# The Aaron Klug Centre for Imaging and Analysis

## Lecture 1: Intro to EM for Biologists

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Understanding of biological systems is derived from a knowledge of the spatial and temporal arrangement of structural entities ranging in size from organisms to macromolecules.

The simplest route to obtaining this knowledge and communicating it to others is often some form of microscopy. In general one can describe microscopy as the exploitation of physical principles to render small things visible.

In this course you will be introduced to a few of the standard techniques of light and electron microscopy. You should be aware that there are a very large number of techniques and technologies that we will not be covering and which may indeed be more appropriate to your specific needs. However those that we do cover will generally provide you with a starting point from which you will be able to advance independently

The purpose of microscopy is insight not images -- R.W. Hamming

## What is microscopy?

Microscopy covers a wide range of different techniques - what they have in common is that they produce images of objects at greater than life size - i.e. magnification is involved.

Magnification, M, is defined as the ratio of the size of a feature in the image to the size of the corresponding feature in the object :

M = D/d
(where D is the size of feature in the image and d the size of feature in object).

## What is a microscope?

In the abstract sense a microscope is a device which records the interaction of a probe of some sort with the object. The probe may be a some form of radiation or even a pointed tip. Different forms of microscopy utilise a variety of physical principles - light microscopy utilises the the interactions of photons with the specimen, electron microscopy utilises the interaction of electrons with the specimen and scanning probe microscopy utilises a variety of different interactions of a fine tip with the specimen.

The most widely used microscope uses light to create images of light transmitting specimens which are projected onto either the eye or a camera. The key components are a light source, an optical system, a specimen holder and a recording medium.

Diagram of a light microscope

The purpose of the optical system is to collect the those photons that have interacted with the specimen and form them into an image. In the case of light this is done by means of lenses, shaped from light transmitting materials. A large number of materials are transparent to photons in the visible part of the electromagnetic spectrum - this makes the fabrication of microscopes using visible light simple and cheap. The process of image formation for other types of radiation is not so simple and what is achieved by a lens in the case of light needs to be achieved in other ways.

## The process

In any microscopic study there are at least five phases:

1. The preparation of the material
2. Visualisation
3. Recording
4. Analysis
5. Communication

It is the function of the EM Unit to assist you as much as it is able during all these phases.

1. Preparation refers to the steps that occur between the sample being a living organism or part of a living organism and its visualisation in the microscope.

It is of course of key importance that the preparation of the material be properly controlled. Much of the preparation can have a key bearing on the subsequent steps and any preparation technique should be applied with understanding and circumspection. In this course we will only address preparative procedures which are directly related to microscopy. These will often occur after substantial other preparation steps and may be influenced by them. It is important to bear this in mind.

2. Visualisation refers to the process of obtaining an image. We will explore a number tools for obtaining images. In many experiments visualisation is sufficient to obtain the required insight - other experiments may entail substantial work after this step.

It is usually necessary to record the images seen with the microscope either for subsequent analysis or for documentation purposes.

Most instruments use direct digital image capture, in which the image is transferred to a computer readable form which can be viewed on a screen, stored on disc, transmitted electronically or printed on a variety of media. Another advantage of digital images is that they are readily accessible for subsequent computer analysis.

In certain cases silver based photographic emulsions (film) , although expensive, retain advantages over digital technology and are still used. (Check the Kodak electron micrography site).

Film recording is often the only technique available on older equipment.

3. Analysis involves counting or measuring of objects in the images. It may be necessary to do considerable computer processing of the images before the objects of interest can be analysed.
4. There is no point in doing any microscopy unless it is communicated. Communication can take many forms ranging from a simple student report to books of hundreds of pages. It is best to keep the form of communication required in mind from the beginning of the project as this will certainly influence how the work is done.

## The scale of things

Interesting biological objects range in scale from organisms to macromolecules

Our primary concerns in this course are with light and electron microscopy. In transmission electron microscopy images are formed from electrons that have passed through thin specimens (100-1000 nm). This differs from scanning electron microscopy in which surface images are obtained by utilising signals (usually electrons) emitted from the surface of thick specimens.

## Physical limitations

If it is cheap and easy to make microscopes that use light as a probe why do we bother with other forms of microscopy?  The main answer is that the resolution is determined by the wavelength of the radiation used.

The resolution of a microscope refers to the smallest distance between two points in the specimen that can be perceived as separate in the image. Physicists use the Rayleigh criterion devised by Ernst Abbe to calculate the resolving power of a particular system. This says that it is possible to resolve smaller things if you use a shorter wavelength or if you collect more data from your specimen by having a larger numerical aperture.

ΔR = 0.61 λ / N.A.

where ΔR is the resolution, λ is the wavelength of the radiation used, and N.A. is the numerical aperture defined as the sine of half the angle of the cone of radiation entering the lens.

Visible electromagnetic radiation spans the wavelength range from 390nm to 750nm. It is impractical to make lenses with a numerical aperture greater than 1.4 - thus the limit of resolution if there are no aberrations present in the lens is 170nm. There is a great deal of interesting cellular structures which are much smaller than this.

One way to increase the resolution is to decrease the wavelength of the radiation used - however the high absorbance that all materials have for shorter wavelength radiation makes the design of lenses similar to those used for visible radiation impossible. One solution to this problem is the zone plate which can be used to focus electromagnetic radiation but this has not been particularly successful. Another solution is to dispense with lenses altogether and use a computer to perform the function of a lens - this is done in the case of x-ray crystallography and has been the primary tool for the elucidation of macromolecular structure at atomic resolution. However it depends for its success on the existence of three-dimensionally ordered arrays of molecules which are sometimes difficult to make and seldom have any bearing on the natural state.

Another problem is the simple generation of the radiation of interest - a microscope that depends on \$10bn radiation source is unlikely to catch on!

The way forward is to use electrons. They have the following useful properties:

• Short wavelength given by the de Broglie wave equation
• Beams can readily be generated, and,
• because of their charge they can be focused by electromagnetic lenses which do not impose material in their path.

## Wavelength of electrons

The wavelength of an electron is given by the de Broglie equation:

$\large \lambda = \frac{h}{p}$eq. 1

Where h is Planck's constant and p the momentum of the electron. The electrons are accelerated in an electric potential U to the desired velocity:

$\large v = \sqrt{ \frac{2eU}{m_{0}} }$eq. 2

m0 is the mass of the electron, and e is the elementary charge. The electron wavelength is then given by:

$\large \lambda = \frac{h}{p} = \frac{h}{m_{0}v} = \frac{h}{ \sqrt{ 2m_{0 }eU } }$eq. 3

However, in an electron microscope, the accelerating potential is usually several thousand volts causing the electron to travel at an appreciable fraction of the speed of light. An SEM may typically operate at an accelerating potential of 10,000 volts (10 kV) giving an electron velocity approximately 20% of the speed of light, while a typical TEM can operate at 200 kV raising the electron velocity to 70% the speed of light. We therefore need to take relativistic effects into account. It can be shown that the electron wavelength is then modified according to:

$\large \lambda = \frac{h}{ \sqrt{ 2m_{0}eU } }\frac{1}{\sqrt{1 + \frac{eU}{2m_{0}c^{2}}}}$ eq. 4

c is the speed of light. We recognise the first term in this final expression as the non-relativistic expression derived above, while the last term is a relativistic correction factor. The wavelength of the electrons in a 10 kV SEM is then 12.3 x 10-12 m (12.3 pm) while in a 200 kV TEM the wavelength is 2.5 pm. In comparison the wavelength of X-rays usually used in X-ray diffraction is in the order of 100 pm (Cu kα: λ=154 pm)

Electrons can be generated using an electron gun. The kinetic energy they acquire is given by

$\large E = Vq$

Where V = the accelerating voltage and q = the charge on the electron Electromagnetic lenses are coils of wire wrapped on a soft iron form. The magnetic field generated is inhomogeneous and acts as a converging lens. The numerical apertures of such lenses have to be restricted to about 0.005 in order to produce images of reasonable quality. Combining the above two equations we can calculate the wavelength for electrons being accelerated by a particular voltage V, i.e:

$\large \lambda = \frac{hc}{q} \frac{1}{V}$

The value of the constants are such that for 60kV electrons the wavelength is 0.003nm. (Note however that the above treatment ignores relativistic effects and should not be used in actual wavelength calculations). Taking into account the low N.A. of an electromagnetic lens we can see that the resolution of an electron microscope is about 0.4nm. Actual resolutions achieved are in the 0.1-0.2nm range. The following diagram shows the spectrum of wavelengths highlighting those that are of interest in structural studies.

Note that the physical constraints governing the design of light, x-ray and electron microscopes are different and result in vastly different instruments.

## Electron microscopes

Electron microscopes are essential tools in the determination of the structures, of cells, organelles, macromolecular complexes and large macromolecules. Transmission electron microscopes are similar in design philosophy to light microscopes, as shown in the diagram below:

The electron gun is analogous to the light source and the lenses are arranged in three groups

• A condenser to control the quality of the illumination.
• An objective lens to produce the initial magnified image of the object
• Projector lenses analogous to the eyepiece to produce further magnification.

A difference is that in the case of the light microscope most of the magnification is produced by the objective lens, whereas the objective lenses of electron microscopes are designed to have low magnifications with most of the magnification occurring in the projector lenses. It should also be obvious that projection of the final image onto the eye is not an option in the case of the electron microscope - the image is realised either by film or by projection onto a fluorescent screen.

An alternative design of microscope is possible in which a finely focussed beam of electrons or light is scanned across the specimen, exciting a signal which is then detected electronically and then formed on a display monitor. Instruments of this design were first realised using electrons resulting in the scanning electron microscope and later in the confocal scanning light microscope.

## Constraints on the use of electrons

The properties of electrons impose severe constraints on the design of instruments and the way in which the sample must be prepared. Electrons are charged and interact strongly with matter - this must be considered.

The absorbance of a material for electrons depends on its density. Moderate energy (60-200keV) electrons have a path length of a few mm in air at STP. Electron optical systems require a path of the order of a metre and thus the path must be evacuated. A key component of any electron microscope is thus the vacuum system. The need to maintain a vacuum also impinges on the sample which should not be volatile or change significantly when it enters the vacuum. This constraint excludes wet specimens.

The high absorbance of most materials for electrons also restricts their thickness. The practical limit is less than 500nm for 200kV electrons.

The charge on electrons also imposes constraints. If electrons are allowed to accumulate in the specimen it will acquire a charge and repel the electrons in the beam. Thus arrangements must be made to conduct electrons that remain in the specimen to earth.

Electrons in the beam have considerable kinetic energy. If this energy is transferred to the specimen the temperature of the specimen will rise, potentially resulting in structural changes. Arrangements must be made to conduct away this heat.

## Interaction with the specimen

It is perhaps obvious that in order to obtain meaningful information from a specimen that the incoming radiation must be modified in some way. The relative brightness of any point in the image thus depends on the differential interaction of the radiation with the corresponding point in the object.

In the case of light the processes used to produce differential contrast are absorbance, fluorescence and phase change.

Specimens can be stained with light absorbing dyes which either react differentially with the various tissue components producing regions that differ in colour and intensity.

Specimens can also be visualised as a result of their intrinsic fluorescence, or as a result of the introduction of a dye that can easily be excited to fluoresce.

Fluorescent stains can also be attached to antibodies which can be used to precisely locate cellular components. (You can access a site which displays a number of beautiful fluorescent images).

When light waves pass through transparent objects of different refractive index and re-emerge their phase is changed relative to an unperturbed beam. Frits Zernike (1888-1966) discovered a simple way to convert these changes in phase to changes in amplitude - i.e. to render transparent objects visible - called phase contrast. You can find out how it works by reading his Nobel lecture on the Nobel site. The image below shows a comparison of a bright field and a phase contrast micrograph.

Phase contrast microscopy is a simple method for biologists to assess a specimen without having to stain it.

There are two other important ways of visualising transparent objects, Nomarski interference contrast and Hoffmann modulation contrast.

Specimens can be stained with light absorbing dyes which either react differentially with the various tissue components producing regions that differ in colour and intensity.

Metaphase chromosomes stained with acridine orange – a dye which emits a green fluorescence when it binds to DNA.

Specimens can also be visualized as a result of their intrinsic fluorescence, or as a result of the introduction of a dye that can easily be excited to fluoresce.

This image shows a comparison of a bright field and a phase contrast micrograph.

Fluorescent stains can also be attached to antibodies.