in greater detail and in a better three-dimensional way. The primary cilium is a very tiny organelle. Its 200 nanometres thick, and it can be about a micron or two long, so it’s a very thin sort of long structure. And this makes them impossible to see in a light microscope, virtually impossible. Even in an electron microscope, you have to hunt to find them.

With normal electron microscopy, you cut very, very thin sections – about 80–90 nanometres. With electron tomography, you can cut much thicker sections, more 300–500 nanometres. This means you can get the entire cilium in one single section.

Once you’ve generated that dataset, you can turn it upside down and look at it from this end, you can turn it end on, you can zoom in, you can zoom out, you can do a huge amount but you’re not cutting any more sections. You’ve only cut one single section, and the way the tomographic dataset works, it allows you to then take very, very thin slices through your dataset – thinner slices than you could cut and look at in a conventional microscope.

It’s the first time we’ve ever been able to look, at very, very high magnifications, at the relationship between this primary cilium and the environment that it interacts with.

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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

Video: 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.

Video: 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

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