Electron microscopy - a perspective

From magnifying at just 10x in the 1960s, electron microscopy is now a valuable tool in the art of forensics

If I were to tell you that the first electron microscope was hailed a breakthrough in 1931 when it imaged copper and gold surfaces at a magnification of 10x, you could be forgiven for wondering what the excitement was about.  After all, light microscopes were imaging at 1,000x.  Also, it had been developed in Germany by Max Knoll and Ernst Ruska adhering strictly to what was, even then, a design classic – the transmission light microscope.  It merely substituted a focused beam of electrons rather than photons to "see through" the specimen.

To understand the fuss that the crude image made, we need a little understanding of physics.  Electron microscopes were developed due to the limitations imposed upon light microscopes by the physics of light.  In the 1870’s, Ernst Abbe laid down the scientific basis for the design and manufacture of microscopes.  By the late 1920’s light microscopes had reached their theoretical limits of 1,000x magnification and 200nm resolution, a limit imposed by the wavelength of light itself.  Using electrons dissolved this barrier and set scientists on their way to magnifications measured in millions.

Knoll and Ruska acted on the scientific desire to visualise the fine details of sub-cellular structures, like the nucleus and mitochondria.  Their achievement founded a discipline, and an industry, and within 5 years the first commercial TEM (Transmission Electron Microscope) was manufactured in the UK.  Now, just 70 years later, atomic scale resolution better than 0.1nm is achievable.

A decade after Knoll and Ruska, an electron equivalent of the reflecting light microscope was developed in the UK.  Ridiculed in some scientific quarters, Professor Oatley and his team of PhD students persisted with their research and gave the world the Scanning Electron Microscope or SEM.  Since commercialisation in 1965 it has forged a place within a wide range of disciplines, including materials science, semiconductors, forensics, archaeology, food, biology, and pharmaceuticals.

The RMS (Royal Microscopical Society) has always placed great emphasis on the teaching and sharing of information.  To this end, we run a number of EM courses every year, including our week -long EM School, and have a specialist EM section of the Society.  Here, Dr Jeremy Skepper and Dr Debbie Stokes share some of the interesting experiences and applications in which they have routinely used electron microscopes.

Working in a Vacuum
Which none of us do, of course!  Although we all interact in however small a way with fellow scientists or workers, the world of high vacuum was a familiar one for electron microscopists for most of the past 70 years.

Effective electron transmission and collection depended in the first electron microscopes on the absence of molecules within the ‘lightpath’ of the instruments.  Just like a TV or PC monitor, whose Cathode Ray Tubes were evacuated to enable the electrons to pass unhindered to the front screen, so electron microscopes required an evacuated chamber so that they could be directed at the sample. 

This is changing and many scanning microscopes now ‘leak’, allowing small amounts of gas and water vapour into the chamber.  The laws of physics can’t be avoided and the molecules that are introduced do interfere with the passage of the electrons.  However, advances in electron column design and detector technology allow us to work around the variances that are introduced and use them to good advantage. 

The effect is to allow the observation of uncoated non-conducting materials, such as polymers and ceramics, and moist and biological samples.  We can study plants and animals, food and pharmaceuticals.  We can even watch paint dry – it’s more exciting than you think!

The Art of Forensics
Electron microscopy has proven to be an invaluable tool in forensic analysis over many years.  Perhaps the most familiar aspect of this application is in ballistics, where gunshot residue analysis and bullet marking comparison has been made more familiar through crime novels and TV programmes.  These two areas are primarily the domain of SEM.

For instance, it is common knowledge that bullets fired from the same gun bear characteristic markings.  No two guns have the same ballistic fingerprint, and microscopic comparisons of bullet markings can help to determine if they were fired by a particular weapon.  The examinations tend to fall into two areas: marks picked up from the action of the firing pin on the cartridge case and marks on the bullet imposed as it is propelled through the gun barrel.  Both leave distinctive markings that under SEM analysis can help determine whether a particular bullet was fired from a particular firearm.

Less well known is the fact that, when a gun is fired, tiny shards of metal and charge residue are scattered in the crime area.  These may also land on the shooters hand and clothing and may be collected on swabs.  If these were detected in a sample from the hand or clothing of a suspect, and could be matched to similar material collected from the crime scene, it would be direct evidence of the suspect’s presence and involvement at the scene of the shooting.

The SEM is the forensic scientist’s weapon of choice in this detective story since, when the instrument’s electron beam is scanned across a sample's surface, it interacts with atoms in the sample to produce x-ray emissions.  The emitted x-ray has an energy characteristic of the parent element and detection and measurement of the energy permits elemental analysis (Energy Dispersive x-ray Spectroscopy or EDS).  EDS can provide rapid qualitative or quantitative analysis of elemental composition to a sampling depth of 1-2 microns.
In the case of our gunshot residue, the exact elemental composition of those from the suspect and the scene may be determined.  A significant match and the link is made.

These two facets of SEM, detailed imaging combined with elemental analysis, are also valuable in the examination of paint particles, metal residues, fibres or forged documents.  For example, one case involved suspicions about a man who took a large amount of cash into a bank.  Suspected of having come from a bank robbery, the bank notes were examined by SEM where it was found that they had tiny burn holes.  Investigation of the holes and elemental analysis of the surrounding paper showed that metal residues, identical to the metal from which the safe was constructed, had been deposited around each hole.  The courts accepted that the holes had been created by metal sparks from the bank robbers welding torch as they burnt their way into the safe.

It’s not every case that has overtones of ‘The Bill’.  The use of SEM was investigated in a compensation case where a woman considered suing a foundry company after her husband died of lung cancer.  She believed that her husband, a lifelong non-smoker, had been made ill due to his working conditions around airborne heavy metals.  Post-mortem evidence was provided from wax embedded lung tissue, which showed under SEM elemental analysis that metal traces could be observed that corresponded to the ‘x-ray fingerprint’ of one of the heavy metals used in the factory.  Unfortunately in this case the aggressive environment in the human body had changed the nature of the particles such that a precise match was impossible and the case was withheld.

Finally, SEM and EDS played a positive role in one tragic case involving a young child found drowned in a pond.  The father accused the mother of drowning the child in a bathtub and then moving the body to the pond in an attempt to cover up her actions.  Examination of lung tissue found traces of glass and metal, leading to an early assumption that the father’s claim might be true.  However, further SEM investigation found particles in the lung that matched those taken from the base of the pond, including minerals and diatoms that could not have come from any bathtub (Figures 1 and 2).                   

Frtagments of diatoma in lung (figure 1. left) and highpower (figure 2. right)                                                       

These real-life examples show how crucial electron microscopy can be in the hands of experts in determining people’s lives and in the world of justice. However, forensic techniques can be applied to outside the courtroom.

The Forensics of Art
When a 14th century medieval manuscript was acquired by the Fitzwilliam Museum in Cambridge, the museum wanted to understand and identify the pigment source for the paints.  Of course, that’s not a problem if you don’t mind destroying all or part of the sample.  However, having just spent £1.7 million to acquire the manuscript, the museum was understandably reluctant to treat it this way.  The answer was to place the entire manuscript into a ‘leaky’ SEM and perform x-ray microanalysis to determine the elemental composition of the paints.  The manuscript was not damaged and the museum had its analysis.

In a related experience, samples from 4,000 year old cave paintings from South Africa were analysed in a similar way by investigators to determine the composition of the paint and, therefore, the origins of the pigments.  Once again, SEM elemental analysis can non-destructively gain this information from the sample and show how the different elements are distributed within the microscopic paint particles.

No longer the preserve of a dedicated EM department, the SEM in particular has been refined and developed to become a flexible, simple-to-operate analytical instrument. There is in no doubt that the refinement of the electron microscope’s abilities will continue to add new application areas to its swelling portfolio.  For instance, the so-called ‘Environmental’ or variable pressure SEMs are beginning to make a significant contribution to the imaging of biological samples (Figures 3 and 4).  Furthermore, the combination of electron and ion beams is transforming the study of a wide range of materials and even allowing the fabrication of tiny structures crucial to the development of nanotechnology.

Figure 3 (left). Dividing diatoms. Figure 4 (right) Jack-in-the-pulpit pollen

One thing is for sure.  The RMS and its membership will continue to be at the forefront of training these new users as the Electron Microscope remains so important.

by Rob Flavin, The Royal Microscopical Society