The field of forensic microscopy is more than a century old. Though practitioners say their profession is far less glamorous than its portrayal in popular TV crime and courtroom shows, forensic investigation remains a powerful tool for learning the truth about who may have committed various actions, where they occurred, and what tools or substances may have been used. Because almost all trace evidence is reviewed under a microscope at some point, techniques such as stereomicroscopy, polarized light microscopy and scanning electron microscopy (SEM) have become essential tools of bioforensic investigation. “But forensics is different from most other kinds of microscopy,” said Christopher Higgins, applications manager for digital slide scanning systems and analysis at the Olympus Scientific Solutions Group in Waltham, Mass. “Most people using a microscope start with a well-defined idea of what they are imaging. But with forensics, the user starts with a mystery: There’s a victim and we don’t know how he or she died, or there’s a powder and we don’t know what it is. Forensic microscopists start from the beginning to try to determine what it is they are looking at, and based on that, they move on to try to figure out what may have happened.” Most forensic microscopy laboratories have noted a marked uptick in cases over the past few years, largely driven by explosive growth in homeland security and drug cases (Figure 1). Figure 1. Foreign material in human skin at IV drug abuse injection site, viewed under polarized light. Tissue images often are captured during a medical examiner’s investigation of a death. Courtesy of Marianne Hamel and Nikki Johnson. Biological specimens are particularly complex to image and analyze. The heroin and prescription opioid epidemic is straining the time and budget constraints of many offices, and there are only about 400 practicing forensic pathologists in the United States, said Dr. Marianne Hamel, one such professional based in Pennsylvania and New Jersey. But backlogs can be deadly if a terrorist or active criminal remains at large, so there is continuous pressure for fast turnaround of forensic evidence. Forensic pathology changed dramatically in the mid-1980s when DNA test results were introduced in courtrooms. Today, DNA identification is used in a wide variety of applications, from paternity and immigration cases to criminal investigation. The ability to match evidence to a particular individual by comparing DNA samples has led to much higher conviction rates. But DNA evidence is far from indisputable. Evidence can be called into question because of contamination, lack of scientific scrutiny or failure to use proper procedures. Few crimes are better suited to DNA investigation than sexual assault cases, where perpetrators may leave behind bodily fluids, along with skin and hair, on their victims’ bodies. But while forensic examinations using so-called rape kits have become both more humane and more prevalent over the years, the kits themselves often remain unexamined. One article in 2014 estimated that more than 400,000 untested sexual assault kits were sitting on storage shelves at police warehouses and in laboratory facilities across the U.S. As a result of public pressure, many crime labs now have mandates that require them to process sexual assault kits faster and more efficiently, but the challenges are immense. The DNA of the perpetrator or perpetrators and that of the victim are often mixed in bodily fluids. Sperm are often the clearest evidence of a perpetrator’s identity, and accurate DNA analysis of sperm requires a certain number of cells to be present. To complicate matters, an individual sample from the kit may have a low concentration of sperm. Independent Forensics, based in Lombard, Ill., is an ISO17025-accredited DNA lab that manufactures Sperm Hy-liter, a specialized immunofluorescence kit for human sperm identification. “Historically, laboratories have only had simple histological staining and basic brightfield options for finding sperm,” said Dina Mattes, the company’s technical sales director. “Now, using fluorescence imaging, several new options are available to these labs.” The options include basic fluorescence microscopes, new slide scanners, stereomicroscopes with fluorescence, and complete systems for locating, dissecting and isolating sperm to be sent for DNA analysis. Some of the most exciting developments in collecting DNA from a sample come from cancer research, where laser microdissection was developed to cut out cancerous cells for polymerase chain reaction (PCR) or similar genetic analysis. Full-scale forensic microdissection systems have recently been developed that scan multiple evidence slides, detect the fluorescing sperm, dissect the sperm out of the samples and catapult them directly into PCR tubes for DNA analysis (Figure 2). Figure 2. Sperm cells (outlined in green) and buccal (cheek) cells (outlined in red) as automatically detected in a cell mixture. Image captured using Sperm Hy-liter from Independent Forensics, Lombard, Ill., and the Zeiss PALM MicroBeam laser microdissection system. Courtesy of Carl Zeiss Microscopy GmbH. Such systems often employ a combination of reflected and transmitted light illumination, using Alexa 488 or fluorescein isothiocyanate (FITC) to help image sperm heads along with 4′,6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI) to help differentiate sperm from among the other nonspecific cell nuclei present in the sample. Phase contrast imaging also can be used to help differentiate sperm from other types of cells; using several optical techniques simultaneously can improve the ability to locate the sperm cells even among heavy cell debris. “Systems like this are designed for larger forensic institutes that handle a lot of sexual assault cases,” said Michael Gögler, market sector manager of the Microscopy Business Group at Zeiss in Germany. “They increase reliability and throughput and provide full DNA profiles from just a few dozen sperm cells.” For laboratories that prefer a more manual process, there are other options. For instance, stereo zoom microscopes can be equipped with fluorescence and a motorized micromanipulator with tweezers to hold a sphere that picks up the sperm. The user then directs the sphere to the PCR tube. Another trend in the field is increased scrutiny of methods and findings. After several studies a decade ago called into question the accuracy of hair, bite mark, footprint and other forensic evidence, the National Academy of Sciences issued a scathing 2009 report that called for an overhaul of forensic laboratories and the implementation of stricter and more scientific standards. As a result, increasing rigor is being applied to every aspect of the field, including forensic biology. Comparing images with other images can help enhance the accuracy of findings, and tissue banking can help in the creation of a storehouse of images readily available for comparison. “Digital imaging is one of the greatest areas of change in forensic microscopy because we are now able to put past images into a database and use that to do image recognition and analysis of future samples,” said Chris Higgins of Olympus. “We do this already in cancer diagnostics with digitized reference books; standardization is moving into forensics and clinical diagnostics as well.” At least one state is now considering providing funding to allow coroners in urban areas to document forensic pathology specimens digitally and share image data among regional medical examiners’ offices. Not only would this help standardize procedures, it also would make it easier to cross-check evidence across local regions, in cases where a perpetrator may have offended again in a nearby locale. The proposed system also would provide images for presentation in court to help bolster a jury’s understanding of how an individual pathologist came to a particular finding. The FBI also keeps a growing database to supplement local ones. Virtual microscopy is a related area of interest (Figure 3). The Olympus VS120 virtual slide system, for instance, creates high-resolution brightfield or fluorescence digital images of complete forensic specimens that can be stored on a server for simultaneous remote viewing at a range of magnifications. Multiple high-resolution specimen images are carefully stitched, enabling users to switch between micro and macro observation for swift viewing of regions of interest while referencing the overall structure. Figure 3. Birefringent plastic that disintegrated in a knee replacement is shown on the monitor of the Olympus VS120 virtual microscopy system, which allows imaging of entire tissues or organs via the careful stitching of multiple high-resolution images. Both macro and micro areas can be called up for viewing as needed. Courtesy of Olympus Corporation of the Americas, Scientific Solutions Group. Training and education are another way in which digital image management can help improve forensic accuracy and quality. “People in the field are starting to take more control of their professional development. They want their findings to be rock solid,” said Charles Zona, Dean of Students at Hooke College of Applied Sciences, a division of the McCrone Group, Westmont, Ill., one of the nation’s preeminent institutions dedicated to forensic science. “There is growing interest in certifications and specializations for various aspects of microscopy.” The McCrone Group offers courses in specialized microscopy for forensics. In addition, the McCrone Atlas of Microscopic Particles, now celebrating its 50th anniversary as a reference tool, contains more than 1100 images and is fully available online at no charge. Professional microscopists and trainees use the Atlas to help in materials characterization and particle identification, sharpening their skills by comparing their findings with top-quality images. While the move to digital technologies offers many advantages, it has challenges as well. Title 21 of the Code of Federal Regulations, Part 11, governs everything that has to do with electronic records about people, including digital case files. Compliance procedures can be complex and are still developing. Many of the intricacies of forensic digital imaging involve security issues regarding chain of custody, how many people have access to a particular file, and what changes have been made to the data — by whom, when and why. Pathology is not the only area of forensics that has changed. Many other types of microscopes and imaging systems are now used in forensic investigation. In addition to traditional stereo, brightfield, polarized light and SEM imaging, specialized facilities now use polarized Fourier transform infrared difference spectroscopy, Raman spectroscopy, energy dispersive x-ray spectroscopy, confocal imaging, atomic force microscopy and transmission electron microscopy where warranted. Each state now has at least one mobile vehicle with a well-equipped forensic microscopy laboratory to help in cases of urgent need, such as when a mysterious powder is detected or other weapon of mass destruction is suspected. Fortunately, most such cases are false alarms. As the technology grows more complex and the cost of outfitting laboratories to do the latest biological forensic analysis methods increases, some facilities are becoming specialized. But almost all forensic laboratories, regardless of their setting or parent organization, are seeing growth, as they answer demands to produce more scientifically significant results in less time. New optical techniques, along with emerging capabilities in software and data management, should help forensic scientists answer critical questions and provide better and faster identifications in a wide host of situations. Meet the author Ilene Semiatin has three decades of experience writing about and creating marketing programs for the life sciences, industrial optics and microscopy fields. She holds a bachelor of science degree from Boston University; email: ilene@edge-comm.net.