The Focus on Microscopy

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By Richard Brown

In literature, TV and movies, the popularity of forensic science is driven by depictions of the heroic scientist/investigator who catches the bad guys by analysing dust and microscopic particles left by perpetrators at crime scenes. Whether it is literature’s greatest detective, Sherlock Holmes, or TV’s crime-solving crew on CSI, the microscope-wielding scientist as “hero” has come to represent the general public’s idea of the field of forensic science. However, many overlook the primary methods that serve as the very backbone of the field: microscopy.

Microscopy

Microscopy is the application of the microscope to research and study objects. While microscopy plays a significant role in the high-profile world of crime-related forensic science, less glamorous is the job of the general microscopist, who focuses on the tiny details of everyday challenges. These challenges range from the quality of consumer goods, to the performance of mechanical devices, or the isolation of contaminants in the environment. Regardless of the challenge, the general microscopist relies on the same techniques used by the forensic scientist to investigate real-world problems.

The Microscope

The microscope is the one analytical instrument that has been the workhorse since the beginning of man’s interest in the world of dust and living things too small to see with the unaided eye. Although it is the primary tool of scientists who study dust, debris and the microscopic world of living and non-living things, the microscope is a source of wonder and frustration. Since its invention in the sixteenth century, the microscope has evolved from many forms. What Henry Baker said about the microscope in his address to the Royal Society of London on October 28, 1742, “ . . . Many have been frighted from the use of it, by imagining it required great skill in optics, and abundance of other learning . . . whereas nothing is really needful but good glasses, good eyes, a little practice and a common understanding to distinguish what is seen; and a love of truth to give a faithful account thereof . . . ” is still true today, as scientists tend to shy away from their own skills of observation and interpretation and lean on the analytical instrument or black box solution, falsely believing them to be faster or more reliable.

For modern particle “detectives,” the microscope is an invaluable tool. Investigations into the identity of a dust or small particle contaminating a drug solution or a fuel injector armature require a steady hand and a highly skilled scientific investigator. Not as glamorous as their television contemporaries, they are every bit as in demand and prized by the largest manufacturing companies in the world, for microscopists are the interpreters of the microscopic world. Often the amount of material present in a few small crystals or particles found on a clean surface is insufficient to dilute and shoot into a gas chromatograph-mass spectrometer or other analytical instrument. This approach is especially unwise when the morphology or shape of the particle is an important clue to its identity and its source.

The Microscope Applied

Different microscopes yield different information. Polarized light microscopy (PLM) can identify a particle based on the particle’s optical characteristics. An experienced PLM analyst can readily identify a white powder as the particles observed would display characteristics that may be unique to a particular compound. Ground glass and fine quartz sand may appear similar to the naked eye. Placing a small pinch of each material on a glass slide with a liquid of known refractive index covered with a cover slip enables the PLM analyst to observe one powder to be non-crystalline and the other to be crystalline. This observation can be made, including time to prepare the samples, in less than two minutes. If the question was, “Which of these is most likely quartz sand?” then the microscopist is done, as only one powder exhibits characteristics consistent with quartz sand: the crystalline white powder. If the question was, “What are these materials?” then the microscopist has more work to do. Another half hour and the optical characteristics for the typical or common particles comprising each powder can be determined and documented with a photomicrograph. If particle size is important, a dispersion of particles from each sample can be prepared and individual particles can be measured by automated scanning electron microscopy-energy dispersive X-ray spectrometry (SEM-EDS). Elemental data can also be acquired for each particle if necessary.

This logical progression from one microscope to the next is the way particles of dust and debris of unknown origin are classified and identified in the modern microanalytical laboratory. Microscopes offering different, new and creative ways to analyse and examine objects are produced as research and industry push for more information at the edge of the envelope.

Using a combination of polarized light microscopy (PLM), Fourier transform infrared microspectroscopy (FTIR) and SEM-EDS, the microscopist can rapidly classify particles to provide information that can settle patent disputes, contamination problems, materials failures and customer returns for all types of industries. Microscopy is the only way to examine and identify small particles on, in and around finished products. Many times an exact identification is unnecessary, but classifying the particle to eliminate other potential sources is sufficient. One such case arose when a customer-service-oriented manufacturing firm required the examination of metallic and plastic debris that would occasionally become incorporated into its finished automotive product. From a customer return standpoint, it was necessary to have an outside laboratory perform the analysis and determine if the particles were from the manufacturing process or from misuse by the end user.

The customer submitted samples of as many materials used in the handling, packaging and manufacturing of the product that could be obtained. Using a combination of PLM, FTIR and SEM-EDS, each sample was characterized and stored in a small database to serve as a reference library for unknown particles that were occasionally discovered in their finished product. The sample database was kept up-to-date by new sample submissions whenever a product or process change was implemented. The initial cost of the database was a few thousand dollars. This relatively small investment paid off by having particle analyses that took hours instead of days. Most of the customer returns were initially traced to the manufacturer. This finding enabled the manufacturer to trace the point of contamination and implement procedures that would correct the problem. An added benefit of the database was an increase in production quality resulting in fewer customer returns and higher morale among the manufacturing staff.

Material Identification

Most people can distinguish an apple from an orange. The average person believes what he sees when his observations and conclusions are reinforced through his own experience and through the observations of others. Eventually we are confident in our ability to differentiate between an apple and an orange. However, not many people could prove that this is an apple or that this is an orange. Fewer still could determine the species of apple or orange. The expertise required increases as the need for more and more information about an object increases. To the experienced microscopist, differentiating between calcium carbonate and quartz is as easy as apples and oranges. How much study a particle requires depends upon any given situation. Clues are provided to the investigator about the manufacturing process or other situation that lead to the discovery of the particle. The microscopist must then use all the available pieces of the puzzle to determine the source and the identity of the particle

Identification vs. Classification

A fingerprint and, in some cases, a sufficient number of DNA fragments are determined to be identical to the suspect and could not have come from any other person. This level of individualization is considered identity. A blue nylon fibre from the victim’s sweater is determined to be identical to the blue nylon fibres comprising the carpet in the suspect’s van. This, too, would be strong evidence, if not for the added information that this is probably the most common carpet used in the automotive industry. Or, still strong evidence if the carpet was only produced for a few months and was put in only a limited number of vehicles. But a carpet fibre, a hair or a particle of paint can provide characteristics that belong to a class or group of materials that are not unique from one another. The microscopist, after examining the unknown particle, must put into proper context the probative value of his or her findings as they relate to the situation.

Will the need for the experienced microscopist ever disappear? As long as products and materials weather, decompose and continue to revert to smaller and smaller particles, there will be a need for the unique skills of the microscopist. The really fascinating part of this story is that manufacturing processes are creating increasingly smaller products; micromachines, microprocessors, nanopigments and increasingly smaller particles with the potential to cause increasingly larger and more catastrophic failures, relatively speaking. The microscopist and the microscope will most certainly be around for another 400 years!

A former CSI or criminalist, Richard Brown has over 15 years of experience in light and electron microscopy. He has been published in more than 30 publications and has made numerous presentations on topics including forensic analyses, digital imaging and microscopical characterization of environmental and industrial samples. Currently he is an executive director at Atlanta, Ga.-based MVA Inc., a multi-disciplined scientific consultancy and laboratory specializing in the classification of small particles by light and electron microscopy.