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Spectrochimica Acta Part A 68 (2007) 1089–1095

Raman microspectroscopic studies of amberresins with insect inclusions

Howell G.M. Edwards a,∗, Dennis W. Farwell a, Susana E. Jorge Villar b

a Chemical and Forensic Sciences, School of Life Sciences, University of Bradford, Bradford BD7 1DP, UKb Area Geodinamica Interna, Facultad de Humanidades y Educacion, Universidad de Burgos, Calle Villadiego s/n, 09001 Burgos, Spain

Received 19 October 2006; received in revised form 22 November 2006; accepted 24 November 2006

bstract

Raman microscope spectra of specimens of Baltic and Mexican amber resins containing insect inclusions have been analysed using near-infraredxcitation to assess the potential for discrimination between the keratotic remains of the insects and the terpenoid matrix. For the Mexican amberpecimen the insect spectra exhibit evidence of significant protein degradation compared with the insect remains in the Baltic amber specimen.n both cases the Raman spectra of the insect remains are still distinguishable from the amber resins. Despite its better preservation, however, nopectra could be obtained from the inside of the larger insect preserved in the Baltic amber in agreement with the observation that most insectnclusions in amber are hollow. It is noted that the Mexican amber insect is located adjacent to a large gas bubble in the amber matrix, to which

he observed degradation of the insect and its poor state of preservation are attributed. It is concluded that Raman spectra of insect inclusionsan provide useful information about the chemical composition of the remains and that confocal microscopy is particularly advantageous in thisespect. 2006 Elsevier B.V. All rights reserved.

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eywords: Raman microspectroscopy; Amber; Copal resin; Insect inclusion; K

. Introduction

Although amber is used generically [1] to describe a range ofossilized resins which occur in the geological record as “organicinerals” [2–6], amber should strictly be applied to geologi-

ally mature samples whereas the younger resins, usually fromp to about 2 Mya are better described as copals. Much inter-st is now being generated in this fossilized material [7] whichas recently been adopted into the classification of minerals,ven though the latter are strictly crystalline compounds of well-efined chemical composition; other members of this class areuch rarer and include duxite, fichtelite, ravantite, idrialite andelllite. Maturation is a complex process involving mechani-

al and chemical changes which occur in the resins on burial7]; oxidation especially can occur of the labdane-based diter-

enoid structure (Fig. 1) which comprises the main componentf amber and copal resins assisted by thermal breakdown intomall molecules which have increased mobility through the resin

∗ Corresponding author.E-mail address: h.g.m.edwards@bradford.ac.uk (H.G.M. Edwards).

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386-1425/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.saa.2006.11.037

tic degradation

atrices. The effect of pressure and humidity changes on ambernd copal resins in their burial environments can also produceracks, striations and cloudiness in the resins.

Of particular interest to the subject of this paper is the pres-nce of inclusions in amber resins which have been identifiedy specialists as plant detritus (seeds, leaves and bark frag-ents), bubbles of trapped air and water which can make the

mber appear milky white in colour—the so-called bone amber,ineral crystals such as pyrites and insect, reptile and animal

emains. The latter in particular have excited much generalublic interest for many years and large specimens of amber con-aining insects or reptiles are now considered extremely valuableommercially as well as scientifically [1]. Contrary to popularelief, however, insect remains in ambers are often hollow andence only the shell or carapace of hard keratin remain [8,9].herefore, the fond expectation that the analysis of insect andlant inclusions in amber would provide a time capsule of theora and fauna which were extant tens of millions of years ago

s not generally realisable; the extraction of DNA from theseemains for their regeneration is not normally achievable andven in the most favourable cases will generally result in theomplete destruction of the specimen.

1090 H.G.M. Edwards et al. / Spectrochimica

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ig. 1. Molecular structure of the labdane skeleton, the major diterpenoid con-tituent of amber.

Because of the wide range of colours found in ambers andopals, ranging from a pale yellow-white to a very dark brown,nd their ease of working they have been prized for jewellerynd decorative work for thousands of years. However, somelever forgeries have been perpetrated which have called for aeans of non-destructive forensic analysis for suspected items

nd Raman spectroscopy has been applied in this direction10].

Previous reports of Raman spectra of ambers and copals inhe literature have identified some characteristic features andignatures which are indicative of resin maturation [10–12] butt is apparent that it is difficult to attribute specimens to specificeographical locations because of sample degradation with age.t was noted, however, that Borneo amber, dating from the mid-le Miocene ca. 17 Mya, could be identified uniquely from itsaman spectra because of the presence of triterpenoids in addi-

ion to the labdanes that normally comprise the resin matrices.ther Raman work in the literature [13,14] has concentrated on

he relative intensity measurements of the two strong bands inmber and copals at about 1646 and 1450 cm−1, respectively,rising from C C conjugated stretching and CH2 deformation.hese data suggest a definitive method for the determination of

he extent of resin maturation, and hence the age of the resins,sing calibrated samples for the analyses [11,13–15]. In con-rast, the infrared spectra of ambers and copal resins [16,17]ndicate that the presence of a “Baltic shoulder” due to a suc-inic acid component in the 1250–1175 cm−1 range on the strong–C stretching mode at 1150 cm−1 can be used to discriminateetween resins this location and analogues from other Europeanources, such as Sicily, Poland and Lithuania. Unfortunately, theresence of this feature also in several North American ambersimits the usefulness of this result for diagnostic geographicalurposes.

In this present study we have investigated the potential ofaman spectroscopy for the observation of insect inclusions inmber resins for the first time which sets a specific challenge forhe following reasons:

The insect remains are expected to be keratotic and of an

unknown state of preservation; the protein signatures fromthese inclusions are predicted to be very close in wavenumberto those of the supporting amber matrix, namely, 1660, 1450and 1220 cm−1 compared with 1650, 1440 and 1260 cm−1.

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Acta Part A 68 (2007) 1089–1095

In older, matured resins the chemical degradation of the ker-atotic protein in the insect inclusions and of the labdanes inthe supporting amber matrix will be expected to result in aband broadening of the significant spectral features which areessential for the discrimination between the inclusion and thesurrounding amber.The instrumental conditions under which the spectral dataare obtained are perceived to be critical for the discriminationbetween inclusion and the surrounding matrix; for example,wavelength selection for the excitation of the spectra to min-imise fluorescence emission (which is known to increase foraged and degraded archaeological samples), and the influ-ence of the focal cylinder dimensions on the discriminationbetween the protein and resin.

As a result of these experiments, it should be possible to assessor the first time the potential for Raman microspectroscopyor the identification of the molecular composition of the inclu-ions with the view to understanding their state of preservationnd also from a possible forensic aspect whereby the fraud-lent incorporation of artificial material as an inclusion in anmber resin to greatly enhance its value would be detectableon-destructively.

. Experimental

.1. Raman spectroscopy

Excitation of Raman spectra in the near infrared at 1064 nmas accomplished using a Bruker IFS 66/FRA 106 instrumentith a Ramanscope dedicated Nikon microscope attachment andNd3+/YAG laser operating at a maximum power of 25 mW at

he sample to avoid degradation and thermal damage. To effectnhanced signal-to-noise ratios 4000 scans were accumulatedt a spectral resolution of 4 cm−1 using a 100× lens objective;n this arrangement the focal cylinder waist is normally about0 �m in diameter. Each accumulated spectrum required abouth to record and several sample regions were examined for

he amber matrix and insect inclusions; for each insect inclu-ion the laser was imaged onto the thoracic-abdominal area,hich provided the largest region of keratotic presence. Bandavenumbers of the sharpest features are accurate to ±1 cm−1

r better.A Renishaw InVia confocal Raman spectrometer with a ded-

cated Leica DMLM microscope was used with 785 nm diodeaser excitation and a long working objective of 50× magnifica-ion; in this arrangement 60 accumulations were necessary, eachf about 10 s exposure time to achieve spectra of the desireduality with a laser power of 2 mW. Each accumulated spec-rum therefore required about 30 min of scan time. The focalylinder waist in this arrangement is formally about 1–2 �m in

In each case the spectra were essentially fluorescence-freend it was possible to record vibrational bands of amber resinsver the wavenumber range 100–2000 cm−1 without evidencef background emission.

H.G.M. Edwards et al. / Spectrochimic

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ig. 2. Insect inclusion in Baltic amber (Jurassic–Late Eocene, 195–38 Mya).

.2. Specimens

Although a range of ambers and copal resins have been stud-ed by Raman spectroscopy hitherto [10–15], the main featuref the present study is the assessment of the spectral data thatould be obtained from insect inclusions in the amber matrices.or this purpose two specimens were analysed here:

Baltic amber (Jurassic–Late Eocene, 195–38 Mya), orange incolour, with an insect inclusion (Fig. 2) of length approxi-mately 4 mm located very close to and parallel to the upper

ig. 3. Insect inclusion in Mexican amber (Upper Oligocene–Lower Miocene,0–20 Mya).

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a Acta Part A 68 (2007) 1089–1095 1091

surface; the insect is of the “stinging” type (bee, wasp) andmay be classified [1] as either Parasitica, Mymarommatid(Chalidoidea) or Hymenoptera, Apoidea (Apis mellifera). Anespecially interesting feature of this insect under visual micro-scopic examination is the presence of a diffuse mass in thethoracic region of the body.Mexican amber (Upper Oligocene–Lower Miocene,30–20 Mya), a pale yellow in colour, containing a large-winged insect inclusion (Fig. 3) of approximately 0.5–1 mmin body length which is situated close to the upper surface ofthe resin which has been characterised as an alder fly/cranefly in type, Mecoptera/Neuroptera. Additional features makethis inclusion interesting for spectroscopic study in that thebody is angled away from the upper surface, the head ismissing and there is an adjacent elongated gas bubble whichhas probably been created by the agitation of the entrappedinsect on coverage by the fluid resin. The absence of theinsect head and the added complication of the wing foldingprevents a more detailed description of the insect species.

. Results and discussion

Both long wavelength laser excitations produce very simi-ar Raman spectra of good quality from the amber matrices ofhe two specimens provided for analysis. As determined previ-usly, excitation of amber resin spectra using lower wavelengthasers is severely compromised by background emission whichs sufficiently great to swamp the weaker Raman spectral bands.

.1. Spectral assignments of resin matrices

The wavenumbers and band assignments of the Raman spec-ra from Baltic amber and Mexican amber resin specimens haveeen described in the literature [11–15].

The Raman spectra of the Baltic and Mexican ambers arehown in the spectral stackplots in Figs. 4 and 5, respectively, andre tabulated in Table 1; here, the spectra excited using the 1064nd 785 nm laser lines are seen to be of comparable quality withinimal interference from broad-band fluorescence emission

ackground. The two wavenumber regions selected in the figuresescribe primarily CH stretching between 2700 and 3100 cm−1

nd skeletal functionality modes in the 1700–200 cm−1 region.he vibrational band assignments follow from our own previousork and others in the literature. It is clear that the Raman spectraf the Baltic and Mexican ambers are indeed very similar to eachther superficially but there are several rather minor differencesxhibited, which can be summarised as follows:

Baltic amber has a weak feature at 3080 cm−1 which can beassigned to unsaturated C CH bonds, whereas this feature isnot seen in the Mexican analogue. It has been suggested in pre-vious work that this band is characteristic of an immature resinand is the C CH2 exocyclic methylene symmetric stretch-

ing mode on the C7–C18 position of the labdane skeleton[11–15]. Its presence in our sample of Baltic amber, there-fore, is strongly suggestive of a younger resin and belies itsformal classification to the Jurassic–Late Eocene period as

1092 H.G.M. Edwards et al. / Spectrochimica

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ig. 4. Fourier-transform Raman spectrum of Baltic amber; 1064 nm exci-ation (a) wavenumber region 2500–3500 cm−1, (b) wavenumber region0–2000 cm−1.

first believed. This result is very closely similar to a “youngBaltic” North German amber specimen which we have pre-viously studied [11]. In contrast, the Mexican specimen isdevoid of a spectral band in this region and must be considered

a mature resin.The major Raman bands in the CH stretching region for boththe Baltic and Mexican specimens occurring at 2930, 2869,2849 cm−1 and 2922, 2867, 2847 cm−1, respectively, are not

ig. 5. Raman spectrum of Mexican amber: 785 nm excitation, wavenumberegion 150–3200 cm−1.

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Acta Part A 68 (2007) 1089–1095

definitive enough to provide an unambiguous geographicalsourcing protocol from other specimens considered hitherto.The doublet bands at 1659 and 1645 cm−1 in the Balticamber are replaced by a singlet at 1651 cm−1 in the Mexicansample, which again reflects the higher C C content fromnon-conjugated moieties of the Baltic amber specimen andits attribution to a younger resin.Other band wavenumber differences between Baltic and Mex-ican ambers are the CH2 deformation at 1472 cm−1 in thelatter which has no counterpart in the former, a band at1362 cm−1 with an analogue at 1354 cm−1 for the Baltic andMexican specimens, with CH2 scissors modes at 1295 and1284 cm−1, CH2 rocking modes at 979 and 973 cm−1, CCstretching modes at 746 and 721 cm−1 and ring deformationmodes at 552 and 520 cm−1 all provide some means of dif-ferentiation between the two specimens to greater or lesserextent. In this respect, the CC modes near 700 cm−1 offer areasonably good discrimination between ambers from differ-ent sources since Colombian and East African samples haveanalogous bands at 697 cm−1, whereas Burmese amber has aband at 716 cm−1.

From these data the Raman spectral characterisation of thember matrix in each specimen is defined.

.2. Insect inclusions

A particular focus of this paper is the demonstration of theotential of Raman spectroscopic techniques for the analysisf inclusions in biomaterials; although these have been applieduccessfully to the analysis of organic cyanobacterial strata inocks and minerals [18,19] and also to the presence of inorganicomponents in an organic matrix, namely desiccative salts inummified skin [20], it is believed that this is the first time

hat Raman spectroscopy has been used to study organic inclu-ions in an organic host matrix. A key instrumental requirementor the successful accomplishment of this project will proveo be confocal microscopy with near infrared laser excitation,here the depth penetration of the focal cylinder can be carefullyonitored and controlled.Currently, there is some controversy over the effective spatial

esolution actually achieved in confocal Raman microscopy andt is appropriate to consider this situation here as it is vital to thenterpretation of the results obtained from the insect inclusionsn amber.

.3. Laser focal dimensions

Raman spectra are collected from a focal cylinder whoseimensions are dependent upon the wavelength of the excitingadiation, the focal length of the objective lens and the diameterf the laser beam; the key parameters of length of focal cylin-er and the diameter of the focal waist are defined by the lens

umerical aperture and its magnification (Fig. 6) [21]. However,t was soon realised that scattered radiation originating fromut-of-focus planes situated above and below this focal cylin-er contributed significantly to the observed Raman intensity.

H.G.M. Edwards et al. / Spectrochimica Acta Part A 68 (2007) 1089–1095 1093

Table 1Raman wavenumbers for amber specimens and insect inclusions

Insect inclusion

Baltic amber Mexican amber Baltic amber Mexican amber Assignment

3080 w C CH2 stretch2930 s 2922 m CH stretch2869 m, sh 2867 m CH stretch

2849 m, sh 2847 m, sh CH stretch1665 mw C O stretch1652 mw C O stretch

1659 s C C stretch1651 s C C stretch

1645 s C C stretch1602 mw C CH aromatic1583 mw C CH aromatic

1472 m CH2 bend1450 w 1442 w, br NH bend

1362 mw CH2 bend1354 mw CH2 bend

1332 w, br CH2 bend

1295 m 1284 m 1290 w, br CH2 bend1260, 1240 m CH2 bend

1201, 1182 mw CCH bend1003 w 1001 s CC aromatic

979 mw 973 mw 963, 855 mw CH3 rock815 w 793 mw CC stretch

746 mw 721 mw CC stretch620 m CC bend aromatic

556 w 520 w CCC bend

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abaksblat et al. [21] found that for a sample comprising par-iculates of 40 �m, some 20% of the total scattered radiationollected through a 300 �m pinhole for 514.5 nm laser excita-ion came from scatter planes that were >5 �m from the focalaist plane.This report gave rise to much controversy about the true

imensions of the focal cylinder in Raman microscopy which areritically relevant for confocality and actual dimensions of focal

ylinders were suggested [22–24] to be considerably in excessf the theoretically calculated values, often by as much as 100%.major factor in the poorer axial resolution achievable seemed

o be the refraction at the air-specimen interface but the situa-

ig. 6. Laser focal cylinder dimensions, defining the beam waist and depth ofocus.

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ion is made even more complex because the axial resolutionas dependent on the focal distance and depth of focus within

he specimen [19]. In subsequent work it was confirmed that thealculated depth of focus dimensions were underestimated forarger cross-section specimens but approximated very well forhin films [25,26]. Additional distortion produced by sphericalberration resulting in a diffusion of the focal cylinder is depen-ent upon sample refractive index and is critical for large lensumerical apertures [27–29].

The conclusion is that one cannot be definite about the preci-ion of the depth of focus actually achieved in confocal Ramanicroscopy and that Raman spectra recorded from subsurfaceaterial in transparent matrices are subject to contributions from

djacent layers, which are not formally appreciated theoreticallyut which are highly significant experimentally.

.4. Raman spectra of insect inclusions in amber

In the light of the reservations cast upon the actual spatialepth resolution achieved, it is relevant to assess the importance

f confocality in the recording of Raman spectra from the insectnclusions in the amber specimens studied here, since both areituated several mm below the polished surface of the specimens.t will be especially critical to be able to detect the presence of the

1094 H.G.M. Edwards et al. / Spectrochimica Acta Part A 68 (2007) 1089–1095

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ig. 7. Fourier-transform Raman spectrum of insect inclusion in Baltic amber:064 nm excitation, wavenumber range 50–1800 cm−1. The spectrum indicatesell-preserved keratotic signatures.

xpected keratotic signatures from the insect bodies in the formf the amide I, NH deformation and CH2 deformation bandsf the proteins around 1660, 1450 and 1240 cm−1, respectively,hich are likely to be compromised with the bands occurring

n the amber host at 1660–1650, 1470 and 1280 cm−1. Anotheromplication may arise if degradation of the insect inclusionsas taken place since their deposition in the resin matrix, whichill result in wavenumber-shifted and broadened bands.Using 1064 nm excitation, FTRS and the Baltic amber spec-

men, the spectrum of the insect inclusion is revealed as aell-preserved keratotic signature (Fig. 7); here, the major pro-

ein features in the range 1660–1200 cm−1 are observed withoutny evidence of interference from the surrounding amber matrix.owever, with the insect inclusion in the Mexican amber andTRS with 1064 nm excitation the situation is very different,ince now only the spectrum of the amber matrix is seen andhere are no discernible bands arising from a protein componentn the insect inclusion. The laser focal cylinder in this experi-

ent is estimated at about 10 �m diameter waist and a depth of00 �m.

Using the confocal Raman microscope and 785 nm excita-ion, however, the spectrum of the insect inclusion in Mexicanmber is shown in Fig. 8; clearly, this is not that of the amber hostatrix and can be assigned to protein, albeit heavily degraded.he bands at 1602 and 1583 cm−1 are assignable to the CCHuadrant stretching modes of aromatic species, 1442 cm−1 toH2 deformations, 1201, 1182 and 1154 cm−1 to CCH bendingodes, 1030 cm−1 to C–O modes, 1001 cm−1 to aromatic ring

reathing and 620 cm−1 to aromatic ring deformation. Otherroad bands at 1332, 1290 and 793 cm−1 can be assigned toliphatic CH bending and CC stretching modes.

This result indicates that severe protein degradation hasndoubtedly occurred in this inclusion and that aromatic com-ounds have been produced.

The difference in the discrimination between the inclusion

nd host for the Mexican amber specimen using the two spec-rometer systems is striking and the success of the 785 nmxcitation can be attributed to the tighter focal cylinder dimen-ions of the confocal microscope operation relative to the poorer

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ion, wavenumber range 150–1750 cm . The major features of amber (Fig. 5)nd keratins (Fig. 7) are absent and the spectrum is characteristic of degradedroteins.

ampling effected using the FTRS instrument at 1064 nm. Inontrast, the excellent discrimination effected using the FTRSnstrumentation at 1064 nm for the Baltic amber inclusion,hereby the Raman spectral signatures from the insect inclu-

ion are significantly different from those of the host matrix andan be assigned to keratotic proteins requires an explanation;he probable reason for this must surely be attributed to the rela-ively large size of the insect inclusion in the Baltic amber, which

eans that the focal dimensions of the imaged laser beam cane contained therein, hence permitting the Raman scattering toccur from essentially completely within the inclusion itself. Aess obvious but rather significant factor in the case of the Mex-can amber insect inclusion is the damage that has occurred tohe keratin in the insect body—no signatures remain for proteinands here, as detected using the confocal microscope, and its clear that extensive degradation has occurred relative to thealtic amber analogue. Close inspection of the insect inclusion

n the Mexican amber specimen reveals that a large gas bubble isituated adjacent to and in contact with the insect remains; oxy-en and water vapour in this bubble would therefore have beenn contact with the insect body after its entombment in the hostmber matrix which could have assisted in the complete degra-ation of proteins and DNA in the insect remains; this appearsot to have occurred for the Baltic amber insect inclusion.

The preservation of ancient DNA and proteins in archaeo-ogical biomaterials is a complex issue, but some key work inhe literature relating directly to the preservation of insect tis-ue in amber has highlighted the role of water in the destructionf the proteins and the formation of aromatic compounds suchs syringyl and guiacyl phenols from the depolymerisation ofNA [30,31]. This subject is worthy of further investigationith respect to the preservation of ancient tissues in amber andaman microspectroscopy could have an important analytical

ole in this.

. Conclusions

From the Raman spectroscopic studies reported here, whichepresent the first report of the analysis of relict life inclusions in

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rganic minerals, it can be inferred that the use of near-infraredavelength excitation coupled with a confocal Raman micro-

cope arrangement provides the best discrimination opportunityor the observation of protein and degraded protein signatures inn amber host matrix. Of the two specimens selected for studyn this work, spectral data demonstrate that the excellent state ofreservation of one is in stark contrast to the very poor state ofreservation in the other analogue; this is not immediately obvi-us from a visual observation of the specimens, but a possiblexplanation for the difference noted in the spectroscopic resultsor both specimens could be attributed to the presence of a largeas bubble in close proximity to the insect inclusion in the poorlyreserved sample which could have assisted in the degradationnd decomposition of the proteins in the relict remains.

Although FTRS with 1064 nm excitation is a powerful tech-ique for the generic studies of systems of this type, it possesseslarger focal cylinder from which the scattered Raman radiation

s collected and this can be a problem for smaller inclusions inhe discrimination between the spectral signatures originatingrom the relict inclusion and from the host matrix. The largernsect inclusion in the Baltic amber sample posed no problemor this discrimination and spectra of reasonable quality werebtained from the relict inclusion material with no interferenceeing observed from the amber host.

The potential of Raman spectroscopic techniques for theon-destructive analysis of relict biomaterials in closely sim-lar organic host minerals has been demonstrated and the use ofhese techniques for the screening of specimens for molecularreservation studies prior to the application of destructive char-cterisation techniques or for the extraction of amino acids andNA is advocated.

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