From Research to Diagnostic Application of Raman Spectroscopy in Neurosciences: Past and Perspectives

Abstract

In recent years, Raman spectroscopy has been more and more frequently applied to address research questions in neuroscience. As a non-destructive technique based on inelastic scattering of photons, it can be used for a wide spectrum of applications including neurooncological tumor diagnostics or analysis of misfolded protein aggregates involved in neurodegenerative diseases. Progress in the technical development of this method allows for an increasingly detailed analysis of biological samples and may therefore open new fields of applications. The goal of our review is to provide an introduction into Raman scattering, its practical usage and also commonly associated pitfalls. Furthermore, intraoperative assessment of tumor recurrence using Raman based histology images as well as the search for non-invasive ways of diagnosis in neurodegenerative diseases are discussed. Some of the applications mentioned here may serve as a basis and possibly set the course for a future use of the technique in clinical practice. Covering a broad range of content, this overview can serve not only as a quick and accessible reference tool but also provide more in-depth information on a specific subtopic of interest.

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Klammingeretal
Correspondingauthor:
FelixB.Kleine‐Borgmann∙DepartmentofOncology(DONC)∙84,ValFleur∙1526Luxembourg∙Luxembourg
felix.kleineborgmann@lih.lu
Submitted:31May2022∙Accepted:17July2022∙CopyeditandLayoutby:JerryLou∙Published:05August2022
Abstract
Keywords:Ramanspectroscopy,Neurooncology,Neurodegeneration,Neurosurgery,Neuropathology,Machinelearning
1SaarlandUniversityMedicalCenterandFacultyofMedicine,Homburg,Germany
2NationalCenterofPathology(NCP),Laboratoirenationaldesanté(LNS),Dudelange,Luxembourg
3LuxembourgCenterofNeuropathology(LCNP),Dudelange,Luxembourg
4LuxembourgCentreofSystemsBiomedicine(LCSB),UniversityofLuxembourg(UL),Esch‐sur‐Alzette,
Luxembourg
5DepartmentofCancerResearch(DoCR),LuxembourgInstituteofHealth(LIH),Luxembourg,Luxembourg
6DepartmentofLifeSciencesandMedicine(DLSM);UniversityofLuxembourg;Esch‐sur‐Alzette,Luxembourg
7FacultyofScience,TechnologyandMedicine(FSTM),UniversityofLuxembourg,Esch‐sur‐Alzette,Luxembourg
Inrecentyears,Ramanspectroscopyhasbeenmoreandmorefrequentlyappliedtoaddressresearchques‐
onsinneuroscience.Asanon‐destrucvetechniquebasedoninelascscaeringofphotons,itcanbeusedfor
awidespectrumofapplicaonsincludingneurooncologicaltumordiagnoscsoranalysisofmisfoldedprotein
aggregatesinvolvedin neurodegeneravediseases.Progressin the technicaldevelopmentofthis method al‐
lowsforanincreasinglydetailedanalysisofbiologicalsamplesandmaythereforeopennewfieldsofapplica‐
ons.The goal of our review is to providean introduconintoRaman scaering, itspraccalusage and also
commonlyassociatedpialls.Furthermore,intraoperaveassessmentoftumorrecurrenceusingRamanbased
histologyimages aswell as the search for non‐invasiveways of diagnosis inneurodegeneravediseases are
discussed.Someoftheapplicaonsmenonedheremayserveasabasisandpossiblysetthecourseforafu‐
tureuseofthe techniqueinclinicalpracce.Coveringabroadrangeof content, this overviewcanservenot
onlyasaquickandaccessiblereferencetoolbutalsoprovidemorein‐depthinformaononaspecificsubtopic
ofinterest.
FromResearchtoDiagnosticApplicationofRamanSpectroscopyin
Neurosciences:PastandPerspectives
Gilbert Georg Klamminger1,2,3, Katrin B.M. Frauenknecht2,3, Michel Mittelbronn2,3,4,5,6,7, Felix B. Kleine‐
Borgmann2,3,1,5
Review
FreeNeuropathology3:19(2022)
doi:hps://doi.org/10.17879/freeneuropathology‐2022‐4210

Introduction
As one special method of various vibraonal
spectroscopic techniques, Raman spectroscopy
(RS) has been an integral part in neuroscience re‐
searchforsomemenow,beitinneuro‐oncology
fortumorclassificaon1orforthebiochemicaldes‐
cripon of various protein aggregates in neurode‐
generavediseases2. Currentlyitismaking itsway
towardsa clinicalimplementaon3.Looking at the
numerousadvantagesofRS,thereasonsforanin‐
creaseduseinresearchareobvious:itenablesfast
and user‐friendly (easy to apply) analysis for the
purpose of ssue idenficaon (e.g., idenficaon
ofdifferentbrainregionsin three mice strains4)by
observedchangesinthevibraonalleveloftheun‐
derlying biochemical and molecular composion.
Compared to other advanced molecular techni‐
ques, reproducible results can be obtained with
few requirements regarding sample preparaon.
The insensivity to water molecules predesnes
thetechnologyforitsuseinabiomedicalcontext.
Todate,thevastmajorityofstudiesusingRa‐
manspectroscopy examine unprocessednave,or
frozen ssue/cells ‐ few publicaons make use of
formalin‐fixed or paraffin‐embedded (FFPE) ssue
becauseRaman measurementsremainchallenging
due to the strong contribuon of paraffin wax to
spectralintensity,thin specimens,andadisrupon
of the molecular integrity, which is related to the
preceding fixaon process. The long‐term archiva‐
bility and the large number of available samples,
however,suggestuseofRSFFPE ssue in patholo‐
gyisdesirable,e.g.,fortheanalysisoftumorhete‐
rogeneity, or idenficaon of very small tumor
fragments, which could escape diagnosc high
throughputofhistology samples.Thefollowing re‐
view and perspecve paper is divided into three
parts: a) the basics of RS and the most common
forms of its applicaon in medical research are
presented, b) the use of RS in selected neuros‐
cience disciplines is accentuated with the aim to
present different research quesons – but even
more importantly – the most interesng findings
discoveredwiththehelpofRS,c)afuture outlook
forpotenalapplicaonofRS inresearchbut also
inthe dailyclinical work isprovided.At this point,
the minireview by Payne et al.5 needs to be men‐
oned;itdescribesinaclearwaynot onlyapplica‐
ons ofRSin neuroscience, butalsosetsa special
focuson thetechnical aspectsand benefitsof ad‐
vanced spectroscopy‐based techniques depending
ontheparcularusecase.
Bycontrast,thefollowingworkplacesaspeci‐
al emphasis on topics that will inevitably become
relevant to the praccing spectroscopist at some
point, such as varying ssue sample requirements
in different clinical sengs (surgery department/
pathology department) or common data proces‐
sing methods, to name a few. Whenever it serves
expedienttheaenvereader shall bereferredto
addionalmorein‐depthreading.
Searchforrelevantliterature
A literature search (the search terms
“Raman”, “Raman spectroscopy” were each
combined alternately with the terms “brain”,
“neuro”, “neuroscience”, “brain tumor”, “tumor”,
“neurooncology”, “glioma”, “neurodegeneraon”,
“neurodegenerave disease”, “Alzheimer’s
disease”, “Parkinson’s disease”, “Hunngton”,
“amyotrophic lateral sclerosis”, “prion disease“,
”mulple sclerosis”, “myelin”, “demyelinaon”,
“stroke”“brainischemia”,“braininjury”,“muscular
diseases”, “brain infecons”, “meningis”,
“psychiatry”)wasperformed,andonlinedatabases
PubMed Central® and Google Scholar® were
browsed for relevant reviews and original arcles;
othertypes of literature, such as congress papers,
leers,commentse.g.,wereexcluded.Aersearch
results were idenfied, they were hand‐screened
for eligibility (inclusion criteria: employment of RS
on brain/peripheral nervous/muscle ssue, RS on
extracellular components/cells of the nervous/
muscularsystem,orRSinrelaontoneurological/
oncological/psychological disorders; exclusion
criteria: use of vibraonal spectroscopic
techniques otherthan RS) basedon tle/abstract.
Within the responsibility of the authors, the final
seleconofliteraturewasconductedbasedonthe
arclefulltext.Finally,associatedbibliographiesof
selectedpublicaonswere searchedfor addional
relevant sources that semancally met the search
criteria. Only English language literature was
considered – even though Japanese research
groups describe an employment of Raman
spectroscopyinratbrains,andhumanbrains/
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doi:hps://doi.org/10.17879/freeneuropathology‐2022‐4210
Klammingeretal

brain tumors as early as the 90s6–9. Although
references to historical developments are pointed
out whenever a contemplaon of the historical
context seemed valuable special focus is set on
literature of the years 2021 and 2022, reflecng
ongoing research projects/groups ‐ such as
spectroscopical examinaon of microglial changes
due to SARS‐Cov‐2 exposure10 ‐ using RS in
neuroscience.
PrincipleofRamanscatteringandge‐
neralspectrometersetup
The Raman effect is the process of inelasc
scaering of photons; this effect was first descri‐
bedin1928byC.V.Raman,whoexaminedthecha‐
racteriscs of scaered photons when applying a
lightsourceondifferentliquids11,12.Forhisdiscove‐
ry, the Indian physicist won the Nobel prize in
193013, but despite the discovery of the Raman
effectinthefirsthalfofthe21stcentury,ittookun‐
l late1960sbeforeit wasfirstused inabiomedi‐
calcontext14–17.
The interaconof incident lightwith a mole‐
cule leads to changes in the vibraonal state, so
thatthemoleculefallsintoanexcitedvirtualvibra‐
onal state. When returning to the ground state,
the largest amountof theincident photons is ela‐
scally scaered, which means that the energy of
thescaeredphotonisthesameasthatofthein‐
cidentphoton (=Rayleigh scaering). Only aminor
partofthe scaeredlightexperiencesachange in
its energy compared to the incident light; in fact
whenthemolecule endsupona differentstatein
comparison to the ground state, the photon is in‐
elascally scaered. Depending on the interacon
betweenthemoleculeandthephoton,inelascally
scaered lightcan havea higherenergy (an‐Sto‐
kes effect) or a lower energy (Stokes effect) than
the incident light, whereas in praccal applicaon
mainlyStokesscaerisaributedtoaresulngRa‐
man signal, due to its higher intensity.18 See
Figure1 foravisualizaonofthevibraonalstates
transions.
In order to be Raman‐acve as a molecule,
i.e.,toemitinelascRamanscaering,achangein
polarizability is required ‐ this already shows a
difference to a related and oen confused spec‐
troscopic technique, infrared spectroscopy, in
whichanabsorbedphotonleadstoachangeinthe
dipole moment19. Another phenomenon, also ba‐
sed on absorpon and oen observable as a dis‐
rupve factor in Raman measurements due to its
strongersignalis fluorescence;herethe molecule,
excitedby energyofabsorbedphotons,leavesthe
groundelectronicstateandistransferredtoa hig‐
her electronic state ‐ as soon as it returns to the
groundstate,energy isre‐emiedas fluorescence
light20.
The interacon of photons with their target
molecules resulng in an inelasc Raman scae‐
ringwithadisnctenergydifferencereflectsspeci‐
ficchemical bondsand constuons. Thisspectral
fingerprint can indicate the identy of the target
molecule.A spectrumcan thereforebe definedas
a representaonof the intensity values (based on
the degree of change in polarizability) and the
differing frequencies (Raman shi) in a func‐
on18,20. The x‐axis displays the Raman shi in the
unitwavenumbercm‐1,therebythewavenumberis
reciprocaltothewavelengthandthus directly pro‐
poronalto photonenergy19.Theconvenonalex‐
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Figure 1. Occurring opcal phenomena when irradiang a
biologicalsamplewithaphotonsource(laser).
Le: Vibraonal states (v0, v1, v2) involved in Rayleigh and
Raman scaering. In case of elasc scaering (Raleigh
scaering), incoming photons temporarily change the
vibraonal state of a molecule ‐ aer this excitaon, the
moleculereturnsbacktotheinialvibraonalstate(v0).Inthe
caseof StokesRamanscaering,amoleculegainsenergy due
to the excitaon process and finally ends up in a higher
vibraonalstate(it rises fromv0tov1)–the scaered photon
has lower energy than the incident light. In An‐Stokes
scaering the molecule ends up on a lower vibraonal state
aerexcitaoncomparedtothegroundstate(itfallsfromv1to
v0)–therefore,thescaeredphotongainsenergy.
Right: In contrast, the phenomenon of fluorescence occurs
when a molecule absorbs light and thus is temporarily
transferredtoahigherelectronicstate(v’0,v’1,v’2).

perimentalapplicaonoftheprocessusingthepu‐
re Raman effect is so the called Spontaneous Ra‐
man Scaering (SpRS). Addionally, there are
several derivave methods allowing, for example,
scaering with enhanced signal intensity or redu‐
ced background noise, thus lending themselves to
differentapplicaonssuchasRamanImaging (e.g.,
bycoherentRamanspectroscopy).Table1givesan
overviewofthetechnicalbackgroundandadvanta‐
ges of commonly used variants of RS in neuros‐
cience. For a more detailed insight into the
theorecalaspectsofRStheinterestedreadermay
refertoCialla‐Mayetal.21,whoprovidesacompre‐
hensiveoverviewin thebook“Micro‐Raman Spec‐
troscopy:Theory andApplicaon” by Poppetal.22.
Addionally,Huetal.23,Shietal.24andEvansetal.
25 give a good overview about smulated Raman
spectroscopy (SRS) and coherent an‐Stokes Ra‐
man scaering (CARS); Zheng et al.26 wrote an in‐
strucve review about surface‐enhanced Raman
scaering(SERS).
Theexactstructure of aRaman spectrometer
differs depending on the manufacturer and the
technology used. Only general components and
theirfunconarediscussedbelow;addionalcom‐
ponentssuchasanaddionallaseroraspecialRa‐
man substrate are commonly required in
spectrometer setups of advanced Raman techni‐
ques(Table1).With a focus lens,emied photons
of a laser source are focused on the sample, and
aer interacon with the sample both the elasc
and the inelasc scaered photons are collected
by a collecng lens. The reflected and elascally
scaered light is then separated from the remai‐
ninglight,typicallybyadichroicmirror. A prism or
diffracon grang spaally separates the light ac‐
cording to wavelength, leading it to a detecon
system ‐ a photo paper was employed in the
classicalsetup‐eithersimultaneouslyonacharge‐
coupleddevice(CCD)orthroughamonochromator
onaphotomulpliertube(PMT)(Figure2).
As excitaon source, typically lasers, is used
wherethemannerofphotongeneraonaswellas
thewavelengthdiffer.Commonlyemployedexcita‐
on wavelengths within the biomedical field are
532nm, 785nm, 830nm, or 1064nm ‐ for praccal
applicaonspecificeffectsonthessuetypeofin‐
terestaswellaspotenallyinducedbackgroundsi‐
gnalsmustbeconsideredindividuallyandadapted
according to the experimental setup27. Most em‐
ployed lasers nowadays are diode lasers;w ith the
advantageof portability and favorableenergy effi‐
ciency, they have replaced the gas‐based lasers
(helium neon laser, argon‐ion laser) that were
oenusedinthepast.Thetypeofprotonemission
can be divided into connuous‐wave lasers and
pulsed lasers; the former being more common in
SpRSandthelaerbeingnecessaryinSRS18,28.Itis
necessary to bundle photons both in the suitable
focuson thesample(focus lens)andtocollectthe
scaeredphotons(collecnglens)aerinteracon
with the sample. Next, Raleigh scaered photons
arefilteredby adichroic mirror andseparatedac‐
cordingto their wavelengthusingadiffracongra‐
ng;dependingonthesamplingaperture(exitslits/
pinholes) within the setup, a certain number of
photonsaredetectedinafinalstepbythesensi‐
ve detecon system. While the classical “scanning
spectrometer” employs  a rotatable grid concen‐
trangthephotonsonanarrowexitslitandapho‐
tomulplier tubebehind detecng RS,modern set
ups usually use a CCD detector. This mulchannel
wayofphotondetecon(amulchannelarraychip
consisngofseveralpixels)allowsforsimultaneous
registraon and display of all photons, i.e., the
whole Raman spectra18,28,29. Regular wavelength
calibraon(processoftransferringpixelhitsonthe
CCDdetectortodisnctdisplayedwavenumbers)is
recommendedtoreceivereproduciblespectraover
theenreduraonoftheexperiment28.
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Figure2. Schemacandsimplifiedrepresentaonofa Raman
spectrometersetup.

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Table 1. Summary of commonly used Raman techniques, their physical background and the associated
advantagesanddisadvantages.

Raman spectra can be employed in various
ways.Inaddiontothepossibilityofusingthemas
raw spectra primarily for the idenficaon of bio‐
chemicalcomponentsof a sample,methodscalled
Raman microscopy/imaging use the assignment of
colors to Raman bands (only a limited number of
wavenumbers is acquired or analyzed)30  over a
scannedsampletogeneratecontrast.Whenexten‐
ded to focusing through the depth of the sample,
three‐dimensional Raman images can be built16.
Raman microscopy/imaging techniques31,32 and
computaonal image generaon algorithms have
been advanced to generate Raman images of va‐
rious brain pathologies, e.g., gliomas, stroke and
demyelinaon25 or to image metabolism in the
brain33–35.Usingthisapproachofdatavisualizaon,
itis possibleto obtaina similarlook totradional
H&E‐stained slides on unstained specimen, which
enables histopathological diagnosis36. In Raman
mapping, the whole Raman spectrum for each
pointofthedesiredareaofthespecimenisacqui‐
red(either pointby point orwith anexcitaonla‐
ser forming a line on the sample and measuring
simultaneously); using computaonal analysis
aerwards, a visualizaon of differences in the
spectralproperesofdatapointsisachieved30.
Peakassignment
Raman peaks may occur at first sight in va‐
riousformswithdifferentcharacteriscs. Inaddi‐
ontocertain singlepeaks that appearnarrowand
canbeassignedtoexactlyonecorrespondingfunc‐
onalgroup,an addive effectofseveraladjacent
Ramanacvemoleculesinthesamplecanalsore‐
sult in broad peaks. Furthermore, the presence of
several contribung components, and thus neigh‐
bordependent changesin thevibraonalmode in
onespecimen, mayaffect theactual peak in com‐
parisontoanisolatedmeasurement20.Theapplica‐
on of RS in the biomedical context oen pays
special aenon of the regions within the wave‐
numbers 400‐2000cm‐1 and 2700‐3500cm‐1. These
regions,oen referredtoas "biologicalfingerprint
regions" in the literature, are characterized by a
high proporon of Raman peaks arriving from
funconal groups of a typical biological speci‐
men28.AnintroducontotheuseofRSforidenfi‐
caon ofdifferentmolecular funconalgroupscan
be found in Pezzo et al.37 (RS and cell biology) ,
Czamaraet al.38 (RS and lipids), Rygula etal.39 (RS
andproteins)andWiercigrochetal.40 (RS andcar‐
bohydrates).
ByusingRSonbiomoleculessuchasproteins,
itisnotonlypossibletoidenfymolecularfunco‐
nalgroupsi.e.,differenatebetweendifferentami‐
no acids/proteins, but also spaal confirmaons
can be detected since the Raman signal is influ‐
enced by aromac/non‐aromac side chains and
thebackboneofaprotein.Disnctvibraonsresult
in certain amide bands (Amide band A, B, I‐VII)41;
for example carbonyl stretching modes, N–H ben‐
ding or C–N stretching results in the widely used
AmidI(1600‐1690cm−1),AmidII(1480‐1580cm‐1)
and Amid III peaks (1230‐1300 cm−1). They allow
further examinaon of the pepde secondary
structure. In larger unordered protein measure‐
mentsaprecisepeakaribuonmaynotbepossi‐
bledueitslargenumberofcontributors18,39,42–44.
Lipids are ubiquitous in biological specimen,
asthey form themembranes of cellsand organel‐
les. Depending on the literature, spectral proper‐
es resulng mainly from the hydrocarbon chain
andpartly fromthepolar headgroup can beassi‐
gned to the regions 1050‐1200cm‐1 (C‐C stret‐
ching), 1250‐1300cm‐1 and 1400‐1600cm‐1 (CH2,
CH3 group acvity) or also to the regions below
600cm‐1 and between 1000‐1150 cm‐1 (opposite
moonofcarbonatomsofthehydrocarbonchain).
Consistently,an area within the high wavenumber
region2700‐3500cm‐1(somemessolelytherange
between2800‐3100cm‐1isconsideredinthelitera‐
ture) is reported and aributed in a large part to
stretchingofC‐Hgroups.In‐depthanalyzesofpeak
intensityand distribuon inthe high wavenumber
regionallowconclusionstobedrawnaboutthesa‐
turaon status of fayacidsand the aliphac/aro‐
mac components of steroids18,38,45,46. An
interesng contribuon at this point may come
fromKraetal.45,whoin2005measuredandcha‐
racterized twelve brain lipids and further related
occurringpeakstotheirfunconalgroupsandPez‐
zo et al.47, who employed RS to visualize single
(phospho‐)lipidsinneuronalcells.
Carbohydrates and underlying C‐C and C‐H
structuresgiverisetobondsinvariousareaswithin
the Raman spectrum18. For a long me, minor
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aenonwaspaidto the invesgaonofcarbohy‐
drates.Althoughspecific peak assignmentispossi‐
ble,in comparisonto proteinand lipids itremains
lessspecific40.
About 30 Raman peaks of nucleodes, distri‐
buted over severalareas withinthe spectrum, are
mostly aributed due to purine/pyrimidine ring
modes and phosphate groups (especially peaks
nextto800cm‐1and1100cm‐1).Theyareusefulfor
characterizaon of inter alia DNA, tRNA, and
nucleicacid‐proteincomplexes18,48.
Spectroscopicexaminaonnotonlyallowsfor
examinaon of these specific funconal groups
enumerated above, but also to display their inter‐
acons, such as protein‐protein / protein‐lipid in‐
teracon.Theirchanges inspectralpropertyunder
different condions can also be measured17. On
that note, Lee et al.49 have even managed to use
SRS as a tool in neurophysiology when examining
thespectral properesof neuronalmembranepo‐
tenal.
AlthoughspecificRamanpeakshavebeende‐
scribed for various molecules50–56, one should be
cauous when actually assigning peaks to one's
ownsample.Whilepeaksmaybecharacteriscfor
acertainbiochemicalcompound,theycanalsoari‐
sefromdifferentsources;viz they are not specific.
Inordertocorrectlyassignpeaks/detectthemwi‐
thin a spectrum, itis essenal to reducepotenal
confounders within the sample or the experimen‐
talset‐uppre/post‐experimentally.Apotenalway
toassigndisnctpeakswithhighevidenceisdirect
observaon: Targeted manipulaon of a sample
canhelptoconfirmthesourceofapeak.
Thevibrationalspectroscopicexperi‐
mentalsetup
RSisafast,non‐destrucve,userfriendly,and
easytoapplyontoolprovidingmolecularinforma‐
on with minimal sample preparaon require‐
ments in a reproducible manner. However, a
roune use of RS‐base d tools in neuroscience has
notyetbeen established.Regardlessofthe nume‐
rousadvantagescertainlimitaonshavetobecon‐
sidered not only pre‐experimentally, but also
duringimplementaonofanexperimentandaer‐
wards when visualizing and processing the obtai‐
neddata.
Theoccurrence ofthe physicallyrelatedphe‐
nomenon of (auto‐)fluorescence (photons of the
pump beam are absorbed by molecules of the
sample which are raisedto anotherenergy level‐
whenreturningtothe basicenergylevelaphoton
isemied,see also Figure1)is regularly observed
andtheexpectedintensityinthiscaseiswellabo‐
ve the intensity of the Raman signals. To reduce
wavelength‐ dependent autofluorescence, a dis‐
nct wavelength of the excitaon source can be
selected,orSERScanbeused57,58.Althoughincon‐
trast to other sophiscated laboratory techniques
(e.g.,genec/epigenectesng)therearelessre‐
quirements for a correctly prepared Raman sam‐
ple.Afewthingsneedtobeconsideredinorderto
avoidtheoccurrenceofspectral background noise
and spectral contaminaon: Samples mustbe pla‐
cedonarobustRamansubstratesothattheselec‐
ted measuring point and the focus remain stable.
Dependingontheexperimentalquesonaswellas
the expected background noise and the costs, va‐
riousRamansubstratesareavailable.Inaddionto
goldoraluminum‐coatedglassslides(asafuncon
oftheexcitaonwavelengthglassaloneexhibitsa
strong and broad fluorescence background signal
inthe“biologicalfingerprintregion”),specialslides
(low‐eslides,CaF2slides,quartzslides)canbecon‐
sidered28.Thesearecharacterizedbyalowspectral
backgroundorsinglepeakaribuon. Fullwoodet
al.59 and Kerr et al.60 examined the effect of sub‐
strate choice for spectral histopathology in more
detail.IthasbeenshownthatCaF2slides(exclusive
peak at 321cm‐1 or 322cm‐1 respecvely, depen‐
dingontheliterature)61havetheleastinfluenceon
thespectralbackgroundincomparisontolow‐Esli‐
des and Spectrosil slides. The single background
peakcaneitherbeignoredduetoitsirrelevantoc‐
currence out of the important range of biological
componentswithintheRamanspectrum,orcanbe
subtracted via computaonal analysis aerwards.
As a low‐cost alternave aluminum foil can be
used,whichitselfdoesnotgenerateanysignificant
backgroundnoise62–64.
Furthermore, the sample condion (most
commonly nave/frozen or formalin‐fixed) needs
to be considered pre‐experimentally. Although
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fresh ssue samples allow for a straighorward
aribuon of Raman peaks to underlying bioche‐
mical components, they must be processed and
analyzedwithinacertainmewindowandcannot
be stored for a longer period of me. When wor‐
king with fresh ssue, dehydraon and associated
denaturaon of funconal biochemical groups
need to be prevented e.g., by keeping the speci‐
men hydrated19,65. As an alternave, Raman mea‐
surements of frozen biological samples allow
longerstorageandatthesamemesllgiveanin‐
sightinthebiochemicalcomposionofthebiologi‐
calsample.Nevertheless,itshouldbenotedthata
reducon in certainpeakintensiesandsignificant
alteraon of Ramansignalin comparison tonave
ssue were described when using frozen secons
66,67.
The handling of  formalin‐fixed, methanol‐fi‐
xed,orFFPEsamplesisrouneduringthepatholo‐
gical workflow; even though samples allow long
archivability and are broadly available,t his way of
fixaondamagesthebiologicalRamanspectrumto
acertaindegree since thessueundergoesanag‐
gressive chemical procedure68–72. Both formalin
and methanol fixaon reproducibly alter spectral
ssueproperes and affectRamanbandsassigned
tolipids,proteins,and nucleicacids73. Despitefor‐
malin‐inducedbiochemicalchanges such asforma‐
on of cross‐links in the structure of the amino
acids, spectroscopic assessment and classificaon
of formalin‐fixed biological ssue is possible66; in
contrast,methanol‐fixaonwas reported topoten‐
ally hamper the detecon of ssue malignan‐
cy72,74.
The prominent spectrum of bound paraffin
wax is reflected in certain points at 1063, 1133,
1296and 1441cm‐1, whichmakea manualor digi‐
taldewaxingprocessnecessaryandrequireacare‐
fulinterpretaonoftheobtainedspectra75.Several
condions (aggressive chemical processing, requi‐
redchoiceof specialsubstrateandthefineness of
the ssue) hamper spectroscopic examinaon
whenemployingRSonFFPEssueinthepathology
department, although spaal orientaon on the
sample and proper idenficaon of certain areas
areapotenaladvantage.
IntheliteraturedifferentapproachesusedRS
onprocessedssue;inanycase they allfacesimi‐
lardifficules. Huang et al.68 described theeffects
of formalin fixaon on RS of cancerous human
bronchial ssue, whereas Draux et al.71 described
the influence of formalin and air drying on single
cancer cells and aributed spectral changes to
affecon of nucleic acids and proteins. Even
though not only a loss of the original chemical
composion but also potenal contaminaon due
tothe process offormalin‐fixaon inmurine brain
ssuewas determined byHacke etal.76, several
studies proposed formalin fixaon as a sufficient
and favorable method for subsequent spectrosco‐
picdiagnosc77,78.Asaproofofconcept,Stefanakis
et al.79 demonstrated the feasibility of vibraonal
spectroscopyon formalin‐fixedmalignantbrains‐
sue. Employing vibraonal spectroscopy on FFPE
ssue,aneffectonthelipidcontentduetothede‐
waxingprocesswasreported;nevertheless,Raman
bandsrelatedtocellular andextracellularproteins
weresuccessfullymeasured80.Gaifulinaandcollea‐
gues81 examined large intesne FFPE ssue from
rats and analyzed biochemical signals obtained
with label‐free RS in the processed ssue. Other
groups examined FFPE ssue of rectal cancer to
predict radiotherapy response82, to map/analyze
cervicalssue83,84,oremployed RS onhealthyand
malignantbreast85–88/ovarian89/prostac90 ssuein
variousfixaonstates.Foragoodoverviewonthe
influenceof ssue processing onbiological Raman
spectra the reader may refer to the work from
Faoláinetal.66.
During spectroscopic examinaon, back‐
groundnoiseduetoa nearby photon source(e.g.,
room light) should be considered and reduced by
performingtheRamanmeasurementinadarkened
areaorwithdimmedoperangroomlight91–94.Ad‐
dional methods of spectra quality control during
intraoperave measurement have also been pro‐
posed95,96.By ensuringthat thelaser sengs (wa‐
velength and power, duraon of acquision) are
opmized for the examined sample, the best si‐
gnal‐to‐noiseraocanbedetermined,andthermal
ssuedecomposion can be prevented.This form
ofsampledestruconcanbedetectedbyaburned
areawheretheformerfocusareaofthelaserislo‐
cated,as wellas bythe presence ofan addional
carbon band at approx. 1500cm‐1 in the Raman
spectrum28.
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Dataprocessingandcomputational
analysis
Aer the measurement, the large amount of
data97shouldbe sorted andstoredin a structured
manner (data annotaon) to address the research
queson properly. It is good pracce to start the
data processing with an inial visualizaon of the
data.Inthiswaycleardeviaonsfromanexpected
result such as strong contaminaon or cosmic ray
arfacts (randomly occurring electromagnec ra‐
diaon) andhot pixels(overresponseofa pixelon
the CCD detector to an incoming photon) can be
recognized and corrected28,98,99. For a more detai‐
led reading on potenal anomalies and arfacts
thatmayoccur,seeBowieetal.100.
During data preprocessing, a baseline correc‐
on canbe appliedtothe datatominimalizeresi‐
dualbackgroundsignalandautofluorescence101,102;
a common way to model and subtract the back‐
ground noise to obtain the intrinsic sample spec‐
trum103,104. Addionally, a common way to further
reducethenoiseinthedataisasmoothingtechni‐
que, such as Savitzky‐Golay filtering28,105,106. Both
of the above‐menoned methods must not be
usedwithoutpropercauonasthereisalwaysthe
riskof producingarfacts,as well as equalizing si‐
gnificant data points. In order to correct confoun‐
ders that result from the experiment setup itself
(e.g., slightly different dryness or thickness of the
specimens) data normalizaon methods, such as
min‐max normalizaon or z‐normalizaon, usually
precede the actual data analysis107. Specialized
spectroscopy soware are commercially available
and enable even the inexperienced spectroscopist
touse the acquired data ina structured andcom‐
prehensivemanner108.
Duetothelargeamountofdata,severaldata
reducon methods are used for quick explorave
purposes,aboveallPCA(principalcomponentana‐
lysis)iswidelyemployed.Thisunsupervisedcluste‐
ringtechnique canbe used to determineprincipal
componentsin a big data set, which explainsa si‐
gnificant part of the variance and reduces noi‐
se41,109.
In the last step of computaonal analysis,
classificaon algorithms and machine learning
techniques110,111arecommonlyusedtoclassifythe
spectraldata eitheraccording to pre‐experimental
definedgroups(supervisedclustering)oraccording
tonewgroupsbasedonsimilariesinspectralpro‐
peres(unsupervisedclustering)112.
Awidelyusedtechniqueinunsupervisedclus‐
tering is hierarchical cluster analysis (HCA), in
whichthedataistransferredtoahigher‐dimensio‐
nalspace,clusterinacertainproximitytooneano‐
ther based on similar properes. A number of
cluster variables can be specified individually,
which forms the selected number of similar clus‐
ters103.Unsupervised clusteringisbeneficialforex‐
ploratory research quesons since no prior
knowledge of possible group properes is requi‐
red28.
Commonmethodsusedforsupervisedcluste‐
ringaretrees/randomforestclassificaons(several
decisiontreesinarow)orsupportvectormachines
(search for a hyperplane to disnguish between
classes)91. The groups determined a priori are re‐
ferredtoas "classes" andthe goldstandardhisto‐
pathologyoenservesasgroundtruth.Ingeneral,
thealgorithmistrainedwithatrainingdatasetand
tested with an external validaon data set aer‐
wards.Toavoidoverfing(capabilityofgooddiffe‐
renaon only on the specific training data set) a
validaonofperformancee.g.,k‐foldcrossvalida‐
on or holdout validaon is performed, and metri‐
ces of algorithm performance (e.g., sensivity,
specificity, f1‐score, accuracy, AUROC/AUPR value)
are calculated aerwards based on its output113.
Ralbovskyand colleaguesprovidedan overviewof
machinelearningalgorithmsand their funconsin
Ramanbasedcancerdetecon112.
RSinNeurooncology
Withagrowingnumberofpublicaons in the
lastyears(Zhangetal.114andBanerjeeetal.115de‐
scribed a change in spectroscopic properes of
gliomacellsincomparison to astrocytes already in
the mid‐2000s), the neuro‐oncological field is one
ofthelargestareasofresearchonRS,inwhichthe
therapeucal balancing act betweenmaximum re‐
seconof normal‐brain‐resemblingtumorous resi‐
dues and minimal surgical disrupon of healthy
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brainfunconsprovesparcularlydifficult.
On the subject of RS in (neuro)oncology re‐
views by Auner et al.20 and Hollon et al.117 give a
comprehensive introducon to the respecve to‐
pic;forfurtherreadingonimplicaonsandcurrent
progressofRSinoncologyseealsoSantosetal.117.
Atfirstsight, use ofthisspectroscopictechni‐
quemainlyapplytotwomainresearchfocuses:on
the one hand a spectroscopic detecon of mali‐
gnancy118,119 which in a next steps allows precise,
accurate diagnosis of the tumor ent y intraopera‐
vely withouthavingtowait forfurther tradional
ssueprocessing (pathologicaldiagnosis onfrozen
secons)120,121, and on the other hand real me
surgeryguidance i.e., live feedbackintraoperave‐
ly122,123 aiming for maximal tumor resecon124–126.
Both topics merge and evolve at a certain point;
this may result in new research quesons, e.g.,
whenaimingtodeterminetumorinfiltraonzone/
resecon margin or when aiming for detecon of
tumor genecs on various states of tumor ssue.
Moreover,alsobasicresearchquesonsin oncolo‐
gycanbeaddressedwiththis vibraonalspectros‐
copic technique e.g., monitoring lipotoxicity in
glioblastoma cells127, observing cell response of
U251glioblastomacellsaerinduced apoptosis128,
examining the glycosylaon paern of proteins in
medulloblastoma129, or observaon of redox state
of mitochondrial cytochromes130, just to name a
few. Most research groups use SpRS20 as an easy
toapply,labelfreemethod.MoreadvancedRaman
techniques in neurooncology131are usedpredomi‐
nantlyinanimal models132–134–where Surface en‐
hanced resonant Raman spectroscopy (SERRS)
detecon of tumor margins135 has shown progno‐
sc benefits136,orCARSwas employedfordetec‐
on of different human brain tumors in a mouse
model137.
RSfordetectionoftumorgroup,ge‐
neticalterationandhistomorphology
RS can disnguish between grey and white
maer and (partly)otherbrainregionssuchas ce‐
rebellum, striatum, basal forebrain ‐ both macros‐
copically and on cellular resoluon4,138–146,147.
Interesngly,oneanalysisofthemousebrainusing
SERS revealed a different spectral fingerprint and
thus also different biochemical composion bet‐
ween le and right hemisphere148. Spectroscopi‐
callyfeasiblediscriminaonbetweengliomassue
andbrainssuewasreportedinseveralstudies3,149–
153 as well as between dura mater and meningio‐
ma, which was demonstrated to be based in part
onpeakscorrespondingtocollagenandonthehig‐
herlipidcontentwithin tumorous ssue154–156.Be‐
side these binary classificaon models, several
studies showed the potenal of RS aiming for a
mulc lass classificaon to differenate various tu‐
moreneswithinoneclassifier119,157–166ortode‐
terminetheprimarysiteofmetastasis167,168.
Using Raman mapping/imaging for brain tu‐
morvisualizaon116,169,evenspecial morphological
features of tumors (e.g., necrosis in glioblastoma,
celldensityorindividualcellnuclei)couldbeiden‐
fied170–172. Even though areas of tumor necrosis
are typically characterized by an increased pre‐
sence of proteins such as phenylalanine (around
1032cm‐1,amongothers)aswellascholesteroles‐
ters(1739cm‐1)171,173,one groupproposedtwodis‐
nct spectral properes within the necrosis of
glioblastomacells:“highlynecroc”,showinganin‐
creaseinplasmaproteinsand“peri‐necroc”,exhi‐
bing a higher lipid content174. The
histopathological heterogeneity of tumor ssue
samples was addressed in fresh and frozen brain
secons, although possible confusion between
different tumor components (i.e., tumor hemor‐
rhageandnecrosis)isdescribed36,173.The genomic
heterogeneity in glioblastoma has also been suc‐
cessfullyaddressed175.Otherapproachesmakeuse
of an alternave advanced Raman technique na‐
medSmulatedRamanhistology176–179(SRH),whe‐
re disnct wavenumbers are used for image
acquisionandvirtualH&E‐likeimagesaregenera‐
ted aer computaonal processing. With this ap‐
proach in combinaon with deep convoluonal
neural networks, amongst others Hollon et al. as‐
sessed (pediatric180) brain tumors intraoperave‐
ly1,181,182. In the scope of this imaging approach,
also a tradional pathological diagnosis based on
digital Raman histology slides seems feasible183–
185.
RS could be used to idenfy brain edema186,
tumorrecurrence187ortumormargins188–194butal‐
sotumorinfiltraonzones.195,196Ingeneral,infiltra‐
ve glioma cells showed significant spectral
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differences in the regions of phenylalanine and
Amide III (around 1030cm‐1 and 1230‐1300cm−1),
aswell as the region assignedto C‐C stretching li‐
pids and nucleodes (around 1050‐1100cm‐1) –
justtolistafewwavenumbersofinterestexempla‐
rily197.Jietal.196reportthecellularitywithinasam‐
pleaswellasthedensityofaxonsandtheraoof
lipidandproteincontentsasthebasisforthediffe‐
rence in spectral properes. Even single tumor
cells198were detectable using RS, somethingalter‐
nave imagingmethodsstruggle with. RSwasalso
applied to observe glioblastoma tumor evolu‐
on199,to determine the molecularsubtype ofglio‐
blastoma200, and to give insight in glioma
biochemistry201.
RSwasshowntobesuperiorindifferenaon
of brain tumor and glioblastoma in comparison to
5‐ALA‐induced fluorescence202,203, and capable to
detect IDH mutaon s in gliomas – inter alia chan‐
ges in the spectral protein profile are consistently
reportedincaseofIDHmutaon204–206.Italsosho‐
weddiagnoscvalueintumordiscriminaonwhen
measuringsmall extracellularvesicles207, or poten‐
al when tracking/detecngmetabolicchanges208–
210in braintumors/cancercells,aswellasdrugde‐
livery mechanisms211 and post‐therapeuc chan‐
ges212inglioblastomacells.
Spectroscopicclassificaonofdifferentgrades
ofbraintumors is possible213.Zhou etal.214 disn‐
guishedbetween differentWHO gradesofgliomas
using Raman bands of tryptophane (around
1588cm−1, among others) and carotenoids
(1008cm−1, 1157cm−1, 1521cm−1,  2320cm−1, and
2667cm−1) as well as the peak intensity rao bet‐
ween proteins and lipids in the high wavenumber
region (2934cm−1/2885cm−1). The group of Morais
et al.215 and Lilo et al.216 differenated between
differentgradesofmeningiomas.Zhangetal.217as‐
sociatedan intensityraointhehighwavenumber
region with different meningioma grades. While
gliomas/neuroepithelial tumors and meningiomas
have been described218 and morpho‐chemically
analyzed219,220 extensively221, some work also exist
on neuroblastomas. One group differenated bet‐
weendifferentneuralcrest‐derivedtumorsinfresh
and frozen ssue222,223, and Ricciardi et al.224 used
RSto examinechangesinthebiochemistryofneu‐
roblastoma cells aer exposure to radiaon. Me‐
dulloblastomas225, biopsies of the pituitary
gland209,226,seeds of renoblastomas227, andcarci‐
noma metastases228 have been spectroscopically
studiedaswell.
Early,intraoperative,andneuropa‐
thologicaldiagnosticsusingRS
Perioperave  ex vivo ssue assessments al‐
low for direct and early treatment decision, e.g.,
when examining smear brain tumor samples94 or
discriminang between primary CNS (central ner‐
vous system) lymphoma and glioblastoma based
on biopsies229. RS can also be applied intraopera‐
vely (in vivo) ‐ recently even in dogs230 ‐ using a
hand‐held probe for tumor classificaon231–237,
where a real‐me auditory feedback mechanism
has been proposed to guide the neurosurgeon238.
Transcranial RS, leaving the skull intact, has been
proposedanddemonstratedinamousemodel239.
Using opcal spectroscopy applied on FFPE
ssue,Devpura et al.240and Gajjaret al.159exami‐
nedapossibleapplicaonofRStovariousbraintu‐
morsalreadyin 2012/2013. Shortly aer,Fulwood
etal.241disnguishedbetweenglioblastoma,meta‐
stasesandnormalbrainusingimmersionRSonFF‐
PE samples. Livermore et al.204 have been able to
carryouttheabove‐menonedanalysisoftheIDH
mutaon deteconinglioblastomatumorsalsoon
FFPEssue.Differenthistological areas can bedis‐
nguished in glioblastoma in FFPE ssue, with a
sound separability between the peritumoral area
andtheareaofnecrosis242.
To enable early and non‐invasive cancer dia‐
gnosis, some approaches aim for idenficaon of
meningioma243andglioma244paentsbasedonse‐
rumsamples andresulngspectroscopicbehavior.
UsingRSasanaddivetechnique,LeResteetal.245
combinespectroscopicdataandtranscriptomicda‐
ta for machine learning analyses on glioblastoma
subtypesandrelatedclinicaloutcomes.
RSinNeurodegenerativeDiseases
Misfolded proteins and aggregates in various
diseases246–248,e.g., Alzheimer's(tauand amyloid),
Parkinson's (alpha‐synuclein), Hunngton's (poly‐
glutamine),are in generalaccessible to vibraonal
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spectroscopictechniques249.Usageofthesetechni‐
ques ranges from tracking and characterizaon of
misfolded proteins41, to potenal new diagnosc
methods250,251,especiallyin biofluids252–254. Studies
on the pathological hallmarks of neurodegenera‐
vediseasesused avarietyof RS techniques;most
frequently employed techniques are  SERS, TERS
(Tip‐enhanced Raman spectroscopy), as well as
DUVRR255,256(deepUVresonance Raman),wherea
wavelengthinthe rangeofUV (200nm)is usedas
excitaonsourcewhich resultsinanincreased in‐
tensity. Another common technique named ROA
(Ramanopcalacvity)makesuse oftheprinciple
that a chiral molecule scaers le and right han‐
ded polarized photons at different intensies and
soisparcularly useful to analyzeproteinaggrega‐
tes257,258. Furthermore, also IR (infrared)‐spectros‐
copyand related/modifiedvibraonalmethods are
common, and a combinaon of techniques could
lead to an increased diagnosc ability and gain of
knowledge2,259–262. Several ways of increasing the
detectabilityofasample via RShavegainedpopu‐
larity in the neurodegenerave field. Bringing in a
labelled isotope into the backbone of a pepde
shiscertainamidbandsandenablesa demarca‐
onfromthe exisng amidebands emanang from
theunlabeled proteinsin thesample, althoughan
overlap of Raman peaks of interests may oc‐
cur263,264. Another similar approach integrates ex‐
ternal probes such as unnatural amino acids with
vibraonal potenal into the sample, which can
aerwards be traced by specific Raman peaks,
oen in the range between 1900‐2900cm‐1where
theinterferencewithotherpeaks of thespecimen
is minor264–266. For further reading, Devi et al.2
providesadetailedinsightintotheuseofRSinthe
fieldofneurodegeneravediseases.
Around 20 years ago convenonal RS was al‐
ready capable of disnguishing between AD brain
ssueandhealthycontrolbrainssue(in2022ma‐
chine learning algorithms are useful to do the sa‐
me267) and to determine the presence of
amyloid‐beta‐sheetsin senile plaques268–270. Short‐
ly aer, Raman signals of the hippocampus of AD
rats were proposed to aid diagnosis of AD271.
Kurouskietal.44giveanoverviewoftheapplicaon
ofRSin the courseofplaque formaonandstruc‐
ture; Wilkosz et al.41 provide a comprehensive list
ofwavenumbersassociatedwithproteinaggrega‐
on. Detailed examinaons of the (secondary)‐
structure of beta‐amyloid in various experimental
set ups have been carried out using DUVRR272–275
orROA44.Cunhaetal.276usedacombinaonofRa‐
man techniques for amyloidplaque characteriza‐
on.SERShasbeenusedtoidenfytauproteinand
(soluble) amyloid beta277,278, and to detect amylo‐
id‐beta1‐40monomersandamyloid‐beta1‐40fibrilsin
soluon279 as well as in brain ssue280. Aβ40 and
Aβ42281 were shown to be disnguishable. TERS
was used to characterize natural Aβ1‐42 fibrils and
idenfytoxicoligomericforms282,283.
RS was capable of visualizing amyloid in AD
brains post mortem and of displaying neuric
plaques and neurofibrillary tangles284 – even
though the laer findings were quesoned and
measurement of lipofuscin granulates instead of
plaques was proposed285 Raman imaging also de‐
termined the presence of hemoproteins in senile
plaques286 and allowed for reconstrucon of the
evoluon process of differenttypes of amyloid be‐
ta plaques287. Based on RS measurements, AD‐as‐
sociatedastrogliosis288andlipiddepositsinvicinity
of fibrillary plaques were idenfied and further
morphologicallydescribed289.
Beside the idenficaon of amyloid beta290–
292,forexampleinthesurroundingofneuronalspi‐
nes293,Ramanimaging294,295hasbeenusedtocom‐
pare the concentraon of Aβ in hippocampal
regions and eye lens ssue296 and to determine
cholesterol‐andsphingomyelin‐richstructuressur‐
rounding amyloid plaques, thought to represent
dystrophic neurites297. Another research group
used CARS to determinea higher content of lipid,
collagen and amyloid fibers in Alzheimer‐affected
brainsamples298.
Searching for biomarkers as an early diagno‐
sc toolinAD299–302, humantears303,saliva,304 ce‐
rebrospinalfluid305(differentstatesofamyloidbeta
confirmaons could be detected in cerebrospinal
fluid already in 2008306), renal imaging307 and
bloodsamples308–318havebeenevaluatedforapo‐
tenal diagnosis of AD using spectral differences
arriving from platelets319 or the concentraon of
theneurotransmiers Glutamate (GLU) and γ‐ami‐
nobutyric acid (GABA)320. Inthe courseof this ap‐
proach, it has been shown that corcal cataract
maynotbeasufficientpredictorofAD296. The de‐
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tecon of neurotransmiers using RS has been
shownandfurtheranalyzed,byArdinietal.321,Lee
etal322,Moodyetal.323–325 (i.e. RSfordetecon of
neurotransmiersthroughtheskull),Caoetal.326/
Zhou et al.327 (neurotransmier detecon in se‐
rum), Ciubuc et al.328 (RS for dopamine detecon
andanalysis), Silwalet al.329 (dopamineand dopa‐
minetransporterinteracon), Manciuetal.330(do‐
pamine – serotonin interacon) and Shi et al.331
(quanficaonofnorepinephrine).
Inaddion,RSisalsosuitabletoexaminethe
interacon of beta‐amyloid with metal ions332–337.
Interesngly, detecon of tau335–338 and insulin342–
345hassofarbeenstudiedtoalesserextent;ozone
exposureas a knownrisk factorhasbeen foundto
lead to spectroscopically measurable changes of
thehippocampusinaratmodel346.
In Parkinson’s Disease (PD), a main focus of
theapplicaonof RS isthe characterizaon of the
secondary structure of alpha‐synuclein338,347–349 as
wellas theidenficaon of alpha‐synucleinaggre‐
gaons, feasible not only in the brain but also in
the gut350. Mensch et al.351 used ROA to examine
the spectral properes of α‐synuclein during tran‐
sion to its secondary structure. Another group
spectroscopically characterized the striatal extra‐
cellularmatrixin a PD mouse model352. Sinceearly
lossofdopaminergicneuronsisanearlychange in
paents with PD, differentapproachesaim forde‐
tecon of dopamine353–355, e.g., in striatum of mi‐
ce356, or in blood samples of paents with
anpsychoc drug‐induced Parkinsonism357. Other
efforts to establish early diagnosc tests for PD,
such as examinaon of erythrocytes and blood
coagulaoninPDpaents358,werecarriedoute.g.,
byCarlomagno etal.359 usingsaliva of PD paents
and Schipper et al.360 who combined RS and NIRS
(near infrared spectroscopy) to disnguish bet‐
ween blood samples of PD paents and a control
group through different spectroscopic properes
correlatedwithoxidavestress.Mammadovaetal.
361usedRSinaPDmousemodeltodetectpatholo‐
gical renal changes as a method to disnguish
betweenhealthyanddiseasedsamples.
AnalyzingperipheralnervousssueinALSmi‐
ceandautopsiesofpaentssufferingfromALS,Ti‐
an et al.362 showed that Raman imaging was
capable of visualizing anddetecng early patholo‐
gical changes. Different approaches disnguish
betweenaltered lipids and proteoglycans in spinal
cordssueofALS miceand healthycontrols363,or
testtheprognoscvalueofSERSinALSpaents364.
InaddiontothemanyapproachestodiagnoseAD
andPDpaentsbyRS,othersfocusonALSaswell.
Fordiagnoscpurposes, Zhang et al.365 used SERS
onplasmasamplestodisnguishbetweenALSpa‐
ents and  a healthycontrol group; Morasso et al.
366proposedvibraonalspectroscopyandextracel‐
lularvesiclesasapotenal biomarkerandanother
research group spectroscopically examined saliva
fromALS,PD,andADpaents,showingdifferences
inthespectralproperesofeachgroup367.
InthecontextofHunngtonDisease (HD), RS
has been used for quanficaon and visualizaon
ofaggregatedpolyglutamine368andfortheassess‐
mentofitsstructure369,370.Huefner et al.371 found
significantchangesin thespectrarelatedto disea‐
se progression, as well as differences correspon‐
dingto genotypeand genderin serum samples of
HD paents and healthy controls. In another ap‐
proach, membrane composion of HD‐affected
andcontrolperipheralfibroblastswereseparatable
using RS, suggesng that cell membrane damage
mayserveasfuturediagnoscbiomarker372.
RS has also been used for research on Prion
Diseases373–378; one research group employed the
methodtoexaminethediagnoscvaluewhenana‐
lyzing blood samples of sheep to detect the alte‐
ringfromofPrPCtoPrPSc379.
Spectroscopicexaminationofmyelin
compositionintheCNSandinperi‐
pheralnervetissue
RSproves useful togain adeeper understan‐
dingofthemolecular myelincomposion;Pezzo
etal.380 examinedthe physical chemistryof cocul‐
tured neuronal and Schwann cells. In addion, RS
may be advantageous to detect pathological pro‐
cesses of demyelinang diseases in the CNS or in
peripheral nerve ssue. Carmona et al.381 studied
thespectroscopichallmarksof lipid chainsinmye‐
lin membranes as well as the secondary structure
of associated proteolipid proteins (PLP). Some pu‐
blicaonsreportthepossibilityofdetecngmyelin
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invivousingRaman microscopy382,383;Huang et al.
384 described different composions of myelin
structures, whereas Wang et al.385 used CARS mi‐
croscopytodetectnot only myelinbutalsoaxons,
the node of Ranvier, and the Schmidt‐Lanterman
incisure. Fu et al.386 visualized fiber tracts in mice
brain by imaging the myelin along the axons. In
2021Lucasetal.387 usedCARStodeterminemyeli‐
naon deficits in a fragile‐X‐syndrome mouse mo‐
del.Out of pure academic interestthe publicaon
of Poulen et al.388, in which Raman scaering on
spinal cord myelin disnguishes between three
different species (human, mouse, lemur), shall be
menonedatthispoint.
Few Raman experiments deal with Mulple
sclerosis(MS)389;theprocessofmyelindegradaon
canbeaddressedwithRSnotonlyquantavely390
but also qualitavely. To tackle alteraons in the
biochemical composions in human brains post‐
mortem,Poonetal.391–393measuredvariouspatho‐
logic features and showed that even normal
appearing white maer next to MS lesions inclu‐
ded spectroscopically measurablechanges. Imitola
et al.394 correlate the presence of microglia (on a
side note: even the acvaon of microglia is tra‐
ceable using RS395) and axonal injury/demyelina‐
onusing CARS microscopy.Fu etal.396 appliedthe
same method to examine different me points of
experimental autoimmune encephalomyelis in
miceandGasecka et al.397 usedCARStodetect in‐
ducedautoimmunedemyelinaoninspinalcordof
mice. Another approach was carried out by the
teamof Alba‐Arbalatet al.398;they detectedspec‐
tral changes of defined molecules in the rena
(evenaninvivouseofRSappliedonhumanrena
isin line withlaser safetyregulaons399) ‐ associa‐
tednot onlywith differentphasesof MS,but also
age‐relatedinhealthypaents.
Raman‐based research of myelin composion
andpathologyisnot limited to MS, italsoextends
to the study of demyelinaon and its biochemical
changesinperipheralnervessue400‐evenpatho‐
logical401 and age related402 changes. Using diffe‐
rent Raman techniques the remyelinaon process
in the spinal cord of rats aer iatrogenic induced
demyelinaon403, as well as remyelinaon in rat
sciac nerve404, and biochemical changes during
nerve injury405,406 can be tracked. Another ap‐
proachusedCARSimagingtointerprettheinterac‐
on of different macrophages (resident and
recruited)aerWalleriandegeneraon407.
UpcomingnovelfieldsforRS‐from
stroketomusculardiseasestopsych‐
iatry
RS has been applied in combinaon with in‐
frared spectroscopy and atomic force microscopy
tocharacterizedifferenttypesofthrombiin ische‐
mic stroke408 or to characterize atheroscleroc
plaques409,410. Changes in fibrin concentraon in a
blood clot aer zonal thrombolysis with urokina‐
se411,orthe metabolic regulaon ofarterytone412
were examined. Other research groups invesga‐
tedspectroscopicchangesinthehippocampusdue
to cerebral ischemia‐reperfusion413, or spectrosco‐
picchanges in theamount ofCu+and Cu2+ions in
brain ischemia414. Russo et al.415 used Raman tra‐
ceablecytochrome cto invesgateeffects ofinsu‐
lin on the hippocampus aer transient ischemic
brain condions, Yamazoe et al.416 used a self‐de‐
veloped Raman approach to detect areas of an
ischemic core area. The group of Caine et al.417
used a combinaon of imaging techniques,
amongstothersRamanimaging,totrackbiochemi‐
cal changes in the peri‐infarct zone aer induced
strokein a mousemodel.As an alternave way of
infarcondiagnosc,Fanetal.418 proposedtearRS
in combinaon with machine learning tools as a
non‐invasivetechnique.
Incontextofbrainhemorrhages,Raman ima‐
ginghas beenused todetect microvesselsandin‐
duced hemorrhage419, as well as to track the
oxygen flow in brain vessels420. Furthermore, RS
wasemployedasamethodin ratbrainswithstria‐
talhemorrhagestoevaluatethebiochemicalcom‐
posion aer rehabilitaon treatment421.
Employing SERS, the subarachnoid hemorrhage
biomarkerglial fibrillary acidproteincan bedetec‐
ted422.SERScanalsobeusedtoassesscomplica‐
ons post subarachnoid hemorrhage, like
vasospasmandhydrocephalus423.
Inssuecondionsofbrainorspineinjury,RS
was applied to ssue of rat models424–426 and on
renae of miceaertraumacbraininjury427.Bio‐
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chemical changes in affected areas arising from
hem or divergent levels of cholesterolwere disco‐
vered428and comparedtoMRIscans429.RSwasca‐
pable of detecng injured motor cortex areas
wherecertain spectroscopic properes were asso‐
ciated with cell death430. Employment of SERS‐ba‐
sedmethods allowfordetecon ofneuron‐specific
enolase(NSE),N‐acetylasparateor S‐100βin blood
samples as biomarkers for brain injury431–435; ai‐
ming for intraoperave assessment of molecular
changes ‐ one group developed a device for intra‐
cranialspectroscopywithinbraininjury436.Changes
in the biochemical and cellular composion of rat
brain aer gamma radiaon have been addressed
by Kočović et al.437. For a further reading the rea‐
dermayrefertoStevensetal.438,whohasrecently
reviewedthecurrentdeploymentsofRamanspec‐
troscopy in traumac brain injury in a detailed
way.
Even muscular diseases are accessible to RS:
Niedieker et al.439 used CARS imaging to visualize
morphological hallmarks such as glycogen storage
and internalized nuclei in various muscular disea‐
ses;Alixetal.440reporteddifferentspectralproper‐
es of mitochondrial and non‐mitochondrial
muscular diseases; and Gautam et al.441 showed
the differences in the spectra of Raman measure‐
ments from muscles of Drosophila with certain
mutaonsaffecngthemuscularsystemincompa‐
risontohealthycontrols.SpRSwasusedforinvivo
idenficaon of Duchenne muscular dystrophy
(DMD)affectedmusclesinamousemodelandhu‐
man muscles affected with the same disease with
ex vivo measurements showing similar Raman
peaks442. Hentschel et al.443 evaluated the use of
fibroblasts together with applicaon of CARS and
othermethods to study theeologyofneuromus‐
cular diseases. Blood sample tesng for the dia‐
gnosis of DMD was proposed and successfully
performedinamousemodel444;thecomparisonof
spectral properes of the erythrocyte membrane
in DMD paents and healthy controls demonstra‐
tedbiochemicaldifferencesduetoproteinanoma‐
ly445.
OneofthepotenaldomainsofRSinthearea
ofinfecous diseasesofthebrainandmeninges is
thediagnoscdeteconofpathogens.Ithasalrea‐
dy been capable of idenfying viral strains446,
changesinbacterialmetabolism447,ordifferenate/
detectdifferenttypesofbacteriarelatedtomenin‐
gis448,449. Although the diagnoses of tuberculous
meningis450 or Neisseria meningis451 as well as
possibledifferenaonof bloodcell types452 using
RSonCSFsamplesisreported,reliabledeteconof
bacterialmeningisin CSFwas notyet sufficiently
sensible; therefore, a combinaon of techniques
wassuggested453.AnotherapproachemploysRSin
neuroimmunology as a tool to monitor apoptoc
changesinhippocampalprogenitorcells454.
RShas also beenapplied in psychiatricdisor‐
ders;e.g.tovisualizethedrugmechanismofase‐
rotoninreuptakeinhibitorinmousebrain455andto
idenfybloodserumsamples based onalteraons
in phospholipids and proteins of paents with
affecve disorders456–458. Recently, Chaichi et al.459
measured changes in brain lipidome spectroscopi‐
cally in post‐traumac stress disorder (PTSD) rats,
but also the vibraonal spectroscopic properes
within myalgic encephalomyelis have been sub‐
jectedtofurtheranalysis460,461.
Conclusionsandoutlook
All studies and literature cited in this review
focusedonpreclinical/clinicaluseofRSwiththein‐
tenon toprovide theinterested reader a general
overview rather than a detailed account of each
parcular topic. Before jumping into acon and
establishingRSasanaddionalresearchmethodin
one’sown laboratory,taking alook on themetho‐
dological reviews by Butler et al.28 (including con‐
crete informaon about the general experimental
setup and requirements for biological ssue), and
Guo et al.462 (analysis of Raman data, machine
learningalgorithms)mayproveuseful.
Upcoming applicaons of RS potenally aim
forin vivopredicon of progressionrisk463 orem‐
ploymentofvibraonalspectroscopyfordetecon
ofepileptogenicbrainregions464.AdvancedRaman
techniques such as Spaally offset Raman Spec‐
troscopy (SORS)465 may potenally permit live in‐
sight into ssue biochemistry of deeper brain
structures.Alternavely,a futureestablishment of
intraoperaveRaman imaging(in parcular it may
evenbeperformedinvivo466)willpotenallyallow
fastdeteconof bothhistomorphological features
and tumor genecs; therefore producing an inte‐
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grateddiagnosis467 atan early stageofthediagno‐
sc workflow468.  Extensive clinical studies aiming
for approval of RS in neuroscience by regulatory
authories are sll missing, even though a clinical
needandapaentbenefithasbeendemonstrated
by a broad range of groups and laboratories. To
translatepromisingresultsintoclinicalpracce,se‐
veral challenges should be considered. When vi‐
braonal spectroscopy is tested as a diagnosc
method in a mulcenter approach, experimental
workflowsofspectroscopicexaminaonneedtobe
standardized and facilitated; consensus within the
spectroscopic community on a collaborave expe‐
rimental setup and procedure prevents potenal
invariances due to different sample preparaon
protocolsandhiddenarfacts91.Tomaximizespec‐
traloutput andenhance spectralintensity ina cli‐
nical seng, handheld probes / spectrometers
with opmized design and in vivo parameters as
wellasapreferablylowsignal‐to‐noiseandhighsi‐
gnal‐to‐backgroundraoarecurrentlyunderinves‐
gaon by a growing number of companies
stepping up their efforts in the interface of rese‐
archandclinicalimplementaon.117,469
Since the use of RS on FFPE ssue allows di‐
rectcomparison with the diagnoscgold standard
ofhistology,RSisexpectedtoexpandits applica‐
onsin neuropathologicaldiagnoscsin thefuture.
Upcoming studies will not only challenge the cur‐
rentuseofRSonunstainedFFPE ssue(isreliable
diagnosis also achievable on H&E stained samp‐
les?)butalsodiscussapotenaluse ofvariousRa‐
man substrates in a cost‐oriented manner470. To
reducethe costfactor(id est expensivesubstrates
suchasCaF2orlow‐Eslides)futureemploymentof
RSonglassslidesseemsworthwhile;therefore,oc‐
curring autofluorescence during measurement
needs to be addressed.Within that approach,the
use of a certain excitaon wavelength or the de‐
tecon ofonlyasmallspectralwavenumberrange
havebeen proposed60,471. Inthis sense,Ibrahimet
al.472 aimed to use glass as Raman substrates by
employingadigitalprocessingmethod.
In the field of neurodegenerave diseases, a
majorandhighlyancipatedimpactofRScouldbe
theearlyandnon‐surgicaldiagnosisofdisordersin
a reproducible manner. Despite promising results,
this applicaon area is only beginning to develop.
To maximize diagnosc reliability, a deeper under‐
standing of Raman features and their correspon‐
ding biochemical origin in biofluids is key. Within
thehuge amount of obtained data, itremains ne‐
cessary to address paent dependent spectral va‐
riaon as well as variaons related to a concrete
experimental set up. Close cooperaon between
different research groups and ensured data sha‐
re470 potenally accelerate the development to‐
wardsclinicalimplementaon.
Anexemplarysuccessstoryofclinicaltransla‐
onwasreportedinthefieldofdermatology,whe‐
reRShadalreadybeenestablishedas a diagnosc
methodforearlydeteconofskincancer;ahand‐
held device was commercially produced in Cana‐
da463,473,474. To speed up translaon from research
labs to commercializaon and clinical use, several
networks have been founded, e.g., Internaonal
Society for Clinical Spectroscopy (ClirSpec, clir‐
spec.org)andRaman4Clinics(raman4clinics.eu),all
aiming for exchange of experse and creaon of
researchcollaboraon117.
Toconclude,itishighlylikelythatRSwillcon‐
nueto evolve asa methodin theinterseconof
applied biophysics and medicine – and potenally
make its way deeper into the field of life science,
such as detecon of plasc in zebrafish brain ho‐
mogenatesasaresultofexposuretonanoplasc475
and even more clinical applicaons. Where the
journeywill finally lead remains to be seenin the
nextyears.
Acknowledgements
Figure 1and Figure 2werecreated withBio‐
Render.com.MM thanksthe FNR for funding sup‐
port(FNRPEARLP16/BM/11192868grant).
Funding
Luxembourg Naonal Research Fond, FNR
(FNRPEARLP16/BM/11192868granttoM.M.)
ConflictsofInterest
Wehavenoconflictsofinteresttodisclose.
FreeNeuropathology3:19(2022)
doi:hps://doi.org/10.17879/freeneuropathology‐2022‐4210
Klammingeretal

Free Neuropathology 3:19 (2022) Klamminger et al
doi: https://doi.org/10.17879/freeneuropathology-2022-4210 page 17 of 32
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