William Illsey Atkinson

Infrared microspectroscopy proves to be a powerful detection tool.

A murderer is identified when a shirt he had thought was totally destroyed yields subtle proof it belonged to his victim. A policeman survives a gunshot, and irrefutable evidence sends a suspect with an otherwise ironclad alibi to jail. A cocaine abuser is caught after a few nanograms of the illegal substance turn up in his office.

The resolution of these recent cases fell to a new technique called infrared microspectroscopy (IMS), and the evidence is overwhelming that it’s the hottest thing in infrared (IR) spectroscopy since fast Fourier transform-IR (FFT-IR) spectroscopy. IMS is rapidly revolutionizing IR spectroscopy for the analysis of both organic and inorganic samples.

See box, How Does IMS work?

The Case Is Made
What makes the new technique hot is its combination, in one instrument, of two centuries-old techniques: spectroscopy and microscopy. The microscope uses visible light; the spectroscope uses IR.

Initial inspections are morphological as IMS users act as classic microscopists, locating interesting structures and examining their physical relationships. But then, at the click of a button, IMS users can launch a broad-spectrum IR analysis of whatever has caught their attention, over a spot as narrow as 5 µm.

The real power of IMS, then, lies in this fusion of two techniques that had previously been independently applied by completely separate disciplines. In biology, the new technique has recently derived IR spectra for living cells in the process of mitosis and for individual structures within cells—findings unattainable a decade ago.

Table 1. IMS Instrument Manufacturers and Suppliers
SensIR Technologies	www.sensir.com
PerkinElmer Inc.	www.instruments.perkinelmer.com
Spectra-Tech Inc.	www.spectra-tech.com
ChemIcon Inc.	www.chemimage.com
S.E.E. Incorporated	www.see-incorp.com
Nicolet Instrument Corp.	www.nicolet.com
Mattson Instruments	www.mattsonir.com
Bruker Optics	www.bruker.com/optics
(Web sites were accessed on October 14, 2000.)

Strangely, news of the technique’s capabilities has been slow to disseminate. Recently, for example, a Yale neurophysiologist contacted an IMS technical expert to enquire about obtaining IR spectrographic data for brain tissue. When questioned about his method of sample preparation, the neurophysiologist said he used a standard kitchen blender and, as the only spectra he could obtain were for the whole brain, he prepared his sample by homogenizing the brain tissue into a puree. He was astonished to learn that reflection, transmission, and grazing-incidence IR spectra could be generated, often with minimal sample preparation, not only for individual brain cells, but even for subcellular organelles. He now prepares his samples with a microtome instead of a blender.

Another case of serendipity led John Reffner, technical director for SensIR Technologies (Danbury, CT), one of a handful of IMS instrument manufacturers (see table 1), to an IR source 1000 times greater than the most intense source he had previously found. A mutual friend suggested that Reffner contact a physicist at the National Synchrotron Light Source at Brookhaven National Laboratory (Upton, NY). At Brookhaven, powerful electromagnets accelerate electrons to relativistic velocities, then deflect them. In changing course, the electrons emit an intense, broad-spectrum photon burst, from X-rays through near- and far-IR and down to radio frequency. This pulse emerges from a window so small that it is effectively a point source.

IMS using synchrotron radiation enjoys a high signal-to-noise ratio as its IR spectra are extraordinarily clear. But wouldn’t such an intense IR pulse barbecue an organic sample? When asked, Reffner quickly stated that he has never recorded sample damage from a synchrotron’s IR beam line.

“The area to which the energy is applied is small, 25 µm² or so, and the radiation is not coherent,” says Reffner. “A synchrotron is not a laser, and its IR is perfectly safe for nondestructive investigation of organic material, even living tissue. All it does is make molecules vibrate in a revealing way.”

New synchrotron light sources are currently being built all over the world—funding was recently announced for a facility in Saskatchewan—and up to half of the IR beam lines at these installations will be dedicated to IMS investigations. IMS seems poised to eclipse alternative techniques of IR investigation, relegating even standard workhorses using diffraction gratings, such as FFT-IR spectroscopy, to marginal use. Although technology is always changing and it is impossible to forecast what new techniques may emerge, it seems a safe bet that IMS will dominate IR-spectrographic investigations for the next 5 or 10 years. For example: at a 1999 conference in San Antonio, TX, on the use of IR spectroscopy in criminal forensics, a scientist who trains FBI operatives in forensic spectroscopy announced that “IMS has already established itself as a required technique for every forensics laboratory.”

Like pulling ink off paper, IMS pulls an ink spectrum from a business card. (Courtesy of SensIR Technologies.)

Indisputably, the discipline that’s driving the development of IMS most intensely these days is forensics, both criminal and noncriminal. Noncriminal forensics includes sourcing andcharacterizing trace contaminants when tracking down problems in industrial products. In one case, IMS showed that cellulose blotches deposited at a precise location in the manufacturing line caused the malfunction of a run of computer hard disks. In another case, the technique showed that unintended variations in coating thickness were responsible for ruining a batch of silicon microchips that all other testing methods showed to be perfect.

For pharmaceuticals, forensic IMS has the ability, unavailable with standard techniques such as polarized-light microscopy or X-ray diffraction, to deduce changes in a drug’s molecular chemistry as its temperature changes. More controversially, IMS can facilitate reverse engineering, permitting firms to duplicate a competitor’s product.

The Witnesses Are Called
It is in criminal forensics, however, that IMS has so far had its most stunning successes.

“In the first forensics case on which I was consulted, a young lady was abducted and murdered; her body was found floating in a lake,” recalls Reffner. “She had disappeared while on her way to her job, where she wore a company T-shirt. Shortly after her disappearance, the suspect in the case was observed tending a fire under a nearby bridge. A small swatch of partially burned cloth was retrieved from the fire site. Its knit pattern and color matched that of the company shirt, and FBI Forensics proved that the sample contained cotton. But the FBI could not confirm the presence of polyester, which the shirt yarn also contained. Although the fire had melted the threads, I was able to confirm the presence of polyester using IMS. The data proved crucial in obtaining a conviction.”

In another case involving IMS, a policeman in Maine had been shot, but luckily, he was wearing a bullet-resistant Kevlar vest and was not killed. A spent bullet was recovered from the scene, and it matched a suspect’s gun. However, the suspect maintained that he often went shooting in the area. Using the bullet itself as a reflective substrate, IMS revealed a thin layer of Kevlar on its nose. The bullet was in effect a sample, self-prepared when it hit the vest. Another conviction.

A third case shows IMS’s ability to illuminate problems opaque to other methods. Colleagues accused a businessman of cocaine abuse. A glass plate was recovered from his office and closely inspected. Although it had been scrupulously cleaned of all contaminants, microscopic examination showed the presence of some shallow scratches. Sufficient material was obtained—a few tens of nanograms—for IMS to demonstrate conclusively the existence of cocaine.

A final case reflects less well on the law. A man who had been arrested later complained of excessive force during his arrest. IMS analysis of a smudge on his pants matched the IR spectrum of shoe polish on the arresting officer’s boots. A black eye for the justice system; a gold star for IMS.

Criminal forensics may have human interest, but the most lasting and revolutionary changes from IMS will probably originate in fields that have so far exhibited little interest in IR spectroscopy. Reffner thinks the best way to facilitate this change is not to create a new cadre of IMS specialists but to educate scientists from all disciplines in IMS techniques. “It’s not enough to train new experts,” he says. “IMS must be taught as a new skill to individuals that already have their own specialization.”

“Consider the pathologist reviewing cells from a Pap smear. His trained eye is needed to select those cells that appear to be cancerous or precancerous. Then, using IMS, he can immediately derive IR spectra for those cells to support his informed opinion,” Reffner continues. “I can’t do that. Only working scientists will know where to apply IMS.”

The Defense Is Made

The small size of SensIR Technologies’ Travel IR system allows IMS measurement to be performed immediately at the scene of a crime. (Courtesy of SensIR Technologies.)

Of course, no technique is perfect, and IMS has its drawbacks. One of these is cost. As Reffner admits, “IMS can be expensive if you want to do everything.” That’s no problem for industrial testing facilities, and a surmountable barrier for public forensic labs. But it can delay or prohibit acquisition by cash-strapped university undergraduate programs, which are the best places for young scientists to pick up IMS skills. Other drawbacks are the size and weight of a top-line IMS instrument, and the usual complaint that “good enough”never is. Says Reffner: “If you get spectra from an area 10 µm across, someone will want to get it down to 1 µm.” The industry is already working on these problems, although IMS technology may be close to the limits of physical optics.

Instead, the big news in IMS instrumentation may be a new generation of smaller, more portable units with easier sample prep. These may couple spectroscopic and microscopic functions with video technology. Such portable instruments would be ideal for quick and accurate characterization of unknown organic chemicals in a hazardous waste spill. Coupled with an onboard digital library of IR spectra, the new IMS instruments could provide fast answers to questions involving flammability, toxicity, and disposal. With portable IMS, fire fighters would know immediately whether they were dealing with something they could simply flush away with a fire hose.

As for sample prep, for many routine IR analyses that may soon be a thing of the past. SensIR has recently developed a diamond sensor that can be put into direct contact with an unknown substance. Diamond has a high index of refraction, is transparent to IR wavelengths, and is the hardest and most inert substance known. These properties make it an ideal material for direct-contact sampling.

In summary, the big plus for IMS is its ability to reveal new facts by relating morphology and chemistry, letting scientists examine form and function in very small regions. Look for robust growth in this new technology—and a lot more surprises.

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William Illsey Atkinson is a Seattle-born science writer who lives in Vancouver, BC. Prototype, his nonfiction book on current technology, will be released in 2001 by Thomas Allen Publishers (Toronto). Comments and questions for the author may be addressed to the Editorial Office by e-mail at tcaw@acs.org, by fax at 202-776-8166 or by post at 1155 16th Street, NW; Washington, DC 20036.