ORIGINAL PAPER

Potential application of Raman spectroscopy for determiningburial duration of skeletal remains

Gregory McLaughlin & Igor K. Lednev

Received: 6 May 2011 /Revised: 11 August 2011 /Accepted: 14 August 2011 /Published online: 26 August 2011# Springer-Verlag 2011

Abstract Raman spectroscopy was used to study trendsin chemical composition of bones in a burial environ-ment. A turkey bone was sectioned and buried for shortintervals between 12 and 62 days. Buried sections wereanalyzed using Raman microspectroscopy with 785 nmexcitation. The results indicate that chemical changes inbone due to soil bacteria are time-dependent. Spectro-scopic trends within buried bone segments were corre-lated to burial duration. A preliminary model wasconstructed using peak integration of Raman bands.Data collected within buried bone segments fit verywell in this model. The model constructed is sensitiveto changes in bone composition in a scale of days. Thisstudy illustrates the great potential of Raman spectros-copy as a non-destructive method for estimating theburial duration of bone for forensic purposes.

Keywords Raman spectroscopy . Forensic anthropology .

Forensic science . Collagen . Cortical bone . Time sincedeath

Introduction

Forensic science is a quickly expanding and maturingbranch of analytical chemistry [1]. The analysis of bones asforensic evidence is seen in cases of missing persons,disaster, or war. The analysis of skeletal remains is a highlyspecialized field. A forensic anthropologist is usually

tasked with determining the sex, age, stature, and race ofremains that have been identified as human [2]. Morpho-logical features in the skull and pelvis can help to answerthese questions [3]. A more difficult parameter to estimateis the length of time that a bone has been buried. With thisknowledge, investigators would be able to accuratelyestablish a timeline of events with respect to the victim.Hypothetically, this knowledge could establish the timesince death, or postmortem interval (PMI). A method thatextrapolates the exact burial duration of skeletal remainswould allow for easy discrimination of modern bones withpotential forensic relevance from ancient bones of potentialarchaeological relevance.

There are several methods used by forensic pathologiststo estimate the postmortem interval. For example, techni-ques such core temperature measurements can be used toestimate the PMI with reasonable accuracy. Algor mortis, orpostmortem cooling, occurs at a rate of approximately 2.5–1.5 °C h−1 for the first 12 h after death [3]. However, whenall soft tissue decomposes and only a skeleton remains,these techniques are inapplicable. Complete skeletonizationof a buried corpse can vary from a scale of weeks to years,depending on burial and environmental variables [2].However, where scavenger animal activity is high, softtissue can be removed and the remains buried in a matter ofdays [4]. In cases where no soft tissue remains, datingmethods become very problematic. As quoted from Nagy etal., “Dating human skeletal remains is one of the mostimportant and yet unreliable aspects of forensic medicine.The identification of an unknown individual is a complexpuzzle made up of many parts and one of the mostsignificant of them is the dating of skeletal findings [5].”

Some chemically based techniques that have beendeveloped include radiocarbon dating and spectroscopicanalysis. The following are examples of a few available

G. McLaughlin : I. K. Lednev (*)Department of Chemistry, University at Albany,1400 Washington Avenue,Albany, NY 12222, USAe-mail: lednev@albany.edu

Anal Bioanal Chem (2011) 401:2511–2518DOI 10.1007/s00216-011-5338-z

methods; this is not a comprehensive review of skeletaldating methods.

A common technique for dating skeletal remains usesthe luminol reagent. Luminol has been widely adopted byforensic investigators for the ability to presumptively detectthe presence of blood [3]. The luminol chemiluminescent(CL) reaction is specific to the hemoglobin in blood, butfalse-positives are possible [6]. Introna et al. investigatedusing luminol to date skeletal remains [7]. It was observedthat recent (1 month to 3 years) bones exhibited a strongCL, easily detectable by the naked eye. Older bone samples(25 to 35 years) exhibited a weaker CL while the oldestbones (80 years) sampled exhibited no CL. The generaltrend was that bones with increasing PMI exhibited weakerchemiluminescence.

A recent blind evaluation of this technique byRamsthaler et al. demonstrated that the luminol test issusceptible to significant observer classification error [8].Two examiners were asked to classify bones as either recentor non-recent based only on visual observations of theluminol reaction. In the case of recent samples, the false-negative rate was 15% and the overall correct classificationrate was 89%. In addition, there were three false-positiveoccurrences for a bone sample over 100 years old.Although simple and cost-effective, the luminol test is nota comprehensive technique to date skeletal remains.

A more robust chemical dating method for skeletalremains is carbon isotope analysis. However, the 14C halflife is 5730 years, which makes the dating of modern(younger than 200 years) bones troublesome [9]. Thebiological turnover rate for bone collagen is long, makingradiocarbon dating of collagen in modern bone inherentlyinaccurate. For these reasons, this type of analysis is usuallyonly appropriate with archaeological bones. However, astudy by Wild et al. significantly advances this technologyfor forensic purposes [10]. The contemporary 14C atmo-spheric concentration profile shows a distinctive peakcaused by nuclear weapons testing. A recent 14C calibrationcurve can be reliably constructed starting from year 1950due to this “bomb peak”. Lipids in bone or bone marrowwere seen to be more reliable material for recent carbondating using this method. The results of this study indicatethat estimation of year of death using these materials ispossible.

A recent study by Nagy et al. attempts to develop a high-throughput screening method based on a vibrationalspectroscopy to distinguish recent forensic skeletal remainswith archaeological remains [5]. This approach used Four-ier transform infrared spectroscopy to grade the mineralphase of bone samples using the crystallinity and carbon-phosphate indexes. This method was seen to provide anefficient method to discern modern and ancient bonesamples. Other skeletal dating methods which specifically

target the inorganic apatite portion of bone include theanalysis of fluorine [11], silicon [12], carbonate [12], anduranium [13] contents. Generally, these chemical speciesmigrate between the soil and the sample in buried boneover time making them suitable for archaeological datingpurposes. Most recently, burial duration estimation has beenexplored using citrate content [14].

An investigation into the archaeological dating potentialof Raman spectroscopy revealed a spectroscopic trend withtooth burial duration. Bertoluzza et al. sampled a moderntooth and various teeth specimen dated between 150–6,000 years before present [15]. The intensity ratio of theRaman peaks at 960 and 2,941 cm−1 (v1 PO4

3− and v CH2

band, respectively) in tooth enamel were correlatable toburial time. There is a gradual decrease in Raman peakintensity ratio with burial duration. We hypothesized thatsimilar trends could appear in short burial duration intervalsof bone tissue.

Chemically, bone tissue can be considered a compositematerial. An inorganic carbonated hydroxy apatite mineral,commonly referred to as bioapatite, is embedded in anorganic matrix. This organic matrix is mostly composed oftype I collagen. Wet bone is composed of roughly 10%water, 20% organic, and 65% mineral portions by weight[16]. For simplicity, these components are often referred toas phases. Working in concert, these phases give bone itsbiological function. The mineral phase is extremely strongand capable of bearing compression while the collagencontributes slight elasticity, resisting fracture [16]. Bone is awell-studied material in the forensic sciences due mainly tothe tissues resiliency. Bone and other mineralized tissuesare more likely to survive postmortem than any otherbiological tissue [16].

The secondary structure of α collagen chains isdominated by the left-handed helix. Three left-handedhelices together form the native collagen triple helixquaternary structure stabilized in part by hydrogen bonding[17], Van der Waals forces [18], and tightly bound watermolecules [19–21]. The exact stabilizing mechanics of thecollagen triple helix is an active research area. The proteingelatin is the irreversible denatured product of collagen,typically caused by hydrolysis [22].

When a bone is introduced to a soil environment,chemical and physical alterations, or diagenesis, of thetissue occurs [23]. The diagenesis of bone tissue is acomplicated, multivariable process. With respect to thechemical nature of bone, processes that occur affect bothmineral and organic phases. The mineral phase can undergomineral precipitation, ion exchange, recrystallization, dis-solution, and leaching [5, 23]. The diagenesis of the organicphase is primarily associated with collagen loss due tochemical breakdown or microbial attack followed byleaching [23, 24].

2512 G. McLaughlin, I.K. Lednev

Soil bacteria often exhibit collagenase activity [25]. Theenzymatic digestion of collagen via this enzyme will formgelatin and free amino acids, which leach from the bone [22].Since the microbial alteration of bone is an irreversibleprocess, the degree of alteration should be dependent on theburial duration. We believe that alterations to the organicphase will occur first and are therefore potentially moreinformative in a forensic context [23]. The mineral phase ofbone should remain relatively intact for modern bones buriedfor short intervals.

Raman spectroscopy is a technique that has great potential inthe field of forensic science. Within this discipline, Ramanspectroscopy has been used to study body fluids [26–30],gunshot residue [31], explosives [32–35], and condomlubricants [36, 37]. When light is incident to an object, asmall portion of photons are scattered which have a differentenergy than the light source. A Raman spectrum is obtained bydetecting these inelestically scattered photons resulting fromthe laser irradiation of a sample. The energy difference, orRaman shift, between the laser source and the scatteredphotons correspond to molecular vibrational modes. A Ramanspectrum can be used to ascertain information about theidentity, structure, and properties of various materials based ontheir vibrational transitions [38]. Because virtually all organicmolecules exhibit a unique Raman spectroscopic signaturebased on molecular vibrational transitions, this technique iswidely regarded as confirmatory for the identification ofunknown materials. This technique is non-destructive and non-contact and typically requires little to no sample preparation. Byutilizing a Raman microscope, as little as several micrograms ofmaterial are needed and samples can be in the liquid, solid, orgas phase. Raman spectroscopy is also very efficient atmonitoring both organic and inorganic phases of bone [39].For these reasons, we feel the forensic analysis of bone tissueusing Raman spectroscopy is exceptionally appropriate.

Our hypothesis is that predictable spectroscopic trends inbone exist in the dynamic burial environment. Furthermore,these trends should correlate to burial duration. This experi-ment serves as a proof of concept for the application of Ramanspectroscopy to monitor the chemical changes in bone withburial. We report on the development of a preliminary modelbuilt on spectroscopic data that could be used to extrapolateburial duration. Our data fits the model very well withinburied bone samples. In addition, this model is sensitive tochanges that occur to bone during burial in a scale of days.

Materials and methods

Samples

A turkey leg bone was obtained from a grocery meatmarket. A turkey bone is a convenient proxy for human

bone for this preliminary study. Any external muscle or softtissue on the bone was manually removed, and then thebone was washed in tap water. Initial measurements wereobtained from several spots on the surface of the bonebefore burial. The bone was then cut into five segments,each approximately one inch in length. Standard pottingsoil was chosen, and the pH was measured at 6.8. Eachbone segment was individually buried in a small containerand placed in an office environment. The range oftemperature throughout burial fluctuated between 60–70 °F (16–21 °C). The soil was kept moist through periodicwatering with deionized water. Fragments were unburied inroughly 2-week intervals (Table 1). Once unburied, thefragments were chemically bleached using a publishedprotocol. After bleaching, the fragments were kept in afreezer for preservation, only being removed for sampling.Five spectra were obtained using a Raman microscope fromarbitrary areas for each fragment.

Chemical bleaching

When each fragment was unburied, they were manuallywashed with tap water followed by a bleachingprocedure using 30% hydrogen peroxide and pureacetone. Samples were soaked in hydrogen peroxidefor 2 h, soaked in acetone for 2 h, and finally, hydrogenperoxide again for 2 h. This process served both toreduce spectral background and stop microbial action.This chemical bleaching has been reported to signifi-cantly reduce background noise while preserving theinorganic and organic portions [40].

Digestion of bone with collagenase

A 1-g sample of collagenase type 3 (130 μ/mg) wasobtained from Worthington chemical company. A solutionof collagenase was prepared by mixing 22 mg ofcollagenase with 35 mL of tap water. A small bone segmentfrom the same turkey specimen was immersed in thissolution at room temperature. Raman measurements wereobtained periodically.

Table 1 Burial duration of bone segments

Fragment class Time buried, days

I (initial) 0

A 12

B 26

C 41

D 55

E 68

Potential application of Raman spectroscopy 2513

Raman microscope

A dispersive Renishaw inVia Raman spectrometer equippedwith Leica confocal microscope with 785 nm excitation and×50 objective was used to obtain spectra. WiRE 2.0software was used to operate the instrument. Accumulationtime was set at 35 s for five scans. The scan range was3,200–100 cm−1.

Data treatment

GRAMS/AI 7.01 software was used for data treatment.Spurious spikes were manually removed. Peak integrationwas performed between points using this software and theranges defined in Table 2.

Results and discussions

The purpose of this study is to investigate buried bonetissue for forensic purposes. Since the dynamic processeswhich affect bone and soft tissue are different, we removedsoft tissue from the bone before burial. Our hypothesis isthat a model can be built based on Raman spectroscopicdata and the known digenetic patterns of bone to extrap-olate burial duration. Five segments of a turkey leg bonewere buried in a common potting soil and unburied inroughly 2-week intervals. Once unburied, segments werechemically bleached and sampled five times at arbitraryareas using a Raman microscope. The buried portions werecompared with a fresh, unburied segment of bone. A burialenvironment is anaerobic, and thus the dynamics should bespecific to that environment. We expect processes for abone exposed to ambient air for a similar amount of time toundergo different processes due to the abundant supply ofoxygen. All measurements were obtained with a ×50objective on an arbitrary area of bone in the outer corticalregion. Unburied bone provided the most consistent set ofspectra whereas buried samples were somewhat morevaried. For the purposes of comparison, six spectra werechosen for figure representation. These spectra are repre-sentative of their constituent group.

Spectroscopic changes with burial

The spectral comparison between an unburied bone with thatof a bone buried for 12 days is shown in Fig. 1. As expected,Raman bands assigned to mineral vibrational modes arenearly identical. The v1 PO4

3− , v2 PO43− , and v4 PO4

3−

bands are similar in shape, relative intensity, and position,evidence that the mineral fraction remains essentiallyunchanged with short periods of burial.

Counter-intuitively, there is significant enhancement ofseveral organic bands, including CH2 region, amide I, δNH, and amide III in the buried segment spectrum. This isunusual because the diagenesis of bone material is typicallyassociated with collagen loss [2, 23]. There is also theappearance of two shoulder peaks at 2,852 and 1,295 cm−1

in the buried segment spectrum. These peaks have beenassigned to the v CH2 and δ CH vibrations of lipids andphospholipids, respectively. Bone has a small, yet some-times significant fat composition. This is particularly aproblem in archaeological bones of mammals with highbone fat content [41]. The fat seeps from the bonepostmortem, negatively affecting the public display of suchitems.

Amide I and amide III regions are highly sensitive tochanges in protein secondary structure. The amide I band isprimarily due to C=O stretching within the collagenpolypeptide. Because the collagen protein structure isatypical, trends in this region do not reflect the commonβ-turn, α-helix, β-sheet trends seen in most other proteins[42]. Fourier transform infrared spectroscopic deconvolu-tion studies of the collagen amide I band suggests that thisis actually a combination of at least three components,located at 1,630; 1,656; and 1,670 cm−1 [21]. The intensitychange of these overlapping components in the amide I

Table 2 Peak integration parameters

Peak Component Integration range, cm−1

v1 PO43− Mineral 991.5–907

v2 PO43− Mineral 485–400

v CH2 Organic 3,040–2,810

Amide I, v C=O Organic 1,715–1,610

Amide III, δ N–H Organic 1,358–1,217

δ NH Organic 1,500–1,415

1600 1400 1200 1000 800 600 400

Raman Shift (cm-1)

Inte

nsity

(ar

bitr

ary)

Amide IAmide III

NHv2 PO4

3-

v1 PO43-

v4 PO43-

A) Buried 12 daysI) Unburied

Fig. 1 Normalized comparison spectra of unburied (blue solid) andburied (red dashed) bone sample

2514 G. McLaughlin, I.K. Lednev

band is certainly evidence of a structural change incollagen. The amide I maximum redshifts 12 wave numbersfrom 1,668 to 1,656 cm−1 with burial. Albeit interesting, itis beyond the purpose of this experiment to fully charac-terize the structural state of collagen in bone after burial.

To the best of our knowledge, enhancement of CH2 and δNH organic Raman bands with bone burial has not beenreported in the literature, but a possible explanation hasbeen presented by Nielsen–Marsh et al. [43] The enhance-ment of these organic Raman bands could be due to theproposed “enzyme exclusion” hypothesis [43]. In thismodel, mineralized collagen is protected from enzymaticdigestion by the presence of apatite. During burial, there isa partial loss of the mineral phase, exposing the collagen tohydrolysis. The observed enhancement of CH2 and δ NHorganic bands is consistent with this hypothesis. There isinitial enhancement of these organic Raman bands withburial due to the partial mineral phase loss. However, it isstill unclear why this trend is seen in artificial enzymaticattack (section Spectroscopic changes with collagenasedigestion) in neutral pH, which tends to protect the mineralphase.

The dynamic Raman spectroscopic trends that occurduring collagen gelatinization have not been reported yet tothe best of our knowledge. It is possible that when thecollagenase enzyme unwinds the native collagen triplehelix, there will be a change in Raman cross section forthese corresponding organic vibrational modes. Ramancross section increases for protein conformational changeshave been reported in the literature. For example, in a studyby Wojtuszewski et al., an intensity change in amide modesis attributed to the increase in Raman cross section resultingfrom α-helix to β-sheet transition [44–46].

Trends with burial time

The visual comparisons of buried bones show a consistentdecrease in organic Raman bands with increasing burialtime. Figure 2 is a normalized stack plot showing fivesample spectra from each burial interval (between 12–68 days). This plot shows several steady spectroscopictrends. Most noticeable is the broadening and diminishingamide I band. This trend is also visible in the CH2 region(3040–2810 cm-1) and δ NH band. On closer inspection, thetwo lipid peaks appear to have the same diminishing trend(Figs. 3 and 4). Conversely, there appears to be no trendabout the PO4

3- vibrational modes.To prove our hypothesis, peak integration was per-

formed. Peak area was chosen over peak height because itwas seen that Raman bands can shift with burial time, asseen with the previously discussed amide I band. Inaddition, peak area integration was seen to be morecorrelative to burial duration than peak height. Peak area

between points was measured so that there is no contribu-tion from the slight background.

It was discovered that between buried samples, a highdegree of correlation exists when integrated peak area ratioswere plotted against burial duration. The best fit (R2=0.93)was seen when peak area ratio of the v2 PO4

3- and CH2

region were used (Fig. 5). This trend could be caused byenzymatic digestion of collagen via soil bacteria(section Spectroscopic changes with collagenase digestion),although further study is required to confirm this hypoth-esis. As this digestive process continues, the intensity oforganic Raman bands measured from the surface of aburied bone decreases. Spectra taken from bones that werenot buried do not fit to this trend line probably due to theaforementioned dissolution burial dynamic. Although the v1PO4

3− (centered at 960 cm−1) band is the most intense andprominent, it did not show as high a correlation. This maybe because this band overlaps with a collagen peak at920 cm−1.

1600 1400 1200 1000 800 600 400

Inte

nsity

(ar

bitr

ary)

Raman Shift (cm-1)

Amide I Amide IIINHv2 PO4

3-

v1 PO43-

v4 PO43-

D

E

C

B

A

Fig. 2 Raman stack plot of buried bone spectra (A–E). Burial durationin days A 12, B 26, C 41, D 55, E 68

3050 2950 2850 2750

D

E

C

B

A

Raman Shift (cm-1)

Inte

nsity

(ar

bitr

ary)

Fig. 3 Normalized Raman spectra of the CH2 region of buried bonesegments (A–E). Segments buried between 12 and 68 days; A 12, B26, C 41, D 55, E 68

Potential application of Raman spectroscopy 2515

The cluster ranges for each sampled group are suffi-ciently narrow such that a given point could be confusedwith its neighbor, but not two groups away. This dataanalysis is shown to be highly sensitive to changes in burialduration in the scale of days. In addition, no advancedstatistical manipulations were required, increasing thechances of widespread practical use. It should be notedthat unburied bone (t=0) does not fit onto this trend line.As discussed in Spectroscopic changes with burial, thebone segment for this period (0–12) exhibited an increasein organic Raman bands, which is contrary to the trend seenlater. Whether or not the burial duration of bone samplesburied between 0–12 days could be accurately extrapolatedusing this approach is unclear.

A non-monotonic change in Raman spectra found fortwo time periods, first 12 days and 12–64 days could causea possible limitation for practical applications, in particular,the determination of the burial duration. However, twopoints are noteworthy here. First, we plan to investigate indetail Raman spectroscopic changes for 0–12-day period (itwas out of scope for this initial study) and evaluate whetherthere is a possibility to discriminate spectra obtained for thetwo periods of time. Secondly, in a practical setting, we donot imagine that a bone that has been buried for a few dayswould likely be confused with one buried for a few weeks.First, there are visual changes to bone both macroscopicand microscopic. Our bone turned a sulfur yellow tint withweeks of burial. Microscopically, pores and cracks form inthe bone super-structure after months of burial. Chemically,there are several irreversible processes which could beprobed to discriminate such bones, like citrate content,collagen loss, and ion exchange. Furthermore, such a recentburial site could be identified by the disturbances in soiland vegetation in contrast to a burial site months old. Areview of bone diagenetic processes which could be usedfor dating are reviewed by REM Hedges [23].

Spectroscopic changes with collagenase digestion

The observed trend of initial enhancement of organicRaman bands followed by slow diminishing organic bandswas an unexpected result. Specifically, the initial enhance-ment of organic bands is unusual because the diagenesis ofbones is associated with collagen loss. We believe this trendcould be a result of simultaneous processes effecting thechemical nature of bone such as (but not limited to) mineralphase dissolution, lipid migration, or enzymatic attack ofcollagen leading to leaching and/or protein denaturing. Ofthese processes, the enzymatic action on bone is easiest toexplore as a possible mechanism.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 20 40 60 80

PO

4v 2

\C

H r

egio

n

Days Buried

Fig. 5 Preliminary model which was compiled using integrated peakareas. CH 3,040–2,810 and PO4 v2 485–400 cm−1. Equation of best fitline is Y=0.0072 X+0.12, R2=0.93

1350 1300 1250 1200

1295

Amide III

Raman Shift (cm-1)

Inte

nsity

(ar

bitr

ary)

D

E

C

B

A

Fig. 4 Amide III region of buried bone segments A–E. Vertical linerepresents Raman shift position of 1,295 cm−1. Burial duration in daysA 12, B 26, C 41, D 55, E 68

1600 1400 1200 1000 800 600 400

Raman Shift (cm-1)

Inte

nsity

(ar

bitr

ary)

Amide I Amide IIINH v2 PO43-

v1 PO43-

v4 PO43-

Fig. 6 Raman stack plot of unburied bone spectra (bottom blue),buried bone segment (red middle), and bone artificially digested incollagenase (green top)

2516 G. McLaughlin, I.K. Lednev

To investigate the mechanism of this trend, enzymaticdigestion of bone tissue over time was monitored usingRaman spectroscopy. A small segment of turkey bone wasimmersed in a collagenase solution. Immersion in anenzymatic solution is not analogous to the burial of bonetissue; however, it could help explain the burial trend.Spectra obtained from this segment were compared withspectra from buried bone segments.

The bone fragment immersed in collagenase was sampledafter a period of 1, 2, and 5 days. A spectrum obtained after5 days of immersion is shown in a stack plot in Fig. 6. In thiscomparison between an unburied bone, a buried bone, and acollagenase digested bone, there are many similarities seenbetween the buried sample and the sample artificiallydigested. Both show an enhancement of the (CH2 region,amide I, δ NH, and amide III) organic Raman bands. Theenhancement of the amide I band in the artificially digestedsegment is not as dramatic as segment that was buried anddid not shift in position as seen in the buried segment. This isevidence that the enhancement of these Raman organicbands is, at least in part, a result of microbial digestion.

It should also be mentioned that the spectrum immersedin collagenase is absent of the previously assigned lipidshoulder peaks. The nature of bone lipids migration in asoil environment has not been adequately described in thescientific literature. Lipids in bones can be informative forarcheologists using carbon isotope analysis [47].

Continuing work and development of a predictive model

Our laboratory is devoted to continue this research to developa predictive Raman spectroscopic model to extrapolate boneburial duration which factors in a range of natural burialparameters such as soil pH, burial temperature, humidity, andthe presence of soft tissue. These variables could dramaticallychange the rate of bone diagenesis. Therefore, it will berequired to further experiment with these factors in addition tomore realistic burial settings with human bone. Since this is amultivariable system, advanced statistical modeling wouldneed to be implemented. The exact mechanism for the trendsobserved needs to be pinpointed. It is also necessary to extendthe experiment to ensure the observed trends continue withincreased burial time. With further research in this direction,information derived from Raman analysis could be used tocomplement existing techniques for the dating of skeletalremains. This would be a significant contribution to the fieldand a valuable tool for forensic investigators.

Conclusions

The application of Raman spectroscopy to study dynamicchemical changes in bone tissue during short burial

intervals was successful. Short burial durations are perhapsthe most forensically relevant, however, few chemical-basedmethods target this time period. Dramatic spectroscopicfeatures that reflect collagen structural conformationalchanges were observed in the amide I region upon burial ofa bone fragment. Most notable of these changes were theenhancement of several organic bands. This enhancement isconsistent with a previously published hypothesis for themechanism of enzymatic action on buried bone tissue [43].This trend is also seen with artificially digested bone. Withinburied bone, spectroscopic trends proved to be highlycorrelative with burial duration. With such positive results,the potential of Raman spectroscopy to extrapolate anestimated burial duration in a practical sense seems possible.

Acknowledgments We would like to thank the University at Albanybenevolence fund for providing financial support for this researchproject and Aliaksandra Sikirzhytskaya for designing the onlineabstract figure.

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2518 G. McLaughlin, I.K. Lednev