Fast Fluorescence Imaging Followed b

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A Novel Two-Step Method for the Detection of Organic Gunshot Residue for Forensic Purposes: Fast Fluorescence Imaging Followed by Raman Microspectroscopic Identification Shelby Khandasammy, Alexander Rzhevskii, and Igor K. Lednev Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02306 • Publication Date (Web): 22 Aug 2019 Downloaded from pubs.acs.org on August 26, 2019

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Analytical Chemistry

A Novel Two-Step Method for the Detection of Organic Gunshot Residue for Forensic Purposes: Fast Fluorescence Imaging Followed by Raman Microspectroscopic Identification Shelby R. Khandasammy, 1 Alexander Rzhevskii, 2 and Igor K. Lednev1,* 1

Department of Chemistry, University at Albany, SUNY, 1400 Washington Avenue, Albany, New York 12222, United States 2 Thermo Fisher Scientific, 2 Radcliff Rd., Tewksbury, MA 01876, United States *[email protected]

ABSTRACT:

Gunshot residue (GSR) is potentially key evidence during a criminal investigation of a shooting accident. Current standardized forensic science methods target the detection of inorganic GSR (IGSR). In this proof-of-concept study, a new two-step method for the detection and identification of organic GSR (OGSR) is proposed. This method utilizes highly sensitive fluorescence hyperspectral imaging of a sample area to detect potential GSR particles, followed by confirmatory identification of the detected particles using Raman microspectroscopy. In this study, two different GSR samples on adhesive tape substrates were created. One sample was made by manually placing a known amount of OGSR particles onto an adhesive tape substrate. The second sample mimicked a real crime scene situation and had an unknown number of GSR particles mounted onto an adhesive tape substrate using a most common tape-lifting procedure for the recovery of GSR from the skin of a suspect and other surfaces. These two samples were subjected to the developed two-step analysis method. It was found that this method was accurately able to detect and identify all OGSR particles. Representative spectra of OGSR particles showed characteristic Raman peaks at 850 cm-1, 1287 cm-1, and 2970 cm-1. This methodology offers a promising means to meet current needs within the framework of GSR analysis by providing a way to accurately detect and identify OGSR.

INTRODUCTION Forensic investigators often rely upon the detection and analysis of trace evidence to elucidate the details surrounding a criminal case. Gunshot residue (GSR) is a potentially important type of evidence during violent crime investigations. GSR particles are produced when a weapon is discharged, and are comprised of two distinct subclasses— inorganic gunshot residue (IGSR) and organic gunshot residue (OGSR).1 IGSR originates from the primer of an ammunition cartridge; meanwhile, OGSR stems from the propellant component of a cartridge.1-3 The length of time that GSR remains on an object or person is highly variable, because it is more likely to be physical movement and not particle decomposition which will cause the loss of GSR particles.3 In fact, GSRs have been reported to persist on an untouched cotton cloth for up to two months.3 Therefore, GSR may be considered to be a persistent form of trace evidence depending on the circumstances of a case. The most common and standardized technique for GSR analysis utilizes scanning electron microscopy coupled with energy dispersive X-ray spectroscopy, also known as SEMEDS or SEM-EDX.4-6 This technique focuses upon the detection of the heavy metal elements lead, antimony, and barium. 1, 4, 6 It is notable that the particles analyzed using SEM-EDS/X belong to the IGSR class, and thus stem from the primer component of the ammunition.4 In recent years,

forensic science research has increasingly shifted focus from the analysis of IGSR particles using SEM-EDS/X towards the analysis of OGSR particles—this is due in large part to the advent of lead-free or non-toxic cartridges in the ammunition market.4-6 The introduction of lead-free or non-toxic ammunition has made it difficult for analysts to identify IGSR particles using SEM-EDS/X, because the characteristic triad of heavy metals is not present in these samples due to the exclusion of lead.7-8 Thus, focus has shifted to the characterization and analysis of OGSR particles, because they possess the potential to bolster the evidentiary significance of GSR.4, 7-8 When considering SEM-EDX/S analyses another key disadvantage that must be commented on is the fact that it is extremely time laborious, as analysts spend a great deal of time searching for and locating particles.9 This search time is prolonged due to large sample areas, time spent analyzing non-GSR particles, and debris such as skin and fibers which can obscure particles of interest.9 In contrast, our new method of detection for OGSR particles results in short detection times, and also remedies the issue of analysts wasting time examining environmental contaminants. This novel method also has great potential for application to large sample areas which is a key goal for future studies. In addition to the disadvantage regarding IGSR analysis which is presented by lead-free ammunitions, the particles are also very small—ranging from 1 to 10 µm in size.6 In contrast, OGSR can be so large as to be observable with the human eye

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or may be considered a “fine dust”.6 This size difference makes OGSR particles more likely to be recovered by crime scene investigators.6 OGSR particles also have the potential to provide information which can indicate what type of ammunition has been used during a firing event.10 IGSR particles also have several potential false positives associated with them—including fireworks, brake linings, cartridge operated tools, and paints.7 In contrast to this, some of the most commonly detected components of OGSR—namely the stabilizers diphenylamine, ethyl centralite, and methyl centralite—are not commonly found in the environment.7 OGSR analysis has been approached using several different methods. A method which has been investigated by several research groups is the analysis of OGSR using liquid chromatography coupled with mass spectrometry (LC-MS).11 This technique has had success for the detection of OGSR components, however, its destructive nature is disadvantageous for forensic analysis.11 Other trends for OGSR analysis include gas chromatography (GC) techniques paired with electron capture, mass spectrometry (MS), flame ionization, and thermal energy analysis (TEA).3 GC coupled with TEA is most popular with regards to OGSR analysis.3 However, GC analyses are disadvantageous for GSR in general, due to the nature of GSR components.3 For example, nitrocellulose (an explosive component in propellants) cannot be analyzed using GC due to its lack of volatility.3 It has also been found that GC is not suitable for the analysis of components such as the stabilizer N-nitrosodiphenylamine, as it will convert into diphenylamine at high temperatures.3 In addition to GC methods, researchers have also analyzed OGSR using high performance liquid chromatography (HPLC) and capillary electrophoresis techniques.3 For a comprehensive look at current OGSR analysis methods the reader is referred to the review published by Goudsmits et al. in 2018.12 The application of Raman spectroscopy for GSR analysis has been explored extensively. For a comprehensive overview on this topic, the reader is referred to the recent reviews by Khandasammy et al., Mistek et al., and Doty and Lednev.13-15 Notably, Bueno and Lednev applied Raman spectroscopic mapping for the detection and analysis of both OGSR and IGSR on tape substrates and demonstrated the nondestructive nature of Raman microspectroscopy presents a viable alternative to the aforementioned common analysis techniques for OGSR.1 In 2018, Bueno et al. reported on validation studies regarding the analysis of OGSR and IGSR particles on tape substrates using Raman spectroscopic mapping in conjunction with multivariate statistical analysis.16 LópezLópez et al. successfully used Raman imaging microscopy to detect and differentiate GSR particles on cloth and on SEMEDX stubs.4 López-López et al. also conducted a study in which they successfully correlated the Raman spectra of unfired smokeless powders to GSR stemming from the firing of ammunition containing the same powders.10 This study notably focused on the organic components of GSR, and emphasized the ability of Raman to narrow down the type of ammunition that had been fired.10 In the studies conducted by Bueno et al. it was reported that GSR particles possess strong fluorescence under excitation in the visible spectral range.6 This property of GSR particles was exploited in this methodology in order to use fluorescence hyperspectral imaging for the detection and location of OGSR particles mounted on an adhesive tape substrate. The high fluorescence quantum yield provided the

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opportunity to perform fast spectral imaging through which visualization of sample areas containing GSR particles was achieved. This imaging is considered to be “fast” in comparison to the time it would take to complete a Raman spectroscopic analysis of the investigated areas and the time laborious process of analyzing IGSR particles using SEMEDX. 9 Confirmatory identification of the particles of interest in the fluorescence images generated was then achieved via the collection of their Raman spectra. The combination of fluorescence and Raman spectroscopy multi-modal imaging has been utilized as a promising tool for the analysis of biological samples. In particular, surface enhanced Raman spectroscopy (SERS) and fluorescence spectroscopy multi-modal imaging have shown great potential for biomedical applications in cancer detection as reported by Lee et al. (2012), Cao et al. (2015), and Pal et al. (2019).17-19 SERS/fluorescence dual mode nanoprobes have also shown great potential for the detection of cytochrome c in cells as evidenced by a recent work of Zhang et al.20 In 2012, Cicchi et al. demonstrated the ability of a two optical fiber probe system which combined fluorescence and Raman spectroscopic analyses in order to diagnose melanocytic lesions.21 In 2014, Jeong et al. developed a multi-modal endomicroscopic system utilizing fluorescence imaging and SERS analysis to identify pathologic lesions.22 Finally, in 2018 Shipp et al. developed a multimodal imaging technique using fluorescence mapping combined with specific spontaneous Raman spectroscopic identification to detect the margins of tissues excised during breast cancer removal surgery. 23 In this work, a novel two-step method for the detection and identification of OGSR was developed. This method utilizes highly sensitive fluorescence hyperspectral imaging of a sample area to detect potential OGSR particles, which is followed by confirmatory identification of detected particles using Raman microspectroscopy. By using this two-step technique we are able to ensure that debris particles are not misidentified as OGSR and that OGSR particles are correctly identified. This method has the potential to save examiners time in searching for OGSR by using fast fluorescence mapping to quickly detect particles of interest and subsequently using Raman spectroscopy to specifically and accurately identify OGSR particles. Thus, this work demonstrates great potential to be applied as a very effective technique for the detection and identification of OGSR.

MATERIALS & METHODS Sample Preparation The OGSR used in this study was obtained with the assistance of our collaborators at the New York State Police Forensic Investigation Center by firing a Bersa Thunder 9 firearm loaded with CCI Blazer 9-mm ammunition into a cloth target. This was done from an approximate distance of 0.3 meters. Two different samples were created in this study. The first sample was made by selecting OGSR particles from the cloth target using tweezers and placing them manually onto double-sided adhesive tape mounted on an aluminum foil covered microscope slide. Ten OGSR particles in total were placed onto the tape substrate in close proximity to each other. These particles were visually discernable with the naked eye during preparation—thus predisposing them to be OGSR based on their size. As was aforementioned, OGSR particles are much larger than IGSR particles. The second sample was created by pressing the sticky surface of the adhesive substrate

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determine which particle might be classified as debris based on the optical image alone. The instrument parameters (see Materials and Methods) for fluorescence mapping were optimized so that mapping could be undertaken in the shortest period of time possible without

of a similarly fabricated slide as in the first sample onto the cloth target in order to adhere an unknown number of GSR particles to the tape. Adhesive tape is the most widely used collection method in practice for the collection of GSR from the skin of individuals suspected of shooting a firearm.3

Instrumentation The instrument utilized to perform all spectral measurements was the Thermo Scientific DXRxi Raman Imaging Microscope. This instrument features an electron multiplying charge-coupled device (EMCCD) detector coupled with a fast continuously moving sampling stage, which allows for imaging of relatively large sample areas at high spectral acquisition rates. The EMCCD detector allows hyperspectral imaging to be performed by sequential scanning in a continuous image acquisition mode. The samples were preliminarily investigated using 780, 633, 532, and 455 nm excitation wavelengths for generating fluorescence and Raman scattering. It was found that 455 nm excitation provided an optimal balance between the relative intensities of the fluorescence signal and the Raman peaks detectable from the GSR particles. In contrast, the other excitation wavelengths generated either overwhelming fluorescence or a weak Raman response. Thus, the 455 nm laser was utilized as the excitation source for all analyses presented in this study. In the fluorescence mapping step, images were acquired using a 10X objective. A step size between 4-5 µm was selected to approximately match the laser spot size. This ensured that the entire sample surface was scanned, and emission signals from all potential GSR particles were collected.24 A laser power of 2.5 mW was used, with an exposure time of ≤ 0.002 seconds for both samples. A 50 µm confocal pinhole was selected for the analysis. The fluorescence images were generated as maximal spectral intensities, and the fluorescence color maps were created using the OMNIC™xi Raman imaging software from Thermo Fisher Scientific. In the next step of the analysis, the fluorescence color map images were used to determine the locations of the particles of interest and Raman spectra were measured from these locations. A 50X long working distance objective was used to collect all the Raman spectra. Step size ranged between 5-20 µm depending on individual particle sizes. A laser power of 3.0 mW was used, and the exposure time was set to 0.05 seconds. In order to minimize the background relative to the Raman peaks a photobleach time was set to 5.0 seconds for each pixel. The Raman spectra were acquired using a 50 µm confocal pinhole. The parameters for the collection of all Raman spectra presented in this manuscript were optimized such that the signal-to-noise ratio would be above 10 for the main peaks in individual spectra. All Raman spectra were preprocessed in MATLAB 2012b using PLS toolbox to apply smoothing, baseline correction, and normalization.

Results Sample 1: Known OGSR Particles

Figure 1. Optical (left) and Fluorescence Color (right) images. Fluorescence Color Image was created using OMNIC™xi by applying a “Peak Height” profile with peak height at 461.41 nm.

compromising the quality of the resultant map image. This fluorescence image has dimensions of 28 mm2. The fluorescence spectra were collected in approximately 52 minutes. The fluorescence-based color image was generated based on the hyperspectral data using the OMNIC™xi software. This image was generated as the color profile for “Peak Height” at 461 nm with a baseline ranging from 479–533 nm. The optical and fluorescence images for Sample 1 are juxtaposed in Figure 1. Under the applied profile conditions all 11 particles in the optical image were visualized in the fluorescence image. All particles appear in the fluorescence image as reddish orange, save for particle 7, which is a paler orange color. This dissimilarity in color indicated that a distinct difference between the intensity of the fluorescence spectra of particle 7 and the other particles exists. The fluorescence spectra for the particles from Sample 1 2

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A total of 10 OGSR particles were deposited onto the tape substrate of Sample 1. The optical image of the sample seen in Figure 1 revealed that 11 particles were present in the sample. The fluorescence image thus indicated that one particle in the optical image was not GSR, and was a result of environmental contribution. However, it was impossible to

Figure 2. Fluorescence spectra for Sample 1 particles & adhesive tape substrate. Individual particle spectra are representative of averaged fluorescence spectra from each respective particle area determined from the OMNICxi Color Image in Figure 1

and the tape substrate are pictured in Figure 2. It is important to note that the spectrum for particle 7 is much lower in intensity than that of the tape substrate. Meanwhile, all other particles produced fluorescence spectra


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with much higher intensity than the tape substrate. This result illustrates that fluorescence mapping has the potential not only

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possessed such low signal to noise ratio that no distinctive peaks whatsoever could be assigned. Excepting particle 7, all other particles demonstrated some specific Raman spectroscopic characteristics of OGSR in addition to producing distinctively intense fluorescence relative to that of the tape substrate. These results were concurrent with the fact that Sample 1 contained ten known GSR particles, and thus one particle was anticipated to be attributable to environmental debris.

Sample 2 Unknown Particles

Figure 3. Raman spectrum of Sample 1 particle 3. This spectrum is representative of the spectra obtained from OGSR particles. The peaks of note are highlighted at 850 cm1 (blue), 1287 cm-1 (gold), and 2970 cm-1 (pink).

to detect GSR on adhesive tape but also to differentiate GSR from debris particles. Raman data was collected using parameters optimized to obtain Raman signatures from the GSR particles (see Materials and Methods). All 10 of the OGSR particles demonstrated Raman peaks that were consistent with those present in the spectra of OGSR as reported by Bueno et al. and López-López et al. 10, 25 Figure 3 depicts a representative Raman spectrum for an OGSR particle (from particle 3 pictured in Figure 1). This spectrum portrays the type of confirmatory Raman spectra that were collected in the second step of the analysis procedure from OGSR particles. It is significant that the spectrum shown in Figure 3 is inclusive of the entire fingerprint region for organic compounds, and notably the 2970 cm-1 band corresponding to the C-H stretching vibrational mode can be seen.10 To the best of our knowledge, this is the first time a complete Raman spectrum has been reported for GSR mapping, a factor which should improve identification confidence in this case. Based on previously published literature data the observed Raman peaks can be assigned as follows: 850 cm-1 to NO2 scissoring, 1287 cm-1 to NO2 symmetric stretching, and 2970 cm-1 to C-H stretching vibrations.10, 25-26 The peak at 2970 cm1 was found by López-López et al. in their study on the analysis of organic gunshot residues to be an indicator of the presence of the stabilizer ethyl centralite.10 Of the eleven total identified particles, six produced spectra with strong peaks of significance at approximately 850 cm-1, 1287 cm-1, and 2970 cm-1. Two of the particles produced spectra possessing peaks at 850 cm-1 and 1287 cm-1. Meanwhile, two particles produced spectra having only weak Raman signals around 1287 cm-1. Therefore, ten of the eleven particles produced spectra with peaks that were characteristic of OGSR. This correlates with the fact that ten known OGSR particles of the eleven observed in the optical and fluorescence images were placed upon Sample 1. The complete Raman spectral dataset for the eleven particles investigated in Sample 1 can be seen in the Supporting Information section. The Raman spectrum of particle 7 seen in Figure 1 did not produce any Raman peaks associated with OGSR, and indeed

Figure 4. Optical (top) and Fluorescence Color (bottom) images. The Fluorescence Color Image was obtained using OMNIC™xi by applying a “Peak Height” profile with peak height at 538.02 nm

The goal of creating a sample with an unknown number of GSR particles was to test the applicability of the method with a realistic sample. Thus, as aforementioned this trial was meant to simulate the analysis of a real life crime scene sample. In the optical image of the unknown sample a total of


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six particles can be visualized readily in Figure 4 labeled 1, 3, 4,5,6,7. Meanwhile, in the juxtaposed fluorescence hyperspectral image of this area (also pictured in Figure 4) a total of six particles labeled 1,2,3,4,5,7 can be seen based on their distinct variance in color compared to the blue backdrop of the image. Note that the six particles that can be readily seen in the fluorescence image and the optical image are different. This fact will be elaborated on later. The peak height profile was applied to the spectral dataset using the OMNIC™xi software to profile peak height at 538 nm with the baseline from 457–457 nm. This fluorescence image has dimensions of 67 mm2. Fluorescence spectra were collected in approximately 85 minutes. Based on the hyperspectral fluorescence imaging six notable areas of interest were identified due to their distinctive coloring in the profile. As aforementioned, the six areas of interest in the fluorescence image are not the same areas of interest that appear in the optical image. This disparity is due to the fact that particle 6 in the optical image did not fluoresce with as great intensity as the other particles, despite its obvious presence in the optical image. Particle 6 was included in the datasets for both fluorescence and confirmatory Raman spectral characterization because its presence in the optical image merited its investigation. Particle 2 is not labelled in the optical image due to the fact that increased magnification is necessary in order to visualize it and the resolution of the overall optical image does not allow for accurate labelling of its location. The fluorescence and Raman spectroscopic data collected for the unknown particles can be seen in Figures 5 & 6 respectively. The fluorescence spectra in Figure 5 revealed that Particle 6 possessed a low intensity fluorescence spectrum. In fact, the fluorescence intensity was so low that it appeared to have almost the same intensity as the spectrum produced by the tape substrate. 2.5

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Figure 6. Raman spectra of Sample 2 particles. The peaks of note which are characteristic of gunshot residue are highlighted at 850 cm-1 (blue), 1287 cm-1 (gold), and 2970 cm-1 (pink).

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analysis. Particles 2a and 2b produced Raman signatures that were overall not consistent with OGSR. Particle 2a displayed only the peak at 1053 cm-1. Meanwhile, particle 2b displayed peaks at around 2920 cm-1, 2979 cm-1, and 1458 cm-1. Particle 6 produced no detectable Raman signature. It should be noted that based on the fluorescence image, particle 6 was discounted as an OGSR particle, and Raman spectra collected from this particle attest to the validity of this exclusion. This instance succinctly demonstrates the ability of the method to eliminate non-GSR particles from further analysis using only the initial fluorescence screening step. The particles 2a and 2b conversely serve to validate the necessity of the second step of the process—the Raman spectroscopic screening step—in order to ascertain whether or not a fluorescing particle is indeed OGSR.

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Figure 5. Fluorescence spectra for Sample 2 particles & adhesive tape substrate. Individual particle spectra are representative of averaged fluorescence spectra from each respective particle area determined from the OMNICxi Color Image in Figure 4.

The complete confirmatory spectral dataset seen in Figure 6 revealed that all particles, save for particles 6 and 2, produced spectra with peaks at 850 cm-1, 1287 cm-1, and 2970 cm-1, which as aforementioned are Raman signature peaks consistent with OGSR. It should be noted that particle 2 in the fluorescence map was revealed to be made up of two potentially distinct particle fragments which were subsequently named Particle 2a and Particle 2b. These two particles were sampled separately using Raman spectroscopic

Given recent advances in spectral imaging, especially in fast imaging techniques, the proposed method presents a prerequisite for creating a completely automated routine for the detection and identification of OGSR particles based upon their spectral characteristics.24 This is achieved through the utilization of the particles’ fluorescence and Raman spectroscopic properties. The use of fluorescence mapping in conjunction with the collection of Raman spectra for a GSR sample presents a twofold methodology which ensures OGSR particles are detected and correctly identified. As indicated in the Introduction, several groups have applied fluorescence and Raman spectroscopic multi-modal imaging as a tool for the analysis of biological samples. The reported method most similar to ours was developed by Shipp et al. for cancer diagnostic applications.23 In that study, the authors used two separate excitation sources to produce fluorescence and Raman spectral data. In contrast to past studies using similar methodologies, our work applies the method of fluorescence and Raman spectroscopic multimodal analysis to non-biological samples for forensic purposes. We also utilized a single laser source for


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excitation and the same instrument for collecting emitted and scattered light which may allow for simplification and optimization of a future instrument for this specific application. While this proof-of-concept has been applied for OGSR detection and characterization, it also has the potential to be utilized for the detection and identification of other nonbiological samples of interest. This method presents a potential means by which forensic analysts may screen collected samples for the presence of OGSR particles. Both steps in the proposed method are accomplished using a single instruments and provide different levels of screening for the particles. The fluorescence step serves to detect fluorescing particles. The Raman spectroscopic screening step allows for the further characterization of the fluorescent particles and confirms or rejects the particles as OGSR with a level of distinct specificity. The methodology presented herein may serve to provide a complement to data from current SEM-EDX/S methods for the detection and identification of IGSR. It also presents the potential to be applied to large sample areas because of the “fast” nature of the fluorescence mapping step, and the ability of the fluorescence mapping to accurately pinpoint locations of interest for an analyst. Our method also enables obtainment of fluorescence images indicating the location of OGSRs in a relatively short period of time, especially when compared to known SEM-EDX analysis time for IGSRs. Since the fluorescence images are correlated with high quality fluorescence spectra users can also validate the visuals provided in the images with relevant spectral information. As aforementioned our developed method for the analysis of OGSR not time consuming and allow analysts to easily pinpoint potential OGSR particles for analysis. This method also has great potential for application to even larger sample areas than those studied herein. Of course, the reported proof-of-concept requires further method development, including: (1) testing of potential false positives due to environmental contaminants, (2) building a robust statistical model for the analysis of Raman spectral data, and confirming GSR identification based on a complete spectroscopic signature and not based on individual Raman bands, and (3) expanding the sample set to assess the ability of the method to detect and identify OGSR from various ammunitions and firearms including lead-free ammunition types. When considering the testing of potential false positives due to environmental contaminants we will focus on investigating OGSR specific false positives, this is in contrast to previous work in our lab which has focused on investigating false positives associated with IGSRs.16 In the future we also plan to investigate the application of this technique for the identification and characterization of IGSRs. As previously mentioned we also aim to demonstrate the ability of this method to process larger sample areas and OGSR on different substrates. It is noteworthy when considering future improvements that all the measurements were taken in this study using an instrument designed for Raman microspectroscopy. Special optimization of the instrument for combined fluorescence mapping and Raman microscopy can further speed up the overall detection and identification of OGSR on a large sample area. Overall, the continued interest in the detection and identification of OGSR particles presents this novel two-fold approach as a potentially valuable tool for the forensic investigator.

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Complete Raman spectral dataset for Sample 1: Known OGSR Particles is available in Supporting Information file.

Acknowledgment We would like to express our gratitude to Lieutenant Heller and Sergeant D’Allaird for their provision of the GSR samples that were used. We would also like to thank Mathew Boll for his assistance at the early stage of this project. This project was supported by Award No. 2016-DN-BX-0166 awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice (I.K.L.). The opinions, findings, and conclusions or recommendations expressed here are those of the authors and do not necessarily reflect those of the U.S. Department of Justice.

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Fast Fluorescence Imaging Followed b

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