Advancing Forensic Analyses with Raman Spectroscopy

Sep 14, 2017
By Spectroscopy Editors

In recent years, there have been significant advances in the application of vibrational spectroscopy to the analysis of forensic samples. Igor K. Lednev, a professor in the Department of Chemistry at the University at Albany, the State University of New York, has been developing the use of Raman spectroscopy for a variety of forensic applications, including determining the age of blood stains and linking gunshot residues to specific ammunition–firearm combinations. He recently spoke to Spectroscopy about his work. 

What have been the most important recent developments in vibrational spectroscopy, and Raman spectroscopy specifically, as they relate to forensic science (1)?

Vibrational spectroscopy techniques in general and Raman spectroscopy in particular have a lot to offer practical forensics because of their nondestructive nature and because they can be used for rapid, quantitative, confirmatory, and in-field analysis. As we described in our critical review article (1), the most recent developments include a universal, nondestructive, rapid method for detection and identification of biological stains based on Raman microspectroscopy and advanced statistics. At the University at Albany, we use multidimensional Raman spectroscopic signatures to differentiate and identify traces of body fluids (2,3). This novel methodology is expected to replace current in-field biochemical tests, which have several important limitations: They are mainly presumptive, they suffer from cross-reactivity, and a separate test is currently required for each body fluid type. In addition, our test allows for differentiating menstrual and peripheral blood, which can be instrumental for sexual assault cases, differentiating animal and human blood, determining the time since deposition of blood for up to two years, and providing information on phenotype profiling including the sex and race of the donor. All of this information is expected to be available immediately at a crime scene, helping the investigator determine what evidence to collect and to build a preliminary suspect profile based on the discovered body fluid traces. The van Leeuwen research group in Amsterdam, The Netherlands, used near-infrared (NIR) spectroscopy to develop a method to estimate the age of a bloodstain (4). Similarly impressive results have been obtained in gunshot residue research. The García-Ruiz research group (5) in Madrid, Spain, and our laboratory (6) reported independently on a new method to identify ammunition using Raman spectroscopy. An IR imaging procedure to automatically detect gunshot residue particles was also developed (7). Sergei Kazarian and coworkers from Imperial College London have made significant progress recently in using infrared spectroscopic imaging as a label-free method with a high chemical specificity and sensitivity for forensic applications (8). Edward Suzuki at the Washington State Patrol Crime Laboratory Division has used IR spectroscopy to identify pigments used in automotive paint (9). Jürgen Popp and coworkers in Jena, Germany, have used Raman spectroscopic techniques to detect pathogens, which is an extremely important concern for biosafety disciplines (10–12).

I would like to give a special emphasis to the impressive advances made recently in the area of spatially offset Raman spectroscopy (SORS). Pavel Matousek and coworkers from the Rutherford Appleton Laboratory pioneered the application of SORS as a novel noninvasive approach for a variety of forensic and biomedical applications (13,14). They founded Cobalt Light Systems in 2008 (recently acquired by Agilent Technologies) and have built a family of compact, fast, and relatively inexpensive instruments for airport security (testing sealed containers including bottles and cans) and the pharmaceutical industry. According to a recent press release, Cobalt's customers include more than 20 of the largest 25 global pharmaceutical companies and over 500 of their devices are deployed at airport checkpoints. I believe that this is the most significant practical application of Raman spectroscopy today with the highest potential impact on everyday life.

How did you come to be involved with forensic analysis?

I did not plan to. In 2003, our chemistry department launched a program in forensic chemistry, offering both bachelor’s and master’s degrees. We received a $1.5 M grant from New York state to build a new teaching laboratory, which was designed to mirror the crime laboratory at the New York state police department, located across the street from the university campus. As a member of the analytical chemistry faculty, I was charged with providing research opportunities for our new students. Mark Dale served as the director of the Northeast Regional Forensic Institute at the University at Albany at that time, and he kindly took me to the National Institute of Justice (NIJ) Conference in Arlington, Virginia. Listening to talks on the current methods in forensic science, I realized that there was huge gap between practical “modern” forensics and advanced analytical chemistry in general and spectroscopy in particular. On my return from Arlington, we immediately started working with Kelly Virkler, a PhD student at that time, on the application of Raman spectroscopy for the identification of body fluid traces. Kelly’s first papers on this topic, published in Forensic Science International in 2008 and 2009, are among the most cited and downloaded articles from this top journal in the field. (15,16).

Dr. Virkler now is a supervisor of forensic toxicology lab at the NY State Police Forensic Investigation Center. We received our first grant from the NIJ in 2009.

The annual NIJ Conference was discontinued in 2013, while research and development activity in forensic science in the United States and the rest of the world has increased significantly during the last decade. Among other meetings, NIJ has organized an annual full-day session at the American Academy of Forensic Sciences (AAFS) annual meeting. This brings the work of researchers already engaged in work relevant to forensic problems to the attention of forensic practitioners, but doesn’t necessarily draw broad attention from the wider scientific community. There remains a great demand for a meeting where researchers from academia, government agencies, forensic laboratories, and industry who are interested in the development of new analytical methods for forensic application can get together. In an effort to fill this gap, NIJ is launching the first National Institute of Justice Forensic Science Symposium at Pittcon 2018 in Orlando, Florida. This two-day event is designed as a major annual research and development forensic science event of the NIJ. Further information can be requested from [email protected]

Gunshot residue (GSR) analysis (1) has been the focus of many studies using vibrational spectroscopy. How has Raman spectroscopy been used for these studies? Has it been combined with other vibrational spectroscopy techniques, and if so, what are the advantages of that approach?

The most common procedure for GSR analysis is scanning electron microscopy–energy dispersive spectroscopy (SEM–EDS). SEM–EDS has a high affinity for the analysis of the heavy metals Pb, Ba, and Sb, whose presence in spherical particles is considered unique to inorganic GSR. Unfortunately, this technique is not applicable for the analysis of propellant residues (organic GSR) or GSR samples originating from heavy metal–free ammunition (“green” ammunition). Furthermore, particles originating from automotive brake pads and tires may be composed of Pb, Ba, and Sb, and cannot be distinguished from GSR via SEM-EDS analysis. Even the successful identification of inorganic GSR with SEM-EDS offers relatively limited forensic value. The most common conclusions are limited to estimating the shooting distance, confirming that a shooting incident occurred, or that a suspect discharged a firearm or was within the proximity of a discharging firearm.

Because of the drawbacks with tool-mark examinations, the ability to link a GSR sample to a specific firearm or ammunition would be a novel and impactful advancement for the forensic community. We approached this problem using vibrational spectroscopic analysis of GSR. We demonstrated that both Raman microspectroscopy and attenuated total reflection–Fourier transform infrared (ATR–FT-IR) microscopy can be used to link individual, micrometer-size GSR particles to specific ammunition–firearm combination (17,18). For both these methods, we demonstrated the potential of mapping of adhesive tape for detection and identification of both organic and inorganic GSR particles (19,20).  (Crime scene investigators regularly collect GSR particles using sticky tape and conduct further analysis with SEM-EDS.) ATR–FT-IR microscopy has some advantages over Raman microspectroscopy for GSR analysis because some GSR particles are highly fluorescent. We have also discovered that Raman and FT-IR spectra of the same GSR particles capture contributions from different vibrational modes and, consequently, provide complementary information about the chemical composition of GSR. Combining Raman and FT-IR spectra allows for more specific characterization of GSR particles and their assignment to specific firearm–ammunition combinations (21). We received our first funding for GSR studies from the NIJ in 2017 and hope to make significant progress in the near future.

In another recent paper about bloodstain aging estimation (22), it was shown that biochemical kinetic changes in bloodstains can be nondestructively probed for year-long periods using Raman spectroscopy. What were some of the challenges in using this technique to gain information that would be useful in a crime scene investigation?

It turns out that blood aging is quite a complex process involving several different biochemical changes at the molecular level, including the chemical transformation of the hemoglobin heme group followed by hemoglobin denaturation, aggregation, and degradation. The kinetics and mechanism of these processes can depend on the environment, including temperature, humidity, and exposure to sun- or room light. We conducted our study for indoor conditions, including room temperature, normal (uncontrolled) humidity, and with no direct sunlight. Should the conditions be different, we would need to build a new model for predicting the time since deposition. It is our target for future research to expand our method to work within typical environmental conditions. At the moment, we can offer the following approach: If a crime scene investigator recovers a bloodstain aged under certain conditions, we can characterize the sample with Raman spectroscopy and conduct an aging experiment under the same conditions. The newly built aging model will be applied to the initially recovered evidence to determine the time since deposition. The newly developed model for the specific conditions could be used for upgrading the method for future usage.

What are your next steps using Raman spectroscopy for forensic analyses?

Over the last 10 years, we made significant progress in the development of Raman spectroscopic tests for the identification and characterization of body fluid traces thanks to significant support from the NIJ. Our state-of-the-art, novel methodology is such that if crime scene investigator can pick up the smallest trace of body fluid she or he can handle, we have software to automatically identify the body fluid and conduct further characterization (23). We are now working on expanding our methodology to body fluid traces on common substrates. We have already demonstrated that we can overcome substrate interference and detect and identify all main body fluid traces on various common substrates. However, “manual” analysis is required; a universal, automatic approach has yet to be developed. We also need to validate our novel methodology for realistic crime scene samples. We rely on help and advice from our collaborators from the New York State Police Crime Laboratory System and their director, Ray Wickenheiser, who has been a strong supporter of our lab for many years sharing his expertise and experience with us.

As for the gunshot residue project, we plan to conduct a systematic study of the relationship between discharged ammunition and its subsequent GSR. These investigations will include the chemical analysis of the unburnt ammunition material and its comparison with corresponding GSR using vibrational spectroscopy and advanced statistics.


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  2. V. Sikirzhytski, A. Sikirzhytskaya, and I.K. Lednev, Anal Chim Acta 718, 78–83 (2012).
  3. A. Sikirzhytskaya, V. Sikirzhytski, and I.K. Lednev, Forensic Sci. Int.  216, 44–48 (2012).
  4. G. Edelman, V. Manti, S.M. van Ruth, T. van Leeuwen, and M. Aalders, Forensic Sci. Int. 220, 239–244 (2012).
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  8. A.V. Ewing and S.G. Kazarian, Analyst 142, 257–272 (2017).
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  11. S. Stöckel, S. Meisel, M. Elschner, P. Rösch, and J. Popp, Angew. Chem. Int. 51, 5339–5342 (2012).
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  16. K. Virkler and I.K. Lednev, Forensic Sci. Int. 188, 1–17 (2009).
  17. J. Bueno, V. Sikirzhytski, and I.K. Lednev, Anal. Chem. 84, 4334–4339 (2012).
  18. J. Bueno, V. Sikirzhytski, and I.K. Lednev, Anal. Chem. 85, 7287–7294 (2013).
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  23. C.K. Muro, K.C. Doty, L. deSouza Fernandes, and I.K. Lednev, Forensic Chem. 1, 31–38 (2016).


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