Intraoperative Raman

S p e c t ro s c o p y
Michelle Brusatori, PhDa,b,c, Gregory Auner, PhDa,b,c,d, Thomas Noh, MDe,f,
Lisa Scarpace, MSe, Brandy Broadbent, MSa,b,c, Steven N. Kalkanis, MDe,f,*

KEYWORDS
 Diagnostics  Raman spectroscopy  In vivo  Molecular signature  Intraoperative

KEY POINTS
 Surgical excision of brain tumors provides a means of cytoreduction and diagnosis while minimizing
neurologic deficit and improving overall survival.
 Raman spectroscopy is a promising investigative and diagnostic tool for neurosurgery, and pro-
vides rapid, nondestructive molecular characterization in vivo or in vitro for biopsy, margin assess-
ment, or laboratory uses.
 A review is presented of Raman spectroscopy techniques for clinical practice, particularly in distin-
guishing between normal and diseased tissue without the assistance of a pathologist, or to
augment pathologic findings.

INTRODUCTION The estimated incidence of new primary brain

Raman spectroscopy has been identified as a
promising investigative and diagnostic tool in
neurosurgery because it provides rapid, non-
destructive molecular characterization in vivo or
in vitro for biopsy, margin assessment, or la-
boratory use. Spectroscopic information re-
sulting from Raman active functional groups of
nucleic acids, proteins, lipids, and carbohydrates
in tissue allows evaluation and characterization.
This article highlights the need for such an
instrument, summarizes neurosurgical Raman
work to date, and discusses what the future of
neurosurgical Raman spectroscopy might look
like.
and central nervous system (CNS) tumors for
2016 is 77,670 with 16,616 predicted deaths
resulting from malignant tumors.1 With advances
in intraoperative imaging, surgical excision is
becoming more relevant for brain tumors,
providing a means of cytoreduction and diag-
nosis while minimizing neurologic deficit and
improving overall survival. Even in primary malig-
nant tumors such as high-grade gliomas, for
which prognosis is poor, the extent of resection
is associated with improved mortality and out-
comes.2 Glioblastoma multiforme (GBM), an
extremely aggressive primary brain tumor with
an average life expectancy of approximately 12
to 18 months, accounts for 15.4% of all CNS

Disclosure: The authors have no relationship to any commercial company with direct financial interest in the
subject material discussed in this article.
a
Department of Surgery, Wayne State University, 5050 Anthony Wayne Drive, Detroit, MI 48202, USA;
b
Department of Biomedical Engineering, Wayne State University, 5050 Anthony Wayne Drive, Detroit, MI
neurosurgery.theclinics.com

48202, USA; c Department of Smart Sensors and Integrated Microsystems, Wayne State University, 5050 An-
thony Wayne Drive, Detroit, MI 48202, USA; d The Detroit Institute of Ophthalmology, Henry Ford Health Sys-
tem, 15415 E. Jefferson Avenue, Grosse Pointe Park, MI 48230, USA; e Department of Neurosurgery, Hermelin
Brain Tumor Center, Henry Ford Health System, 2799 West Grand Boulevard, Detroit, MI 48202, USA;
f
Josephine Ford Cancer Center, Henry Ford Health System, 2799 West Grand Boulevard, Detroit, MI
48202, USA
* Corresponding author. Department of Neurosurgery, Henry Ford Health System, 2799 West Grand Boule-
vard, Detroit, MI 48202.
E-mail address: skalkan1@hfhs.org

Neurosurg Clin N Am 28 (2017) 633–652

http://dx.doi.org/10.1016/j.nec.2017.05.014
1042-3680/17/Ó 2017 Elsevier Inc. All rights reserved.
634 Brusatori et al
and primary brain tumors. A recent study
reviewed tumor volumetrics in 500 newly diag-
nosed patients with supratentorial GBM and
found a statistically significant difference in sur-
vival with subtotal resections as low as 78% for
survival benefit.3 This work, among growing liter-
ature, shows that early maximal resection while
preserving a patient’s functional status is critical
for optimal patient outcome,4,5 and this suggests
a need for new technologies designed to achieve
maximal safe resections.
Many tools have been developed to aid ne-
urosurgeons with intraoperative tumor identifica-
tion and delineation. Image-guided surgical
techniques include intraoperative MRI, fluores-
cence-guided surgery, neuronavigation, and
ultrasonography. As an example, fluorescence-
based imaging methodologies that rely on endog-
enous autofluorescence are briefly highlighted.
Autofluorescence is a well-known phenomenon
in which a biological substrate is excited with a
suitable wavelength of light. The emission of light
in the ultraviolet (UV), visible and near-infrared
(NIR) range results from the presence of fluoro-
phores that naturally occur in tissues such as pro-
teins (elastin and collagen), flavins, vitamin A, and
fatty acids. However, steady state measurements
that rely solely on fluorescence emission intensity
are affected by numerous factors. These factors
include irregular tissue surfaces, nonuniform sam-
ple illumination, and the presence of endogenous
absorbers in the biological substrate. Fluores-
cence lifetime imaging microscopy (FLIM) re-
solves some of the difficulties in quantifying
fluorescence intensities. FLIM produces spatially
resolved images of fluorophore lifetime. The tech-
nique involves exciting the sample with repetitive
light pulses and observing the fluorescence
response. Sun and colleagues6 performed a pilot
study using an endoscopic fluorescence lifetime
imaging for intraoperative identification of GBM
and showed the contrast between normal and tu-
mor tissue. Time-resolved fluorescence spectros-
copy (TR-LIFS) is also used to extract fluorophore
lifetime but, instead of image acquisition, the
method records the time-resolved spectrum at a
single point. Butte and colleagues7 used
TR-LIFS to detect the fluorescence lifetime differ-
ences between normal brain tissue and glioma
in vivo.7 Both time-resolved fluorescence spec-
troscopy and imaging techniques have complex
instrumental setups making the translation to a
clinical setting difficult. In addition, because of
the presence of multiple fluorescence molecules,
the measured signal from the tissue is a distribu-
tion of fluorescence decays, making the methods
susceptible to background interference such as
blood. Also, there is no clear-cut interpretation
of the data in terms of individual components.
Bridging the gap between maintaining neuro-
logic function and comprehensive tumor cell
removal is critical. Direct histologic evaluation
through techniques such as mass spectrometry
is gaining momentum but many techniques are
severely limited by their time-based nature.8
Raman spectroscopy has potential to be an impor-
tant modality for intraoperative evaluation of tissue
during surgical resection. It is a reagentless,
nondestructive optical technique with demon-
strated ability to detect changes in the chemical
composition and/or molecular structure between
diseased and healthy tissue. Although most
Raman spectroscopy studies are exploratory,
in vitro experimental studies have shown that this
technique can be used to differentiate normal
brain tissue from tissue with infiltrating cancer cells
and dense cancerous masses with high speci-
ficity. Further, this technique has potential to be
used in conjunction with imaging modalities such
as MRI and ultrasonography for more in-depth
intraoperative characterization of residual tumor.
This article provides contemporary Raman spec-
troscopy data and further understanding of the
Raman effect on biological tissues.
RAMAN SPECTROSCOPY
Interest in clinical spectroscopy is increasing
because of the potential of vibrational spectro-
scopic techniques for noninvasive tissue diag-
nosis. Raman spectroscopy and infrared (IR)
spectroscopy probe molecular vibrations associ-
ated with chemical bonds in a sample to obtain in-
formation on molecular structure, composition,
and intermolecular interactions.
The Raman effect was discovered in 1928 by
C.V. Raman when he observed that light traveling
through various liquids scatters differently, in a
behavior distinct from fluorescence.9 The basic
process of scattering is the absorption of and
reemission of electromagnetic radiation by mole-
cules. The mechanism associated with scattering
is induced electric dipole radiation. Light incident
on matter can scatter at the same optical fre-
quency as the incident radiation (ie, the induced
dipole moment in a molecule oscillates and radi-
ates at the same frequency as the incident light).
This process is termed elastic scattering (Ray-
leigh scattering). Light can also scatter at fre-
quencies that differ from the original radiation.
This process is called inelastic scattering and,
unlike the elastic process, it involves an energy
transfer between the incident photons and a ma-
terial. An inelastic process, termed the Raman

effect, occurs in approximately 1 in 107 photon
interactions with matter10 and results from mod-
ulations in the induced dipole moment within
molecules. Changes in polarizability with molec-
ular vibration cause the induced dipole moment
to oscillate at frequencies other than the incident
light.11 Radiation emitted at longer wavelengths
(downshifted frequencies) than the incident light
is termed Stokes scattering, which occurs when
a molecule is promoted from the ground state
to a virtual energy level (an intermediate electron
state with energy lower than a real electronic
transition) then relaxes to a vibrational state. Ra-
diation emitted at shorter wavelengths (upshifted
frequencies) than the original radiation is known
as anti-Stokes scattering. This process occurs
when a molecule is initially in a vibrational state
and relaxes to the ground state after scattering.
With conventional Raman spectroscopy, the ef-
fect is independent of wavelength because no real
energy states are involved (only virtual states), and
this is termed nonresonance Raman. However,
certain substances when exposed to electromag-
netic radiation can produce a strong fluorescence
signal that overlaps the Raman signal. Raman
scattering and fluorescence are competing phe-
nomena that have a similar origin. The Raman ef-
fect corresponds with the absorption and
subsequent emission of a photon via an intermedi-
ate electron state, as shown in an energy level di-
agram (Fig. 1). Molecules are excited to a virtual
energy level for a short period, on the order of pi-
coseconds, before an emitted photon results;
whereas in fluorescence the incident light is
absorbed by a molecule and reemitted from elec-
tronically excited states after a resonance time on
the order of nanoseconds.
In contrast, resonance Raman spectroscopy, a
variant of conventional Raman, measures molecu-
lar vibrations in a wavelength-dependent manner.
When the wavelength of the exciting source
Fig. 1. Energy transition of a molecule.
Intraoperative Raman Spectroscopy 635
coincides with an electronic transition of the mole-
cule a resonance effect is observed and the inten-
sity of some Raman active vibrations can be
increased by a factor of 102 to 106.
There are 4 primary components in a conven-
tional Raman spectroscopy system: (1) the light
source (usually a laser), (2) the spectrometer, (3)
a filter to block the laser line and allow Raman
scatter to pass, and (4) a detector.10 Fig. 2 shows
a basic Raman spectrometer. The recorded in-
elastic scattering results in a Raman spectrum or
fingerprint, which can be used in imaging and di-
agnostics. The x-axis corresponds with the
change in energy of the scattered photon; the y-
axis corresponds with the photon count. Energy
shifts correlate to types of bonds, such as C5H,
C-C, or CH2 twist, based on quantized energy
levels of each molecular bond. The amount, loca-
tion, and intensity of peaks vary based on the num-
ber of vibrational bonds in a molecule and
their ensemble interactions with each other.12
Biological spectra can be complex. For example,
L-phenylalanine (C6H5CH2CHCOOH), an important
tumor marker, shows 63 peaks in the Raman
fingerprint (Fig. 3).13
When using Raman with large molecules, the
group contribution approach is used whereby
the largest peaks come from the bonds that
occur most frequently.12 The distinctive peak for
1
L-phenylalanine between 1000 and 1008 cm re-
sults from C-C bonds and ring breathing modes of
benzene, which are the most prevalent bond vibra-
tions.14 Some molecular peaks may not be
apparent in the spectrum depending on bond
strength and shape.15 If the bond does not change
its polarizability, it is not easily detectable. For
example, water has a weak Raman signal because
of its symmetric polarity. In tissue diagnostics, this
can be advantageous. In contrast, infrared spec-
troscopy signals can be overwhelmed by water
emissions.
The Raman spectrum of tissue induces many
peaks representative of the plethora of cellular
constituents (see Fig. 3). Distinct differences be-
tween pathologic conditions are shown as
different intensities of the same peak or peak
shifting based on binding conformation. Occa-
sionally, there is a unique peak from a different
moiety, such as calcifications or hemorrhage.
Biological tissues are largely made up of the
same molecules, but the quantity and interac-
tions of the molecules vary between tissue types.
For example, tumors use glycolysis to generate
ATP, which produces more pyruvate, which con-
verts to lactate and subsequently to the amino
acid alanine, resulting in stronger protein
peaks.16,17
636 Brusatori et al

Fig. 2. Raman spectrometer. A narrow line–width laser source at a particular wavelength, and optimized to the
material under investigation, is focused on the sample. Scattered light is collected by the microscope lens and
passes through a notch or edge filter, which eliminates light of the original wavelength, leaving only inelastically
scattered light. A grating is used to separate the different wavelengths of scattered light, such that the different
wavelengths fall on different areas of a charged coupled device (CCD) or other detector, such as a photomulti-
plier tube.

EARLY APPLICATIONS OF NEUROLOGIC Similarly, the neurinoma and neurocytoma were

RAMAN SPECTROSCOPY characterized by specific spectral bands, corre-
sponding with carotenoids and hydroxyapatite,
In 1990, Raman spectroscopy was first applied to respectively.20
characterize parenchymal water content in situ.18 Koljenovic and colleagues21 created Raman im-
Normal and edematous brain tissue showed ages of sectioned GBM from 20 patients and
similar peak intensities attributed to protein CH compared them with hematoxylin and eosin
bonds. Although water produces a weak Raman (H&E) staining. They showed an ability to distin-
signal, when coupled with the OH water peak, guish vital tumor from necrotic tissue given the
the OH/CH ratio gives a relative comparison of wa- increased presence of cholesterol and its esters,
ter content.
Later, Mizuno and colleagues used Raman
spectroscopy to differentiate gray from white mat-
ter in a rat model by examining the spectral ratio of
protein to lipid content. In this case, gray matter
has an abundance of proteins, whereas white mat-
ter has increased phospholipid levels because of
its myelination. Numerous studies have contrib-
uted a wealth of information to the molecular fin-
gerprints of biological tissues, and several
Raman peak assignment tables are available.14,19
Table 1 provides a summary of peak information
for brain diagnostics.
Follow-up work showed differentiating spectral
characteristics between excised grades II and III
glioma, neurinoma, and neurocytoma of choroid
Fig. 3. Raman spectra typical of white matter and tu-
plexus.20 Although the spectrum for the grade II mor necrosis show features similar to purified mea-
glioma was similar to that of normal cortical tissue, surements of their known molecular constituents
grade III glioma had unique molecular structural (methyl-docosahexaenoate [methylDHA] and phenyl-
changes, namely a band at 1245 cm 1, an intensi- alanine, respectively). Spectra are vertically offset
fied 1130 cm 1 band, and a band at 856 cm 1. and scaled for visualization purposes only.
 Intraoperative Raman Spectroscopy 637

Table 1
Raman peak assignments from neurosurgical literature

Raman
Peak (cmL1) Assignment Citation
58–61
427–430 Cholesterol/cholesterol ester
59
Higher in white matter than gray matter
61
Cholesterol deposits found in tumor sections
61
429 Bending PO4 in hydroxyapatite, deposits found in tumor tissues
58
457 Melanin, found in metastatic melanoma
62
467–475 Glycogen and polysaccharides seen in PA and MG with SERS
63,64
482 Glycogen, seen in metastatic renal cell carcinoma
60
491 Cholesterol
60,61
498–500 Nucleic acids/nucleotides
65
525 S-S stretch mode of gauche-gauche-trans form, higher in
synaptosomal fraction than myelin
61
538 Cholesterol ester, cholesterol deposits found in tumor sections
58–60
544–548 Cholesterol
59
Higher in white matter than gray matter
65
553 S-S trans-gauche-trans mode, higher in synaptosomal fraction than
myelin
62
558–566 Tryptophan, higher in GBM, ODG tumor when measured with SERS
61
583 Bending of PO4 in hydroxyapatite, deposits found in tumor tissues
58,66
597–599 Melanin, seen in metastatic melanoma
64,67
607–608 607: glycerol
39,58–60,68
608: cholesterol
59
Higher in white matter than gray matter
68
Cholesterol deposits found in tumor sections
39,61
613–622 Cholesterol ester
61
Cholesterol deposits found in tumor sections
60,62–64,68
622/624: phenylalanine (protein) associated with necrosis
62
Present in SERS, higher in ODG tumor
63,64,68
642–645 Tyr (protein) associated with necrosis
62
648–655 C-C and C-S stretch of protein (SERS)
Ratio of 724:655 is significantly different between tumor/normal 62
61,63,64
661–669 666: Heme associated with hemorrhage
61,63,68
667, 669: nucleic acid/nucleotide associated with necrosis, tumor
60,61
680–683 Nucleic acids
37,65
700, 703/704 C-N stretch mode from choline head in phosphatidylcholine and
sphingomyelin
59
720:701 ratio increases in GBM
23,58–61,63,64,68–71
Cholesterol
63
Variable in normal tissue
23,59
Higher in white matter than gray matter
65
Distinguishes myelin fraction from synaptosomal
61,68
Deposits found in tumor sections
67
Cholesterol content decreases from corpus callosum to cortex
Decreased cholesterol/phospholipid content in tumor compared 67,71
with normal
65
C-S stretch from methionine, cysteine, and cysteine residues of
proteins (less likely)
716–719 Choline in head group of sphingomyelin and phosphatidylcholine, 23,58–61,63,64,67–69,71
phosphatidylethanolamine
63,64,67,71
Higher in normal than tumor
59
720:701 ratio increases in GBM
61
Stronger in GBM than MG
(continued on next page)
638 Brusatori et al

Table 1
(continued )

Raman
Peak (cmL1) Assignment Citation
722–728 DNA, RNA base ring breathing mode, C-S of proteins, CH2 rocking 60–62,68,72
of adenine, NADH, FADH (SERS), nucleotides
62
Ratio of 724:655 signifies difference between tumor/normal
72
Seen in cell nuclei
67
Increased DNA/RNA in tumor with respect to normal
58
727 Melanin, seen in metastatic melanoma
23,61,63,64,66
739–759 Heme (blood)
65
746 Phosphorus-oxygen-phosphorus bonds symmetric stretch of
phospholipids (predominant in white matter and myelin
fraction)
61,68
749–751 Nucleotides
60,63,64,71
757–760 Tryptophan
63,64
Associated with necrosis
60–63,68,72,73
781–795 O-P-O stretch of DNA/nucleic acids, seen in cell nuclei
63,73
Associated with tumor
Nucleic acid band with the least overlap with lipid, cholesterol, 68
and protein, making it a strong marker for mitosis index
69
800 Quartz substrate (removed via processing)
63,64,68,71
826–830 Tyr, proline
61
830 Phosphate backbone of b-DNA conformation
63,64,67
Higher in tumor, necrosis than in normal
60
845 Cholesterol
63,64,68
851–852 Tyr, associated with necrosis
850–855 Glycogen, seen in metastatic renal cell carcinoma and high-grade 63,73
tumors
20
856 Polysaccharides
22,61
Collagen
20
Strong in glioma
22
Seen in dura mater but not meningioma
73
865 Phosphatidylethanolamine, lower in tumor than normal
64
867 Glycogen, seen in metastatic renal cell carcinoma
68
873 Phosphatidylcholine
31
875–878 Tyr, higher in astrocytoma cells than astrocyte cells
59,60
Choline group in PC and sphingomyelin
62
905–908 Glucose level, increased in PA, as measured by SERS
67,74
925–926 C-C in peptide backbone
60
Cholesterol
22,61,68,71
935–939 C-C of peptide backbone, collagen
22
Seen in dura mater but not meningioma
63
941 Glycogen, seen in metastatic renal cell carcinoma
956 Carotenoid (present in schwannoma, not present in normal tissue) 20
35,62
C-C vibration of protein, carotenoids, hydroxyapatite, collagen
62
Increased in lung metastases tumor, GBM, ODG, PA, EP, MG as
measured by SERS
958–960 Stretching vibrations of PO4 in hydroxyapatite, deposits found in 20,22,61,66,75
neurocytoma, metastatic melanoma, psammoma body of
meningioma
75
Stronger in necrosis than tumor
74
961 Higher cholesterol content in white matter than gray matter
(continued on next page)
 Intraoperative Raman Spectroscopy 639

Table 1
(continued )

Raman
Peak (cmL1) Assignment Citation
32,61
969–977 Tricalcium phosphate Ca3(PO4) calcification seen in schwannoma,
necrosis
58,66
976 Melanin, seen in metastatic melanoma
33
985 Calcification
60
990 Phosphate ions (in PBS)
20,23,31–33,35,58,60–69,
1002–1006 Ring breathing mode of phenylalanine of protein, heme
71,73,74,76–78
23,31,32,62–64,74
Higher in lung met tumor, GBM, EP (SERS), astrocytoma, necrosis
than in normal
32
Stronger in necrosis than tumor
73
Increased in high-grade tumors
79
Slightly increased after radiation exposure
78
Weaker after brain injury
20
Carotenoid (present in schwannoma, not present in normal tissue)
61
Heme/hemorrhage
32,33,35,60,63,64,68,78
1032 C-H of phenylalanine
32,63,64
Increased in necrosis
78
Weaker after tissue fixation
35
Higher in glioma tissue and cells than normal tissue and astrocyte
cells
62
1042–1049 C-O and C-N stretch of protein, higher in lung met tumor, MG
measured by SERS
69
1050 Quartz substrate (removed via processing)
20,23,32,33,58–61,63–65,
1060–1066 C-O stretch and C-O-C symmetric stretch, C-C stretch of
67–69,72–75,77
phospholipids (side chains specifically) and cholesterol
20,23,65,72,74,79
Strongest in white matter and myelin fraction
32,33,63,64,67,73,74,79
Strong in normal, weaker in tumor/necrosis
77
Higher in Alzheimer hippocampus than normal hippocampus
75
Stronger in necrosis than tumor
61
Cholesterol deposits found in tumor sections
61
1071 Stretching vibration of PO4 in hydroxyapatite, deposits found in
tumors
1080–1090 C-C stretch and PO2 symmetric stretching of phospholipids and 20,32,35,60,63,65,72,
74,75,78
nucleic acids
72
C5O vibration of ester/aldehyde
20,65,72
Higher in gray matter than white matter
32
Higher in white matter than gray matter and tumor
63
Seen in metastatic tumors
72
Seen in cell nuclei
75
Stronger in necrosis than tumor
74
Stronger in gray matter than tumor
62
1088–1097 C-N stretch of protein, higher in GBM, ODG, PA, EP, MG when
measured by SERS
59,64,73,77
1088 Lipid/glycogen
64,73
Seen in metastatic renal cell carcinoma, high-grade tumors
77
Higher in Alzheimer hippocampus than normal hippocampus
33,35,60,61,68
1094–1100 Phosphate backbone of b-DNA conformation
68
Marker for mitotic figure
Higher in glioma tissue and cells than normal tissue and astrocyte 35
cells
63
1122 Glycogen, seen in metastatic renal cell carcinoma
(continued on next page)
640 Brusatori et al

Table 1
(continued )

Raman
Peak (cmL1) Assignment Citation
58
1124 Melanin, seen in metastatic melanoma
61
Heme/hemorrhage
20,32,33,35,58,60,61,64,
1126–1133 C-C stretch of phospholipids (side chains), cholesterol
65,67,69,72,74,75,78
35,60,77
C-C and C-N stretch of protein/glucose
59
CH2 vibration
20,65,72,74
Stronger in white matter and myelin fraction
20,74
Stronger in glioma, suggesting higher concentration of trans
conformation hydrocarbon chains in tumor lipids
10,33
Stronger cholesterol/phospholipid content in normal than tumor
77
Stronger in Alzheimer hippocampus than normal hippocampus
75
Stronger in necrosis than tumor
61
Cholesterol deposits found in tumor sections
37
1142 Lipid
20,32,33,59,60,75
1157–1159 Carotenoid, present in schwannoma and some GBMs, not present
in normal tissue
32,75
Stronger in necrosis than tumor
62,68,74
1174–1175 Protein, higher in lung met tumor (measured by SERS), and in GBM
than normal
78
Cholesterol/lipid band appearing after brain injury
62
1206–1208 Amide III, higher in ODG, MG as measured by SERS
33,63,64,68,71
Phenylalanine
32,63,64
Highest in necrosis, lowest in normal, tumor intermediate
66
1212 Oxygenated hemoglobin
78
1228 Cholesterol/lipid band appearing after brain injury
64
Seen in lung carcinoma met
20,31–33,35,60–69,72–74,
1225–1300 Amide III band (collagen/protein, amino acids)
77,78
20,65
Mainly a-helix conformation in normal brain
20
Shifts to lower wavenumber in glioma, suggesting random coil
structure
62,63
Intensity and position are variable in normal brain
31,62
Higher in lung met tumor, GBM, ODG, PA, astrocytoma cells
32,63,64
Associated with necrosis
32
Shoulder in necrosis, weaker shoulder in tumor
35
Higher in glioma tissue and cells than normal tissue and astrocyte
cells
23,33,61,63,64
1231–1258 Heme (blood)
22,32,33,58,63,64,67,
1260 Overlaps with unsaturated fatty acids/phospholipids
69,74
22
Lipids higher in dura than meningioma
59,66,68,72–74
1268–1270 d5CH unsaturated fatty acids
73
Diagnostically significant for normal tumor and tumor grade
72
Decreases with increasing saturation
59
Higher in gray matter than white matter
79
Higher in white matter than gray matter
79
Slightly increased after radiation exposure
(continued on next page)
 Intraoperative Raman Spectroscopy 641

Table 1
(continued )

Raman
Peak (cmL1) Assignment Citation
20,22,23,33,35,58–61,
1296–1302 CH2 twist and wag of phospholipids, fatty acid, cholesterol
63–69,72–75,77,78

67,73
Not a diagnostically significant/variable intensity/position
72
Increases with increasing saturation
8,22,23,67,72,74
Stronger in white matter and myelin fraction than gray matter
and tumor
31,63
Seen in metastatic tumors, astrocytoma cells
77
Higher in Alzheimer hippocampus than normal hippocampus
75
Stronger in necrosis than tumor
61
Cholesterol deposits found in tumor sections
64
1302–1305 Lipid/glycogen (renal cell carcinoma)
60
Cholesterol ester
32,33,35
1313 CH3/CH2 deformation of lipids and proteins
32
Strongest in necrosis, intermediate in tumor, weakest in normal
62
1320–1330 C-H deformation or CH2 bend (protein) increased in lung met
tumor, GBM, ODG, EP, MG (SERS)
23,32,33,60,61,63,64,
1333–1343 Aliphatic amino acids, including tryptophan, nucleic acids
68,71,74
63,73
Glycogen
63,64
Associated with necrosis
63,73
Increased in high-grade tumors and metastatic renal cell
carcinoma
61
1346 Heme/hemoglobin
78
CH deformation of lipid/protein
62
1366 Higher in GBM (SERS)
60,61,72
1372–1376 DNA bases, seen in cell nuclei
32,33
1397 CH2/CH3 deformation of lipids and proteins
58,66
1400–1404 Melanin, seen in metastatic melanoma
69
Quartz substrate (removed via processing)
68
1422 Nucleotides
20,22,23,31–33,35,58–69,
1439–1455 CH2/CH3 deformation of lipids side chains, proteins, amino acids,
71,73,74,76–78
cholesterol/cholesterol ester
67
Intensity and position are variable in normal brain
23,59,74
Higher in white matter than gray matter and tumor
77
Higher in Alzheimer hippocampus than normal hippocampus
22
Lipids higher in normal than meningioma
61,68
Cholesterol deposits found in tumor sections
31,62,63
Higher in ODG, EP, MG renal cell carcinoma met, astrocytoma
73
Variable in malignant tissues
64
Associated with necrosis
35
Higher in glioma cells than astrocytes
61
1454 Heme/hemoglobin
63
1460 Glycogen, seen in metastatic renal cell carcinoma
77
1466 CH2 bend of proteins
60,61,72
1483–1488 DNA bases, seen in cell nuclei
20,33,60,75
1518–1527 Carotenoid
20,59
Present in schwannoma, some GBMs, not present in normal
tissue
32,75
Stronger in necrosis than tumor
37
1540 Nucleic acid, higher in tumor
(continued on next page)
642 Brusatori et al

Table 1
(continued )

Raman
Peak (cmL1) Assignment Citation
60,63,68,71
1550–1555 Tryptophan
32,63
Associated with necrosis, slightly weaker in normal, weakest in
tumor
66,69,71
1546–1568 Hemoglobin
60–63,68,72
1575–1588 C-C stretch of protein, nucleic acids (SERS)
62,63
Higher in ODG, PA, EP, MG
68,72
Seen in cell nuclei, marker for mitosis
78
Sharp band appears after brain injury
33,62,64
1585 Heme (blood)
58,66
1592–1595 Melanin (metastatic melanoma)
61,66
1603–1605 Oxygenated hemoglobin
68
1614 Aromatic amino acids (protein)
23,66
1619–1626 Oxygenated hemoglobin
78
Sharp band appears after brain injury
23,60,66,69,71,73
1635–1640 Water
73
Glioma has higher water content than normal (but normal tissue
may be edematous)
20,23,31–33,37,58,60,61,
1656–1668 Amide I band (protein) mainly a-helix in normal brain
64–69,71,73,74,76–78
20,65
Also assigned to unsaturated fatty acids (nC5C)
22,23,32,33,58,59,63,64,
(1645–1675) Intensity and position are variable in normal brain
66–69,73,74,78
63,67
Associated with necrosis, not as strong in tumor
63,64
Higher in normal than tumor
22
Higher in tumor than normal
31,37
Lower unsaturated lipid content as malignancy increases
73
Lower unsaturation in white matter
73
Higher in gray matter than white
59
Higher in white matter than gray matter
61
Shoulder at 1670 in Alzheimer hippocampus (more deposition of
b-amyloid protein?)
77,78
Weaker after brain injury
23,32,33,59–61,63,68,
1669–1674 Steroid ring of cholesterol/cholesterol ester
69,73
61,68
Cholesterol deposits found in tumor sections
64
1675 Protein (lung carcinoma met)
61
1684 Collagen
62
1688 Higher in EP (SERS)
22,23,32,33,59–61,75,78
1731–1739 C5O of ester groups, cholesterol ester
75
Stronger in necrosis than tumor
22,67
Lipids higher in dura, cortex than tumor
61
Deposits found in tumor sections
78
Not seen in fixed tissues
23,58,63–65,72,73
2850–2853 CH2 symmetric stretch of lipid side chain
65,73
Higher in white matter, lower in gray matter, lowest in tumor
64
Seen in metastatic renal cell carcinoma
23,58,63–65,69,72,73
2880–2886 CH2, CH3 stretch found in lipid (side chain), cholesterol
23,65
Higher in white matter, lower in gray matter
73
Lower in tumor
64
2895 Glycogen/lipid (metastatic renal cell carcinoma)
(continued on next page)

Table 1
(continued )
Raman
Peak (cmL1) Assignment
2929–2938 CH2/CH3 symmetric stretch of lipid side chains, cholesterol
Aliphatic amino acids (associated with necrosis)
2958–2960 CH3 asymmetric stretch
3010–3015 Unsaturated lipids, lower in tumor
3100–3600 OH stretch in water
3300 Weak amide band, water
Lower in gray matter than white matter
Abbreviations: EP, ependymoma; FADH, flavin adenine dinucleotide; MG, meningioma; NADH, nicotinamide adenine
dinucleotide; ODG, oligodendroglioma; PA, Pilocytic Astrocytoma; SERS, surface-enhanced Raman spectroscopy.
carotenoids, and calcification peaks measured in
necrotic tissue. In another study, they differenti-
ated 8 microcystic, 7 transitional, and 2 syncytial
meningioma tissues from 3 healthy dura mater tis-
sues with 100% accuracy using linear discriminant
analysis. Although the spectrum for dura mater
had Raman signatures typical of collagen, the dif-
ference spectrum (meningioma minus dura) was
characteristic of lipids, indicating their relative
abundance in tumors. A partial least squares algo-
rithm was used to examine the real-world scenario
of investigating a biopsy specimen likely contain-
ing mixed dura and tumor tissue.22 This work
showed that given a normal distribution of error
the algorithm estimated with 68.3% certainty that
a specimen was 13% tumor. This certainty in-
creases to 95% if the estimate is 26% tumor.
Krafft and colleagues23 examined nondried,
unfixed brain tissue using Raman spectroscopy
with a laser spot diameter of 4 mm, 80 mW power,
and 15 seconds laser exposure time. Lipid/protein
and cholesterol/protein ratios were more signifi-
cant in distinguishing white from gray matter and
distinguishing normal tissue from glioma and me-
ningioma tumors, consistent with reports that de-
gree of malignancy in gliomas is associated with
changes in the membrane lipid composition.24
Comparative mapping studies showed that
Raman spectra acquired from thawed frozen tis-
sue yielded similar information to fresh tissue.
Contributions from hemoglobin and its byproducts
were the main difference.
Mutations in isocitrate dehydrogenase 1 (IDH1)
and TP53 have been found to be prevalent in
70% to 80% of gliomas, showing a direct relation-
ship with their grading.25 Gajjar and colleagues26
showed that Raman spectroscopy could be used
in vitro to differentiate various grades of glioma.
Gliomas were subdivided into 3 pathologic sub-
types: low-grade astrocytoma (LA), anaplastic
Intraoperative Raman Spectroscopy 643
Citation
23,58,63,65,66,72
23,63,66
65,72
73
23,58,66,80
80
23
astrocytoma, and GBM. IDH1 staining was posi-
tive for 60% of LA with an overall positive rate of
33% for all gliomas. Raman spectroscopic anal-
ysis showed significant (P.01) differences in the
spectra between all 3 grades, suggesting that
spectroscopic biomarkers may provide more
robust delineation of tissue than staining alone.
EARLY APPLICATIONS OF IN VIVO RAMAN
SPECTROSCOPY
Small pilot studies have shown the feasibility of
Raman spectroscopy for in vivo tissue assess-
ment. In vivo instrumentation, facilitated by tech-
nological advancements in spectrometer design,
has enabled rapid Raman collection in a mobile
setup. Standard screening for neoplastic pro-
cesses typically involves gross inspection and
multiple biopsies of aberrant tissue. The develop-
ment of fiber optic Raman probes that fit through
the instrument port of an endoscope has allowed
researchers and clinicians to obtain spectroscopic
information, in a minimally invasive manner, for
in vivo evaluation. Bergholt and colleagues27
showed significant spectral differences between
normal esophageal tissue and cancerous tissue
in vivo with 96% diagnostic accuracy. The Raman
signal capture time ranged from 0.1 to 0.5 sec-
onds. Similar results were shown for benign and
malignant gastric ulcers.28
Further studies evaluated cervical and breast
cancer tissues in vivo. Researchers obtained
similar peak intensities in vivo to prior in vitro
studies for distinguishing between benign, low-
grade, and high-grade precancerous cervical le-
sions with sensitivities of 95.7%, 82.8%, and
81.3%, and specificities of 100%, 92.3%, 88.5%,
respectively, with 1-second acquisition time.29
In a breast cancer study, a Raman probe was
used intraoperatively to evaluate the margins
644 Brusatori et al
following primary tumor excision with 1-second
acquisition and diagnosis time. These margins
were then excised per standard surgical practice.
Using a previously validated algorithm, 1 malig-
nant specimen was identified from among the
other benign breast tissue margins. This specimen
was grossly normal; however, on histopathologic
confirmation reoperation was necessary. This sec-
ond procedure may have been prevented had a
Raman probe been used to guide excision.30
DEVELOPMENTS TOWARD IN VIVO
NEUROSURGICAL DIAGNOSTICS
The applicability of Raman spectroscopy to neuro-
surgery continues to evolve as its effectiveness in
distinguishing disparate tissues is revealed. While
analyzing single cells, Banerjee and colleagues31
showed that Raman spectra were very similar
across the GBM cell lines from 4 different patients.
On a larger scale, recent work from Kalkanis and
colleagues32 further shows the viability of Raman
spectroscopy as a neurosurgical adjunct in tumor
resection. Focusing on GBM, they developed
Raman spectral maps for normal brain, GBM, and
necrosis from 40 tissue specimens, with a total of
17,138 spectra from 95 distinct regions.32 These
were examined along with adjacent H&E-stained
sections. Normal brain tissue samples, consisting
primarily of gray matter, were higher in lipid content
than tumor and necrosis. Necrosis showed higher
protein and nucleic acid content, whereas GBM
was between the two tissue types. Freeze artifacts
affected the discriminant function analysis accu-
racy in distinguishing tissue types in the validation
data set, from 97.8% to 77.5%. However, this fac-
tor is moot in the intraoperative setting. This group
subsequently published studies showing that the
same label-free imaging can distinguish boundaries
of infiltrating tumor and areas of hemorrhage.33,34
Tanahashi and colleagues35 used an ex vivo
platelet-derived growth factor subunit B–induced
infiltrative glioma murine model in their Raman
studies. It was designed to replicate World Health
Organization grade II oligodendrogliomas and the
less distinct margins between infiltrative glioma
and normal tissue. They examined cultured cells
as well as native brain tissue, identifying spectral
peaks that characterized glioma, which were pri-
marily derived from phenylalanine, lipid, DNA, and
protein. Tumor prediction was reported to have a
sensitivity of 98.3% and specificity of 75.0%. The
sensitivity and specificity of whole tissue samples
were 58.8% and 76.4%, respectively.
Recently 2 research groups, in addition to our
group, reported taking Raman spectroscopy
into the neurosurgical suite. In 2015, Desroches
and colleagues36 addressed finding the optimal
camera temperature and lighting conditions for us-
ing a Raman spectroscopy probe during neurosur-
gery. This group showed that the signal-to-noise
ratio increased as the camera temperature
decreased and integration time increased. In addi-
tion, they identified several light sources, including
microscope light, directed surgical lighting (low or
high), and fluorescent light, that prohibited accu-
rate Raman measurement of the sample.36 How-
ever, lighting from exterior lights, indirect low
surgical lighting, and LCD (liquid crystal display)
monitors did not adversely affect the Raman spec-
troscopy measurement.36 Furthermore, this study
and previous work by the same group showed
that a spectroscopic probe can be used to detect
necrotic tissue in vivo using inelastic scat-
tering.36,37 Of note, the probe used the Medtronic
SureTrak neuronavigation system to coregister the
site with preoperative MRI.36 There are reports of
the British Columbia Cancer Agency spin-off com-
pany Verisante performing intraoperative neuro-
surgical diagnostics in collaboration with Imperial
College Healthcare NHS Trust, although published
findings are not yet available.38
In a recent investigation, our group developed a
custom-built portable Raman spectrometer with
probe using a 785-nm laser as the excitation
source to acquire 66 Raman spectra of tissue
samples from 18 patients comprising 9 women
and 9 men with a mean age of 54 years in the oper-
ating room immediately following surgical resec-
tion. From 2 to 5 tissue samples per patient were
examined with Raman spectroscopy in a light-
tight chamber, with 1 site per sample being
recorded. After spectral acquisition, the speci-
mens were submitted to pathology for evaluation.
The Raman spectra were preprocessed to mini-
mize any fluorescence contributions and to pro-
vide better interpretability. Fig. 4 shows the
Fig. 4. Mean Raman spectra of normal tissue, solid tu-
mor, necrosis, and hemorrhage normalized to the
1003 cm 1 band. Spectra are vertically offset and
scaled for visualization purposes only.

Raman spectra of tissue normalized to the peak at
1003 cm 1. This band is the phenyl ring breathing
mode of phenylalanine in the protein and is not
sensitive to conformational changes of protein.
The averaged spectrum from the tumor group con-
sists of spectra obtained from GBM (grade 4), me-
ningioma (grade 1), anaplastic oligoastrocytoma
(grade 3), ependymoma (grade 2), anaplastic oli-
godendroglioma (grade 3), and oligodendroglia
(grade 2). For normal tissue, the spectrum may
have contributions from both gray matter and
white matter. In adults, white matter, which primar-
ily consists of glial cells and myelinated axons, has
higher total lipid concentration, lower water con-
tent, and lower capillary density than gray matter,
which is composed of neuronal cell bodies.
Because white matter and gray matter differ in
their protein/lipid contents, further investigation
(neuropathology identification) is warranted for
further differentiation.
Distinct spectral differences between the mean
spectra of normal, tumor, and necrotic tissue sam-
ples as deemed by pathology as well as hemor-
rhage are evident. In general, characteristic
peaks between 1760 and 1500 cm 1 are attributed
to amide I vibrations (C5O), with contributions
of water, nucleic acids, and lipids (C5C, C5O).
Note that the amide II band at w1550 cm 1
(C-N, N-H) is weak and cannot be observed in
the absence of resonance enhancement. The re-
gion between 1500 and 1400 cm 1 shows spectral
contributions caused by C-H, CH2, and CH3 vibra-
tions characteristic of lipids and protein. Bands in
the region between 1400 and 1200 cm 1 result
from amide III vibrations (C-N, N-H) with contribu-
tions from CH2/CH3 vibrations of protein and
lipids (phospholipids) as well as from nucleic
acids. The region between 1200 and 800 cm 1
contains spectral contributions from nucleic acids,
lipids (C-C, C-O), proteins (C-N, C-C), and C-O-C
stretching of carbohydrates (saccharides/polysac-
charides). The region between 400 and 800 cm 1
is associated with nucleic acids and nucleotide
conformation; however, bands arising from
glucose, glycogen, cholesterol, and lipids are
also present in this spectral area. Not only can
the spectral differences be used for discriminatory
purposes they can provide insight into tumor
growth, compositional changes, and metastasis.
The lipid composition of brain tumors differs
from that of normal tissue. One such difference is
noted. Compared with that of normal and necrotic
tissue, the spectrum associated with tumor shows
reduced spectral features of lipid bands at 1064
and 1296 cm 1. These bands arise from skeletal
trans C-C stretching and CH2 twisting of acyl
chains, respectively, with protein band overlap at
Intraoperative Raman Spectroscopy 645
1296 cm 1. Many lipids found in both gray and
white matter have Raman bands at w1064 and
w1299 cm 1.39 Necrosis, an indicator of malig-
nancy in astrocytoma and common in GBM, re-
sults in cell membrane breakdown. Magnetic
resonance spectroscopy studies have shown
that high levels of mobile lipids (cholesterol ester
and triacylglycerol associated with the presence
of lipid droplets) correlate with necrosis.40 Bands
at 1064 and 1299 cm 1 in the spectra of necrotic
tissue may indicate the presence of triacylglycerol,
which has spectral features at these wave
numbers; however, further investigation is war-
ranted. In addition, the spectrum of necrotic tissue
also shows observable bands at 985 cm 1 corre-
sponding with calcifications and 1155 and
1515 cm 1 with carotenoids. The spectrum from
a region of hemorrhage shows bands at 1003,
1122, 1224, 1458, and 1561 to 1636 cm 1 that
are consistent with hemoglobin and a band at
1342 cm 1 that indicates fibrin.41 This work re-
flects a more direct interoperational study that
closely resembles in vivo measurements.
Although the end goal is for Raman spectroscopy
to have a place in standard clinical practice, con-
ventional Raman spectroscopy has encountered
challenges such as weak signals, long acquisition
times, background fluorescence, and data process-
ing and instrument costs. Advancements in instru-
mentation designed to improve sensitivity and
reduce the size and cost of Raman spectrometers,
as well as strategies to promote signal enhance-
ment (ie, reduce acquisition time) and mitigate fluo-
rescence are currently under development.
In many cases, the measured spectra consist of
a small component of Raman scatter on top of a
large fluorescence background, resulting in a
poor signal-to-noise ratio. Although all light causes
the Raman effect, NIR light reduces interference
from fluorescence in biological samples because
of its lower excitation energy. In contrast, shorter
wavelengths induce larger Raman signals but
stimulate more fluorescent background. Complex
spectral processing algorithms may be used to
assist in analyzing biological Raman data.
Although it is generally believed that NIR
(785 nm) or IR (1064 nm) wavelengths frequently
used in biological Raman spectroscopy impart
no damage to surrounding tissue, assurance of
this technology’s nondestructiveness must be
shown. There is a dearth of research examining
the damage potential on in vivo brain tissue, and
our group is currently studying this aspect.
For improved signal-to-noise ratio, there are
several complementary techniques that have
been developed based on the Raman effect.
Table 2 identifies several of these methods, in
646 Brusatori et al

Table 2
Variations of Raman spectroscopy

SERS
Method Signal enhancement is implemented through the use of metallic surfaces
(nanoparticles/nanostructures). Electromagnetic enhancement is considered
the dominant contributor to most SERS processes and involves the interaction
of surface plasmons (generated by incident light) on metallic nanostructures
with Raman active molecules. The surface plasmons on the nanostructures
transfer energy to the vibrational modes of molecules that are in close
proximity, thus providing a means for signal enhancement53
Advantage vs Significant enhancement of Raman signal is reported by a factor of 1010
standard Raman
Disadvantage for Requires additional steps during surgery, such as adding a nanoprobe molecule
intraoperative to the tissue of interest for enhancement,81 SERS tag must be biocompatible,
use Raman measurement must be in close proximity to tag. The analysis is
confined to tens of nanometers from the nanoparticle or probe. The
variability of the nanoparticles creates nonreproducible results
SORS
Method Scatter photons are collected at the laser spot and laterally offset sites. Using
scaled subtraction algorithms, surface and subsurface spectra can be derived50
Advantage vs Using an offset collection point allows data to be collected from deeper within
standard Raman the area of interest; up to 4 mm was shown.50 By comparison, standard Raman
only penetrates a few hundred micrometers. Reduces tissue fluorescence
Disadvantage for Interrogation and collection offset of at least 3.5 mm are recommended, tumor-
intraoperative thickness detection limitation of 2 mm (breast tissue)51; complex hardware
use requirements
CARS
Method Typically uses 2 pulsed trains, a pump beam at frequency up and a Stokes beam
at frequency us that interact with a sample. When energy difference up us
matches a molecular vibration of the sample, a resonantly enhanced anti-
Stokes signal is generated at a frequency uas 5 2up us82
Advantage vs Background fluorescence does not interfere with the sample, and the signal is 4
standard Raman orders of magnitude stronger than standard Raman82
Disadvantage for Requires tunable pulsed lasers to probe different molecules in the sample.
intraoperative Difficult to effectively couple and synchronize the lasers into a handheld or
use portable intraoperative device
SRS
Method SRS, like CARS, uses 2 pulsed trains (a pump beam at frequency up and a Stokes
beam at frequency us) that coincide on the sample. When energy difference
up us matches a molecular vibration of the sample, the scattering process is
resonantly enhanced, However, unlike CARS, the SRS signal occurs at the
frequency of the excitation beams83
Advantage vs Greater signal strength of approximately 4 orders of magnitude
standard Raman
Disadvantage for Requires tunable pulsed lasers to probe different molecules in the sample.
intraoperative Difficult to effectively couple and synchronize the lasers into a handheld or
use portable intraoperative device84
RRS
Method Signal enhancement with resonance Raman is achieved when frequency of
incident radiation coincides with the frequency of an electronic transition of a
molecule, and provides energy to excite electrons to a higher electronic state.
This technique can selectively augment signals affiliated with chromophores
and other large conjugated molecules
(continued on next page)
 Intraoperative Raman Spectroscopy 647

Table 2
(continued )

Advantage vs Increased signal strength by a factor of 102 to 106

standard Raman
Disadvantage for Provides more limited/selective molecular information. Non–resonance-
intraoperative enhanced bands may seemingly disappear under the intensity of resonance-
use enhanced spectral peaks. Requires a tunable laser to selectively isolate the
contributions from different chromophores. Carotenoids show enhancement
in the visible region of the spectra, whereas DNA is enhanced in the UV
region. UV laser sources can cause cellular damage. Fluorescence backgrounds
can be significant because of excitation coinciding with UV-visible absorption

Abbreviations: CARS, coherent anti-Stokes Raman spectroscopy; RRS, resonance Raman scattering; SORS, spatially offset
Raman spectroscopy; SRS, stimulated Raman scattering.

contrast with standard Raman spectroscopy, and
lists the challenges to incorporating them into an
intraoperative surgical tool.
SURFACE-ENHANCED RAMAN
SPECTROSCOPY
Surface-enhanced Raman spectroscopy (SERS) is
a method to enhance the weak signals in sponta-
neous Raman spectroscopy that uses metallic
nanostructures capable of providing surface-
enhanced Raman scattering with potential
enhancement factors of w105 to 1010. There are
3 mechanisms of enhancement described in the
literature: an electromagnetic effect, molecular
resonance within the molecule, and charge trans-
fer resonance between the molecule and sub-
strate.42 The electromagnetic effect is dominant,
with signal enhancement resulting from the reso-
nant interaction of light with plasmons excited at
the surface of the structure. An enhanced electric
field is created that is localized to a region of a
few nanometers from the surface that subse-
quently can lead to an increase in the signal
of Raman bands for molecules located in the vicin-
ity of the metallic substrate.43 Kircher and col-
leagues44 reported using SERS imaging in a
mouse model for in vivo tumor resection. In
this study, gadolinium-DOTA (1,4,7,10–tetraaza-
cyclododecane–1,4,7,10–tetraacetic acid)–coated
silica-gold nanoparticles were injected into the tail
vein of an orthotopic GBM mouse model. The
nanoparticles were shown to accumulate in cells
within the tumor, but not in normal tissue, without
necessitating a specific targeting mechanism. This
uptake is generally attributed to the enhanced
permeability and retention effect.45–47 Postinjec-
tion visualization of the tumor was achieved with
Raman imaging through the skin and skull of the
live mice because of the unique and enhanced
signal of the nanoparticles. Twenty-four hours af-
ter nanoparticle injection, sequential tumor resec-
tion was performed on live mice. SERS imaging
could detect residual microscopic tumor in resec-
tion beds that were not detectable with visual in-
spection. Another strategy for SERS diagnostics
of tissue involves the use of tailored nanoparticles
labeled with specific antibodies designed to
adsorb to selective antigens in tissue. This
approach has been shown to enable signal-
enhanced Raman measurement of an antigen un-
der investigation. Schütz and colleagues48
showed SERS labels for imaging of the tumor sup-
pressor p63 in prostate biopsies.
Often there are differences in the number of
modes present in SERS spectrum compared
with a traditional Raman spectrum; that is, the
SERS spectrum may have additional bands (or
the absence of bands) compared with those found
in a conventional Raman spectrum. This difference
results from slight changes in the symmetry of a
molecule caused by surface adsorption.
SPATIALLY OFFSET RAMAN SPECTROSCOPY
Traditional Raman spectral acquisition of tissue is
typically obtained using a 180 backscatter geom-
etry and is limited to near-surface measurements
at depths within the first few hundred micrometers
of the surface. Spatially offset Raman spectros-
copy (SORS) enables measurements from subsur-
face layers in diffusely scattering media,49 in
volumes as deep as 4 mm into the sample.50 As
opposed to traditional Raman, in which laser illu-
mination and collection are from the same area
of the sample, SORS involves collecting the scat-
tered light from a point that is away (laterally offset)
from the laser illumination. For a 2-layered sample,
2 measurements are required to recover the
Raman spectra of the individual layers. One spec-
trum is typically taken at zero offset, whereas the
other is taken at nonzero offset. For this case, a
scaled subtraction of the two spectra may be suf-
ficient to recover the spectrum of the sublayer.
However, for a multilayered system, more sophis-
ticated methods may need to be used. Clinical
648 Brusatori et al
applications of this technique can be extended to
bone50 and breast tumor margin evaluation.51
TRANSMISSION RAMAN SPECTROSCOPY
Transmission Raman spectroscopy is considered a
form of SORS, with collection and illumination
points being on opposite sides of the sample. How-
ever, unlike SORS it is not able to provide the signa-
tures of individual layers within the sample. Instead,
it provides information on the entire sample vol-
ume. For tissue interrogation, the coupling of laser
radiation into deep tissue layers is hindered by los-
ses of laser radiation at the surface of the sample
from scattering as well as the diffuse nature of
photon propagation through tissue.52 However,
by using a dielectric filter on the surface of the tis-
sue, Stone and Matousek52 detected Raman sig-
nals from depths of up to 2.7 cm within a breast
phantom made up of porcine tissues.
COHERENT ANTI-STOKES RAMAN
SPECTROSCOPY
Coherent anti-Stokes Raman spectroscopy (CARS)
is a third-order nonlinear process that typically uses
picosecond pulsed lasers, in part because of the
linewidth of most Raman active transitions being
around 10 cm 1. With this technique, a pump laser
(at a frequency up), a probe (at a frequency upr),
and a Stokes laser (at a frequency us) interact with
a sample via a wave mixing process to
generate an anti-Stokes field at a frequency of
uas 5 up us 1 upr. Commonly the probe beam
is the same as the pump, resulting in an anti-
Stokes frequency of uas 5 2up us. To significantly
enhance the CARS signal, the pump beam is tuned
while the Stokes beam is held fixed so that the fre-
quency difference (beat frequency) matches the fre-
quency of a Raman active vibrational mode of the
molecule under study (uvib 5 up – us). This process
results in a signal yield that is 4 to 5 orders of magni-
tude higher than that obtained with conventional
Raman. CARS is typically used for video-rate imag-
ing of single Raman bands, with many studies
focusing on the CH-/OH stretch region of the
spectra for tissue analysis (2500–3500 cm 1). Nar-
row laser bandwidth, speed of laser tuning rates,
and nonresonant background interference limit
this technique to species with high oscillator density
and uniquely isolated Raman peaks.53 This limita-
tion prevents access to Raman biomarkers in the
fingerprint region (500–1800 cm 1) of the spectra.53
Multiplex techniques have been developed to
simultaneously excite multiple Raman transitions,
providing a more complete vibrational picture than
is found with the single-frequency method. With
broadband CARS, the pump pulse has a narrow
bandwidth and defines the spectral resolution,
whereas the Stokes pulse is spectrally broad (usu-
ally in the femtosecond regime). Multiple Raman
transitions within the bandwidth of the Stokes pulse
are excited and are probed. This method allows the
entire spectrum of excited states to be obtained at
once and has been extended in the fingerprint re-
gion of the spectra to allow imaging of biological tis-
sue. Camp and colleagues53 showed high-speed
chemical imaging in two-dimensional and three-
dimensional views of interfaces between xenograft
brain tumors in mice and the surrounding healthy
tissue using color-coded images corresponding
with Raman bands at 2850 and 2944 cm 1 as well
as those in the fingerprint region, 730, 785, 1004,
1547, and 1564 cm 1.
STIMULATED RAMAN SPECTROSCOPY
Stimulated Raman spectroscopy (SRS) signals
are typically generated by the use of 2-pico-
second pulsed trains that coincide on a sample
(a pump beam at a frequency up and a Stokes
beam at a frequency us). By tuning the frequency
difference between the pump and Stokes beams
to match the frequency of a molecular vibration,
uvib 5 up – us, stimulated excitation of the vibra-
tional transition occurs. This nonlinear process
causes an intensity loss in the pump beam and
an intensity gain in the Stokes beam. By modu-
lating 1 of the beams, say the Stokes beam,
and measuring the signal of the pump beam at
the frequency of modulation, the intensity loss of
the pump beam caused by excitation of molecular
vibrations can be distinguished from noise to
generate a high-speed image of a selected
Raman band (vibrational transition). Freudiger
and colleagues54 imaged mouse models of 4
common brain disorders: GBM, metastasis,
demyelination, and stroke. Using 2 colored im-
ages corresponding with CH2 and CH3 stretching
vibrations at 2845 and 2940 cm 1 of lipids and
protein, they were able to delineate brain tumor
margins. For this work, imaging speed was limited
by the speed of the lock-in amplifier with signal
collection in sample transmission mode. Using
SRS, Ji and colleagues55 imaged biopsies from
neurosurgical patients with CNS tumors at 2
Raman frequencies (2845 and 2930 cm 1) to
compare with H&E microscopy and found SRS
to detect tumor infiltration in agreement with stan-
dard H&E light microscopy (k 5 0.86). They devel-
oped a classifier based on cellularity, axonal
density, and protein/lipid ratio in SRS images
that detects tumor infiltration with 97.5% sensi-
tivity and 98.5% specificity.

For margin assessment during surgical resec-
tion, it is desired to obtain high-resolution Raman
spectra over a large sample area. Raster scanning
with traditional Raman has a long data acquisition
time (whours). Coherent techniques such as
CARS and SRS allow much more rapid image
acquisition than is afforded by spontaneous
Raman imaging techniques. However, CARS and
SRS systems are larger and more complex setups
that are difficult to transition to an intraoperative
environment.
RESONANCE RAMAN SCATTERING
With resonance Raman scattering (RRS), the
wavelength of the laser beam incident on a sample
is tuned to (or near) an electronic transition of a
molecule under study. The vibrational modes
associated with a particular transition show greatly
increased Raman scattering intensity (by factors of
102-106). This intensity typically overshadows
Raman signals from all other transitions and can
provide signal enhancement of particular species
under investigation. Even in a complex sample
with numerous vibrational modes, RRS allows
few vibrational modes to be studied at a time.
This limitation can reduce the complexity of the
spectrum to allow easier identification. However,
RRS is often impaired by fluorescence back-
ground, which can obscure the Raman signals
but may be avoided using short (deep UV) wave-
lengths.56 UV RRS has typical application in study-
ing biological assemblies containing DNA or RNA,
whereas visible RRS has typically been used for
analyzing strongly absorbing organic components
such as carotenoids. Using a confocal micro-
Raman system with 532-nm excitation, Zhou and
colleagues57 used RR spectroscopy to discrimi-
nate between normal brain tissues, cancerous
brain tumors, and benign brain lesions. They
used statistical methods of principal component
analysis and the support vector machine to
analyze the RR spectral data collected from
meningeal tissues with a sensitivity of 90.9% and
specificity of 100%.
In addition to methodologies to enhance the
weak Raman signals associated with spontaneous
Raman scattering and reduce spectral acquisition
time, the translation for clinical use also involves
the development of comprehensive spectral data-
bases and tissue classification methodologies that
can be compared with current gold standards.
Validation studies must be performed to confirm
that algorithms developed on ex vivo specimens
are applicable to in vivo tissues. Best-practice
techniques for data processing, acquisition, and
classification need to be developed and adopted.
Intraoperative Raman Spectroscopy 649
The road to clinical application also involves mar-
ket assessment, preclinical validation, first-in-
human, and phase I safety studies; clinical trials to
support regulatory guidelines; regulatory approval;
as well as quality assurance under Good
Manufacturing Practices through the lifecycle of
product development. Because there is no Raman
predicate device for neurosurgery, a careful clinical
study under US Food and Drug Administration
guidance is required. The intraoperative tool under-
going experimental evaluation by the authors has a
small footprint suitable for space constraints in the
operating room and can measure and record
Raman spectra in milliseconds with resolution com-
parable with current bench-top systems.
SUMMARY AND FUTURE DIRECTIONS
Noninvasive or minimally invasive in vivo tools
that can provide rapid tissue assessment and/or
monitor treatment therapies have potential appli-
cations in many fields of medicine. Raman spec-
troscopy can assist in uncovering the molecular
basis of disease and provide objective, quantifi-
able molecular information for diagnosis and
treatment evaluation. Numerous experimental
studies have shown the capability of Raman
spectroscopy for tissue characterization. Raman
spectroscopy may complement or possibly sup-
plant current methods of surgical guidance in tu-
mor resection. The continued development of
Raman spectral databases for various brain disor-
ders, tissue classification methodologies, and in-
strument designs trending toward data with
greater resolution, shorter collection times, and
higher accuracy will ensure that Raman spectros-
copy becomes a powerful in vivo tool in neurosur-
gery. The prospect of near–real-time imaging
combined with accuracy in characterizing malig-
nancies and distinguishing them from normal
brain tissue and tissue with infiltrating cancer cells
can be a revolutionary step forward in how physi-
cians practice medicine.
ACKNOWLEDGMENTS
The authors would like to acknowledge Rachel E.
Kast and James Tseng for their collaboration and
work in this field.
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