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Vibrational Spectroscopy 30 (2002) 3141

Vibrational spectroscopy and medicine: an alliance in the making


Henry H. Mantsch*, Lin-P'ing Choo-Smith, R. Anthony Shaw
Spectroscopy Group, Institute for Biodiagnostics, National Research Council of Canada,
435 Ellice Avenue, Winnipeg, Manitoba, Canada, R3B 1Y6
Received 31 August 2001; received in revised form 17 January 2002; accepted 23 January 2002

Abstract

Both infrared (IR) and Raman spectroscopy are emerging as powerful probes of biomedically relevant properties of tissue and
biological uids. From tentative rst steps, this eld of endeavor is now beginning to mature as the central conceptual and
technical issues come into focus. Using representative examples mainly from our own research, the aim of the present article is to
provide the reader with a brief overview of progress to date.
# 2002 Elsevier Science B.V. All rights reserved.

Keywords: Vibrational spectroscopy; Clinical pathology; Cancer; Clinical chemistry; Imaging

1. Prologue hearts is the effort devoted to the development of


biomedical applications. This theme is gathering
The art and science of vibrational spectroscopy is momentum, as reected by its impact at recent meet-
undergoing something of a renaissance in recent years. ings such as the SPIE-organized BIOS and eBios
This is reected well by the diversity of topics that series in San Jose and Europe, and the inaugural
were tackled at the meeting summarized in this `Shedding New Light on Disease' meeting held in
volume. Not surprising! Although the ICOVS is a 2000 in Winnipeg. Recent encyclopedias and hand-
new conference, it has arisen through the merger of books have devoted a good deal of space to the
two existing, older conferences, one over 30 years old coverage of health-related applications; the Handbook
(ICOFTS) and one just about 10 years old (AIRS), and of Vibrational Spectroscopy includes an entire volume
as such covers virtually all aspects of vibrational dedicated to the life sciences [1,2].
spectroscopy. While the theory of infrared (IR) spec- Although there has been a distinct surge in activity
troscopy is thoroughly worked out, novelties are recently, the idea of applying vibrational spectro-
emerging continuously in the form of new applica- scopic techniques, namely IR and Raman spectro-
tions. A variety of more specialized conferences con- scopy, to address biomedical questions is now more
tinue to thrive, for example those dedicated only to the than 50-year old. As early as 1949 and 1952, Elkan
spectroscopy of biological molecules, and new endea- Blout, Robert Mellors and Donald Woernley reported
vors are emerging continuously. The one closest to our that IR spectra of human and animal tissues provide
information regarding the molecular structure of tissue
*
Corresponding author. Tel.: 1-204-984-4622;
[3,4]. These early pioneers were ahead of their time
fax: 1-204-984-5472. with these visions however; their ideas were not fully
E-mail address: henry.mantsch@nrc.ca (H.H. Mantsch). realized due mainly to a lack of appropriate technol-

0924-2031/02/$ see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 4 - 2 0 3 1 ( 0 2 ) 0 0 0 3 6 - X
32 H.H. Mantsch et al. / Vibrational Spectroscopy 30 (2002) 3141

ogy. Today, a very different scene exists with new ``how much is there?'', and quantitative analytical
characters on the stagefront. The timely development tests play a pivotal role at the front line of disease
of new technologies has provided a cohesive force in detection and monitoring. To that end, the modern
nurturing the relationship between the elds of med- clinical chemistry laboratory makes use of a wide
icine and vibrational spectroscopy; interestingly, the variety of analyte-specic reagents to promote the
key technological advances have emerged from com- development of colors whose intensity is proportional
pletely different realms and have been adopted to to the analyte concentration, and the automated ana-
address biomedical problems. Examples include the lyzers all include spectrophotometers to read the
military/defence elds, with focal plane array systems wavelength prole and intensity of those colors. In
originally designed for weapons guidance now used contrast, emerging analytical methods founded on
for IR microspectroscopic mapping studies; industrial vibrational spectroscopy use the chemical specicity
and agricultural analytical demands have spurred the inherent to the IR (or Raman) spectrum as the basis
development of various sample handling and chemo- for analysis. This is not a completely novel idea,
metric tools for process control; spectroscopic ima- of course, since near-IR spectroscopy is already
ging techniques and algorithms have arisen through employed for routine, repetitive analysis, for example,
both military and agricultural remote sensing efforts; agricultural and process control applications [5]. The
and lastly the optical bres essential to remote spectro- clinical challenge to the analytical vibrational spectro-
scopy have only become available with the explosion scopist is to devise methods that at a minimum must be
of the telecommunications industry. Running in par- readily automated, and suitable for as wide a range of
allel has been the electronic revolution, with faster clinical analyses as possible.
computing power not only permitting the acquisition Several groups, working both in the near-IR and in
and compact storage of massive volumes of data, the mid-IR, have taken up the challenge. For example,
but also rendering practicable the daunting calcula- many of the most common serum assays have proven
tions required for data processing and interpretation. to be amenable to IR-based analysis, including glu-
Indeed, the fusion of various state-of-the-art instru- cose, urea, triglycerides, cholesterol, total protein, and
mental (imaging and spatially localized spectroscopy) albumin [613]. While the various serum studies all
and interpretational methods is paramount in the employed the same calibration method, namely partial
recovery of relevant biodiagnostic information. least squares (PLS) regression, to relate spectral to
This convergence of developments from widely analytical information, each investigation used a dif-
diverse elds highlights the multidisciplinary nature ferent measurement technique. These techniques have
of the marriage of medicine and vibrational spectro- included mid-IR attenuated total reectance (ATR)
scopy. With the active participation and discussion of spectroscopy [6,7], near-IR of the uid [810] and
health care professionals with vibrational spectrosco- mid-IR of dried lms [1113]. The most striking
pists, new questions are being asked with some excit- advantage of mid-IR over near-IR is the very good
ing answers on the horizon. The aim of the present accuracy for glucose quantitation. The same spectro-
article is to highlight, with representative examples, scopic methods that were explored for serum analysis
some of the inroads that both IR and Raman spectro- have been applied to the quantitation of urine creati-
scopy have blazed in addressing relevant medical nine, urea, and total protein [1417]. The accuracy for
problems. Although there is some overlap, it is con- all three assays is similar for the three different
venient for the sake of presentation to partition the measurement techniques.
realm into three segments, namely clinical chemistry, The technique that we have devoted attention to
clinical pathology and medical imaging. makes use of mid-IR spectroscopy of dry lms as the
basis for quantitation [12,15,18]. There are several
reasons to favor this approach; only microliters are
2. Clinical chemistry required for routine measurement, the sensitivity to
certain key analytes (i.e. glucose) is better than is the
The fundamental question that is most often case for near-IR spectroscopy, and the problems asso-
addressed by the clinical chemistry laboratory is ciated with cleaning and maintenance of mid-IR ATR
H.H. Mantsch et al. / Vibrational Spectroscopy 30 (2002) 3141 33

Fig. 1. Scatterplots summarizing the accuracy and precision of a fetal lung maturity assay based upon mid-IR spectroscopy of dried amniotic
uid lms [19]. Panel (A) compares surfactant/albumin ratios as determined by IR spectroscopy to the true values as yielded by a method
currently in clinical use (Abbott TDx uorescence depolarization assay). Panel (B) compares the values as determined by IR spectroscopy of
duplicate lms. The fact that the precision in the IR measurements is so much better than the accuracy when compared to the clinical reference
method suggests that much of the scatter in panel (A) may be due to inaccuracies in the reference method rather than in the IR-based method
(see text).

elements is avoided. The practical implementation of inaccuracies in the reference method? Because this
this approach requires automation of the sample pre- question remains unanswered, we have initiated
paration process, and would also benet from the use further studies to explore the possibility that IR spec-
of an inexpensive IR-transparent substrate to replace troscopy may be more accurate than any existing
the costly barium (or calcium) uoride windows used alternative method for the assessment of fetal lung
for the proof of concept studies. Both of these devel- maturity.
opments are underway in our laboratories. One of the The emergence of biomedical vibrational spectro-
attractions of this general approach to analysis, and a scopy is not limited to IR techniques. Instrumental
driving force behind its further development, is that it improvements are revolutionizing the eld of Raman
may prove more accurate in certain assays than the spectroscopy, with nearly the same impact that the
current state of the art. This possibility is suggested by development of the FTIR spectrometer had in rein-
the scatterplots depicted in Fig. 1, which portray the venting IR spectroscopy. One critical advance has
accuracy and precision of a proposed IR-based fetal been the integration of highly sensitive CCD array
lung maturity test [19]. A method currently employed detector technology, permitting measurements that
widely is based upon an amniotic uid uorescence were previously unthinkable. One example of rele-
depolarization measurement, as implemented on the vance here is a recent investigation exploring the use
Abbott TDx analyzer. The measurement yields a value of Raman spectroscopy for the same clinical analytical
for the ratio of surfactant to albumin, the S/A ratio, role as described above for IR spectroscopy [20,21];
with high values (high fetal lung surfactant level) Raman spectra, in conjunction with PLS models,
suggesting mature lungs, and low values predicting served to quantitate serum glucose, total cholesterol,
respiratory difculties should the child be born imme- triglyceride, urea, total protein and albumin with r2
diately. Fig. 1A illustrates the very good correlation values of 0.74 or higher when compared with accepted
between the proposed IR-based S/A assay, as com- clinical assays provided by commercial analyzers.
pared to the method currently in clinical use. Fig. 1B Parallel studies of these same analytes in whole
which compares the IR-based assay for duplicate blood yielded poorer prediction accuracy especially
measurementsshows a much tighter grouping about for glucose and cholesterol due to the lower signal-
the line of identity, indicating that the IR-based to-noise for the Raman spectra measured for whole
assay is very precise in its estimate of the S/A ratio. blood. Although the analytical performance fell
Could some of the scatter observed when comparing short of the best IR-based analytical results, Raman
IR-based S/A ratios to the reference values be due to spectroscopy has at least one practical advantage in
34 H.H. Mantsch et al. / Vibrational Spectroscopy 30 (2002) 3141

that measurements can be carried out through the


glass wall of typical biouid collection containers.
It will be interesting to monitor further advances in
Raman sensitivity as detector technology continues to
improve.

3. Clinical pathology

Another area of activity that has attracted the


attention of biomedical spectroscopists is the domain
of pathology, which seeks to answer questions regard-
ing ``what is there?'', i.e. the detection, ``is it diseased
or healthy?'', and grading of disease ``how far along is
the disease process?''. This area of investigation has
focused mainly on ex vivo targets in the form of
biouids or tissue samples. A wide range of themes
have been explored, of which we have selected two to
provide a avour of the progress achieved to date.

3.1. Cancer
Fig. 2. Mid-IR microspectroscopy absorption spectra of biopsied
It is not surprising, given the terrible toll that cancer skin sections. Each trace corresponds to the average of at least 150
takes, that the challenge of detecting and grading this spectra acquired with a 20 mm  20 mm pixel size for (a) follicle
dreaded disease has attracted the attention of a large sheath, (b) basal cell carcinoma, (c) epidermis, and (d) dermis.
Representative pixels are highlighted on the image denoting the
group of vibrational spectroscopists [2240]. Several
section from which the spectra were acquired. Spectra are offset
interesting and potentially useful ndings have been along the vertical axis for clarity, and the vertical scale expanded
reported. For example, many of these studies describe threefold for the 28003000 cm per region [22].
characteristic nucleic acid absorption proles distin-
guishing control from abnormal cells and tissues.
Universal distinguishing patterns have proven elusive, The spectra did indeed reveal qualitative differences
however, and there is an on-going debate as to whether among the relevant tissue types (dermis, epidermis,
IR spectroscopy is indeed able to detect the DNA of follicle sheath, and BCC); in particular, the spectra of
cells or whether it is opaque to IR radiation [32]. The the four tissue types revealed characteristic differ-
examples below illustrate some of the successes and ences reecting variations in lipid and collagen con-
pitfalls encountered to date. tent (Fig. 2). Most interestingly, there was no obvious
difference in the nucleic acid absorption positions for
3.1.1. Skin cancer the BCC and epidermis spectra. In order to seek a non-
One application of broad interest is the early detec- subjective, routine basis to discriminate among these
tion of skin cancer, in particular, basal cell carcinoma tissue types, a multivariate classication technique (a
(BCC), which is the most common cancer in Cauca- genetic region selection routine combined with linear
sians. This cancer is often hard to distinguish clinically discriminant analysis (LDA) [41]) was employed to
from other types of skin lesions. By using mid-IR seek out patterns characteristic of the different groups
spectroscopy of biopsied tumor thin sections, one of spectra. With this approach, spectra arising from
research protocol sought to determine whether BCC BCC, epidermis or follicle sheath were classied with
may be distinguished from normal skin (as well as 98.7% accuracy. Most intriguingly, it proved possible
from other skin lesions), and whether different BCC to subdivide the spectra of epidermal tissue into two
grades could be established spectroscopically [22]. subtypes; spectra could be grouped as either (1)
H.H. Mantsch et al. / Vibrational Spectroscopy 30 (2002) 3141 35

normal epidermis overlying BCC or (2) normal epi- spectra plotted therein are from a single preparation
dermis overlying non-tumor-bearing skin, with 98.0% of cervical cells dried onto an IR-transparent window,
accuracy. Furthermore, it emerged that the spectra of and measured using an IR microscope. The lowermost
BCC, squamous cell carcinoma and melanocytic trace represents the average of all 2500 spectra (a
lesions were distinguishable with a classication accu- 50  50 grid was acquired using a 100 mm aperture),
racy of better than 90%. This study serves as a very and the 10 spectra stacked above are measured along a
encouraging example to illustrate the potential 1 mm line within the sample. Given the spectral
strengths of mid-IR spectroscopy in the identication heterogeneity, is it reasonable to expect that the spec-
and screening of cancerous tissue. trum of the bulk sample should carry diagnostic
information? If not, which of the spectra acquired
3.1.2. Cervical cancer via microspectroscopy should be considered in search-
The prospect of an IR-based screening test for ing for spectral information diagnostic of dysplasia?
cervical dysplasiathe IR equivalent of a Pap smear, These are open questions, due in large part to the
has spurred several groups to follow up on the original inherent complexity of the particular problem that is
investigation [31]. Now, a decade later, the challenges being addressed: cervical cell specimens are very
inherent to this pursuit have come into focus. These often confounded by the presence of mucous, inam-
are perhaps best demonstrated by examples that illus- matory cells, blood, etc.; the epithelial cervical cells
trate some of the pitfalls that we and others have themselves may arise from the supercial, intermedi-
encountered. ate, basal, or parabasal layers, and endocervical
The vast majority of vibrational spectroscopists columnar cells contribute their signatures. Any tech-
working in this area come from a background in the nique to recover spectroscopic signatures characteriz-
physical sciences rather than biology or medicine, and ing dysplastic epithelial cells must therefore,
from that viewpoint the complexity of biological surmount the obscuring effects of these non-diagnos-
specimens may not be fully appreciated at rst glance. tic materials. At a minimum, the diagnostic approach
This perspective may lead to dangerous conclusions in must recognize that the cellular specimens are dis-
the world of tissue spectroscopy, for reasons that tinctly heterogeneous by their nature. This fact is now
should be clear from examination of Fig. 3. The widely recognized, and the labeling of individual
spectra as cancer and normal has largely disappeared,
with the recognition that diagnostic patterns, if present
are quite subtle in comparison to other spectral inu-
ences.
Largely because the diagnostic features are subtle,
there is a growing trend to adopt pattern recognition
algorithms to search for underlying spectral patterns
that might distinguish among disease classes. This
approach is predicated on the availability of a certain
minimum number of spectra within each diagnostic
class; the aim is to accumulate enough spectra that
they completely span the natural biological variability.
Once sufcient spectra have been accumulated, sev-
eral options are available to search for discriminatory
features. The approach we have adopted most fre-
quently makes use of a genetic algorithm, in conjunc-
Fig. 3. Mid-IR microspectroscopy absorption spectra for a lm of tion with LDA, to identify a set of N discriminatory
cervical cells from a donor with a normal Pap smear. The 10 spectral subregions. Each spectrum is then represented
uppermost spectra were acquired sequentially along a 1 mm line,
with a pixel size of 100 mm  100 mm, and are offset along the
by the integrated intensities within these subregions,
vertical axis for clarity of presentation. The lowermost trace is the with LDA serving to create partitions that optimally
average of the 2500 spectra measured for a 50  50 grid. separate the spectra into their true diagnostic classes.
36 H.H. Mantsch et al. / Vibrational Spectroscopy 30 (2002) 3141

Our rst experience adopting the pattern recogni- from Bruker Optics, Germany). With these applica-
tion method to the classication of cervical cell speci- tions proven, attention is turning to more sophisticated
mens led to an apparent success rate of >80% tests, for example the task of distinguishing antimicro-
distinguishing 41 control from 41 dysplastic (CIN bial sensitive and resistant strains [4750]. A funda-
III) specimens [37]. However, problems arose when mental element that is key to the success of this
the investigation was then expanded to validate these endeavour and, indeed to virtually all emerging bio-
results. It gradually emerged that a very subtle inu- medical spectroscopic applications is the use of pattern
ence had crept into the early study, leading to arti- recognition algorithms such as cluster analysis, LDA
cially high classication success rates. While the CIN and articial neural networks.
III spectra were accumulated over a period of several Raman spectroscopy is carving out an important
months, the control spectra that were used in the niche in the identication of microorganisms, with the
classication trials were acquired over a period of past 34 years in particular witnessing revolutionary
days. The pattern recognition algorithm had success- strides. The attraction of Raman-based studies is the
fully identied subtle features signifying instrumental capacity to measure spectra directly from microcolo-
drift rather than dysplasia. A more recent study from nies growing on the solid culture plate. The ability to
our group controlled for this possible effect, conclud- measure spectra on hydrated biosamples clearly is an
ing that the IR spectra of cervical cells do indeed advantage of using Raman spectroscopy as compared
provide a basis to identify dysplastic specimens, but to IR spectroscopy, where water must be eliminated
that improvements in sampling and/or analysis meth- prior to spectral acquisition. While the pioneering
ods would be required to bring the accuracy to the studies made use of ultraviolet (UV) resonance Raman
level required for practical application [39]. Parallel excitation, and FTRaman studies have also been
efforts substantiate this view [38], and efforts to reported (using excitation at 1064 nm from Nd:YAG
improve upon the present status quo are bearing fruit lasers), both of these approaches suffer from impedi-
[40]. ments to practical use; UV excitation damages cells
readily, and the long acquisition times (order of
3.2. Pathology of microorganisms 45 min) inherent to FTRaman make it an unattractive
approach for rapid identication [51]. A possible
One of the central roles of the diagnostic laboratory solution has recently been developed, combining
is the identication of the pathogenic microorgan- near-IR multichannel confocal Raman microspectro-
ism(s) often responsible for infection. In life threaten- scopic measurements directly on solid culture plate
ing situations, this is a critical task requiring very rapid with a novel vector algebra method to remove the
turnaround of laboratory results. Vibrational spectro- spectral contributions from the underlying culture
scopic techniques could provide alternatives to con- medium [52]. Successful classications for a limited
ventional microbiological diagnostic methods because set of microorganisms cultured for as little as 6 h have
they are fast, as well as being easy to perform and conrmed the potential of this approach as new rapid
automate. tool in clinical diagnostic microbiology.
Relevant investigations began with IR microspec- Both IR and Raman spectroscopy have been
troscopy of microorganisms [42,43]. Since then, employed as part of a recent comparative study that
numerous studies have been reported describing the combined phenotypic, genotypic and vibrational spec-
spectroscopic differentiation of microorganisms at troscopic techniques (in combination with hierarchical
various taxonomic levels, even down to the subspe- cluster analysis) to type a collection of Enterococcus
cies, strain and/or serogroup/serotype level [4245]. It strains comprising different species [53]. Interestingly,
might come as a surprise to many to learn that IR the classication of these bacteria by FTIR and
spectroscopy is already routinely used to classify Raman spectroscopy revealed discrepancies for cer-
microorganisms in a plethora of non-medically related tain strains when compared with results from auto-
applications such as bioprocess and fermentation mon- mated phenotypic systems in routine use. Of the 19
itoring [46], with automated instruments dedicated strains derived from six Enterococcus species, the
for such purposes (e.g. the MICOR-ID FTIR system spectroscopy-based classications for strains strains
H.H. Mantsch et al. / Vibrational Spectroscopy 30 (2002) 3141 37

Fig. 4. Dendrogram from hierarchical clustering analysis of the FTIR spectra of 19 strains derived from six different Enterococcus species.
The strains marked with an asterisk were not in accordance with the phenotypic identication of the API system. Shading highlights the
identity of certain strains by the API system, see [53] for more details.

did not match the phenotypic designation (API might prove useful in an in vivo setting. In vivo
method) (dendrogram of Fig. 4). These discrepancies measurements are now readily feasible, thanks to
were resolved only by resorting to very elaborate and the wide availability of suitable bre optic probes.
costly genotypic methods to re-examine and reclassify For various practical reasons (robust optical bres,
the relevant strains; the corrected designations were relatively deep optical penetration), most in vivo
entirely consistent with the FTIR and Raman nd- studies employing IR spectroscopy have focused on
ings. This study clearly illustrates the potential the near-IR region. One example is the use of near-IR
strength of vibrational spectroscopic techniques for spectroscopy for the non-invasive screening of skin
the rapid and inexpensive identication of pathogenic lesions [23]. The near-IR spectrum of skin exhibits
microorganisms. strong absorption bands from water and a number of
weaker bands derived from oxyhemoglobin, deoxy-
hemoglobin, lipids and proteins. While an investiga-
4. In vivo spectroscopy and imaging tion seeking to identify spectroscopic cancer markers
revealed no visually obvious qualitative features dis-
The successful demonstration that IR and Raman tinguishing the near-IR spectra of skin lesions from the
spectroscopy of ex vivo tissue samples and biouids corresponding spectra for healthy skin, paired t-tests
can discriminate normal from abnormal samples natu- did reveal signicant differences (P < 0:05) between
rally suggests the possibility that the same techniques control and abnormal tissue at a variety of wave-
38 H.H. Mantsch et al. / Vibrational Spectroscopy 30 (2002) 3141

lengths. A more elaborate pattern recognition techni- vivo near-IR spectra in the 7501100 nm wavelength
que, namely genetic algorithm-guided LDA, yielded range accessible using this instrumentation are com-
very high classication accuracy rates (greater than pletely dominated by absorptions of hemoglobin
80%) differentiating benign and premalignant/malig- and water, in vivo applications have focused upon
nant lesion groups. hemodynamic imaging and hydration imaging. For
Notwithstanding the example just cited, in vivo example, an early application capitalizing upon this
near-IR spectroscopy is nearly always focused upon approach revealed regional variations in tissue (blood)
the absorptions of hemoglobin centered at 920 (oxy- oxygenation in tissue transplants [56,57]. This tech-
hemoglobin) and 760 nm (deoxyhemoglobin). The nique was used to assess tissue viability in an animal
potential to monitor these absorptions in vivo was model involving the reverse McFarlane skin ap
rst pointed out over 30 years ago [54]. There has been surgery on the dorsal surface of a rat. Post-operatively,
a sustained interest in these measurements ever since, the tissue perfusion was evaluated using near-IR ima-
most notably because of the hope that they might ging in order to distinguish nutrient from non-nutrient
provide a window on cerebral oxygenation, for exam- blood ow and to measure oxygen delivery to tissues.
ple during surgeries carried out under cardiac bypass Oxygenation saturation images computed from 760
[55]. This application is conceivable because of the and 800 nm near-IR images provide a measure of
good transparency of tissue in the so-called therapeu- tissue oxygenation across the entire ap. In Fig. 5,
tic window at 700950 nm. a comparison is shown of the post-operative visual
A key new technology has emerged recently to picture of the ap at different time points (1, 6 and
permit the integration of imaging and near-IR spectro- 72 h) after surgery. The discolouration observed in the
scopy in prospective clinical and surgical applications. visual images signal areas of poor tissue perfusion and
The critical element is the liquid crystal tunable lter oxygenation. Also shown is the oxygenation satura-
(LCTF), a continuously variable wavelength selection tion image of the same ap acquired 1 h after surgery.
device with a large circular aperture that makes it ideal It is immediately apparent that the 72 h visual image
for use in conjunction with commercially available closely correlates with the 1 h oxygenation saturation
near-IR silicon CCD cameras. Reectance images image suggesting that near-IR imaging can accurately
(tungsten lamp illumination) may be acquired for a detect tissues at risk of failing in the early post-
set of discrete wavelengths covering the near-IR range operative period. In so doing, near-IR imaging could
to yield a so-called data cube from which both spectral detect changes in tissue status before they become
and spatial information may be extracted. Since the in visibly apparent. This approach allows for continuous

Fig. 5. Visual post-operative images at various time points (panels (AC) from a rat reverse McFarlane skin ap, which serves as a model of
reconstructive surgery. Occlusion of the only artery supplying blood to the skin ap leads to a progressive deterioration in tissue viability,
marked at 72 h by damage that is clear to the unaided eye. The image in panel (D) is a map highlighting regional variations in tissue
oxygenation, derived from near-IR spectroscopic images measured 1 h post-operatively [57].
H.H. Mantsch et al. / Vibrational Spectroscopy 30 (2002) 3141 39

monitoring in a clinical setting thereby, identifying study clearly demonstrates the feasibility of combin-
tissues at risk of failing due to inadequate oxygenation ing Raman spectroscopy with endoscopic methods,
and increasing the likelihood that any intervention and lays the groundwork to encourage further inves-
aimed at saving tissue will be successful. A similar tigations.
methodology has since been exploited to characterize The in vivo studies described were centred on point
burns in a porcine model [58], and to highlight regio- spectroscopic studies. The concept of in vivo Raman
nal variations in tissue hydration [59]. imaging is not nearly as advanced as that of in vivo
Recently, we have begun to explore the possibility near-IR imaging described above. However, several
that near-IR spectroscopic imaging might be useful in studies in the literature report on Raman chemical
surgical applications, in particular as a probe of regio- imaging on ex vivo tissue such as the histopathological
nal variations in cardiac oxygenation. Pilot model characterization of biopsied human breast tissue con-
studies using both porcine and murine hearts have taining foreign polymer inclusions [61,62] and the
shown that the method can detect regional oxygen examination of the phosphate gradients in cortical and
deciencies, for example in situations mimicking a trabecular bone [63,64]. The next challenge is to
heart attack. The model we have employed involves transfer this technology for in vivo Raman imaging.
transient occlusion of the artery that is the main blood
supply to the left ventricle. Spectroscopic image sets
reveal the development (during occlusion) and disap- 5. Epilogue
pearance (upon removal of occlusion) of a distinct
region of poor oxygenation, as characterized by both A brief sampling has been given on some of the
high deoxyhemoglobin and low oxyhemoglobin levels medical applications of IR and Raman spectroscopy.
compared to the surrounding tissue. Now the arena is set for the next act of this ongoing
Along with the number of impressive advances IR saga. The challenge to the vibrational spectroscopy
spectroscopy is making in the in vivo medical domain, community is now to look further beyond the walls of
Raman spectroscopy is emerging as a force to be our domain and to develop new, creative means of
reckoned with (e.g. [26] for a review). The technical acquiring, processing and interpreting those squiggles
obstacles of tissues uorescence, silica Raman and (i.e. our treasured spectra) in such a way that we can
ber uorescence and the weak Raman signal from present useful and user-friendly diagnostic methods to
tissue are being overcome with the use of near-IR complement those already available to the medical
excitation, introduction of lters built directly into the profession. With the blending of ideas from diverse
probe tip and more sensitive CCD detection systems. elds, a new set of perspectives can be arrived at to
By using technology that incorporates these advances, overall improve the health of individuals; in so doing,
in vivo intravascular Raman spectra of high quality these techniques can be transplanted out of the spec-
have been obtained using spectrometers tted with troscopic laboratories and into the medical labora-
dedicated optical ber probes [60]. Comparison of tories, clinician's ofce, operating theaters and/or
spectra obtained in vivo and in vitro demonstrated the patients' bedsides.
in vivo collected aorta spectra to be a summation of
signal contributions of the aortic wall and blood.
Detailed molecular information about atherosclerotic Acknowledgements
plaques (i.e. the presence of lipid pools, cholesterol
and calcications) can be obtained in this manner. While the three authors served as your scribes for
Once again along the lines of cancer research, a this review, due thanks go to all our present and former
near-IR bre optic Raman spectroscopic system has colleagues associated with the Institute for Biodiag-
been used to acquire in vivo Raman spectra of human nostics in Winnipeg, who have contributed their
gastrointestinal tissues during routine clinical endo- efforts to the cause of biomedical vibrational spectro-
scopy; the ber-optic probe was passed through the scopy. They are (in alphabetical order): Khalique
working channel of an endoscope and placed in con- Ahmed, Mike Attas, Maureen Donnelly, Hans Eysel,
tact with the tissue surface [25]. This proof-of-concept Heinz Fabian, Mark Hewko, Kan-Zhi Liu, Lorenzo
40 H.H. Mantsch et al. / Vibrational Spectroscopy 30 (2002) 3141

Leonardi, Mike Jackson, Sarah Low-Ying, Angela [24] T.C. Bakker Schut, M.J.H. Witjes, H.J.C.M. Sterenborg, O.C.
Speelman, J.L.N. Roodenburg, E.T. Marple, H.A. Bruining,
Man, Jim Manseld, Anna Matas, Laura McIntosh,
G.J. Puppels, Anal. Chem. 72 (2000) 6010.
Dieter Naumann, Jeri Payette, Trevor Posthumus, [25] M.G. Shim, L.-M.W.K. Song, N.E. Marcon, B.C. Wilson,
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