Applied Spectroscopy Reviews

ISSN: 0570-4928 (Print) 1520-569X (Online) Journal homepage: http://www.tandfonline.com/loi/laps20

Spectroscopic techniques in medicine: The future

of diagnostics

Ranjit K. Sahu & Shaul Mordechai

To cite this article: Ranjit K. Sahu & Shaul Mordechai (2016): Spectroscopic
techniques in medicine: The future of diagnostics, Applied Spectroscopy Reviews, DOI:
10.1080/05704928.2016.1157809

To link to this article: http://dx.doi.org/10.1080/05704928.2016.1157809

Accepted author version posted online: 25

Feb 2016.

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 ACCEPTED MANUSCRIPT

Spectroscopic techniques in medicine: The future of diagnostics

Ranjit K. Sahu1,2 and Shaul Mordechai1,*

1
Department of Physics, Ben-Gurion University of the Negev, Beer-Sheva, Israel
2
Department of Medicine, University of Virginia, Charlottesville, Virginia, USA
*
CONTACT Shaul Mordechai shaulm@bgu.ac.il Department of Physics, Ben-Gurion University
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of the Negev, Beer-Sheva 84105, Israel.

Color versions of one or more figures in this article are available online at

www.tandfonline.com/laps.

Abstract

Spectroscopic techniques have been finding increasing applications in the field of biomedicine

especially in the field of disease diagnosis and monitoring in spite of the rapid emergence of

several molecular biology based techniques. The significance of spectroscopy techniques and the

possibility of using some of the underutilized regions of the electromagnetic radiations is

discussed in this review. While previous reviews have already dealt with the potential of FTIR

based techniques for clinical applications, the present review addresses the lacunae of the

techniques along with its future trends that may make it a technique routinely applied in clinical

settings.

Key Words

Spectroscopic techniques, clinical applications, FTIR

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Introduction

The requirement of a rapid diagnostic method to distinguish between normal and abnormal cells

and tissues is increasing with the expansion knowledge about diseases and identification of

newer diseases of both pathological and non pathological origin. This has led to the demand of

the medical and clinical industry for rapid and objective techniques that are suitable for clinical
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applications under routine working conditions. The electromagnetic radiations cover a very wide

range of wavelengths and over the years have been utilized in both biology and medicine. While

the use of the more common X-rays and computerized tomography (CT) in medical science is

common knowledge, the advances in the utilization of nuclear magnetic resonance (NMR) and

positron emission tomography (PET) have led to the development of highly important imaging

techniques like CT scan and PET scan which have replaced X-Rays for more complicated

diagnostic uses. Similarly, while the use of light microscope has been common place in medical

laboratories, the utilization of recent advances in techniques like FACS and immuno-

fluorescence has led to their inclusion as well in every day medicine for diagnosing a variety of

pathological conditions. In effect the various properties of light and its associated phenomenon in

biological systems have been widely utilized. For example, the principle of light scattering has

been tested for diagnostic potential (1-3). More often these techniques rely on their ability to

distinguish between normal and abnormal conditions with a very high specificity and sensitivity

which is obtained by narrowing down the markers for the pathological conditions under study

(4,5). Among these techniques an under exploited technique is the FTIR based diagnosis which

utilizes a completely different set of biophysical and biochemical principles for its purpose. In

contrast to other techniques where downstream processing of samples usually results is some

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kind of modification or destruction, FTIR spectroscopy relies on the intrinsic biochemical

composition of the samples, thus eliminating lengthy procedures that often accompany analysis

using other spectroscopic techniques and its applicability has been previously reviewed (6).

In spite of its short comings due to a slow progress in the translation of basic research done in the

field of FTIR and Raman vibrational spectroscopy based diagnosis, these techniques may be the
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ultimate savior in the field of medical diagnosis when it comes to obtaining a rapid diagnosis in

an objective manner. In the present article we review the progress made in the utilization of

spectroscopic techniques in biomedicine while predicting the future of this technique in clinical

applications. This is especially significant considering the fact that there will be a large and ever

growing aging population which will need clinical care and diagnostic at low cost over a long

period of time. Issues like changes in bone conditions may be addressed using this technique (7).

This group also would be susceptible to many of the age related diseases like Alzheimer‟s,

Parkinson as well as different kinds of tumors and cancers that appear with age. Thus, apart from

the diagnosis of microbial and pathogen related diseases like HIV and malaria, clinical systems

will need to gear up for a continuous and routine samples from patients with age related

abnormalities. To accommodate such burden on the health systems, it is imperative that

techniques that are less expensive, non-invasive and cause minimum discomfort and

hospitalization to patients will need to be developed. Additionally these techniques must have a

high specificity and sensitivity to be able to give fool proof results to avoid any ambiguity in

diagnosis. Spectroscopic techniques which are still being examined for their relevance and

potential may be the ones of choice to be relied upon under these circumstances. The present

review examines the progress made hitherto in the application of IR spectroscopic and IR

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imaging techniques individually or as a part of larger complex set up in the last few decades and

its future role in biomedicine.

Applications of spectroscopy in biomedicine

Even though the word spectroscopy may seem alien to many people, radiation based diagnosis

have been at the forefront of modern medicine. While the X-Ray based diagnosis are being
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rapidly replaced by other less harmful techniques like ultrasound where applicable, the advent of

CT scans based on X-Ray is still a popular and often the only way to look at small areas of

organs for diagnosis of different abnormalities including tumors, stones, cysts that may not be

accessible to other methods of probing under in vivo conditions. Similarly while it may seem

common knowledge that histology and immuno-histochemistry are microscopic based

techniques, these too utilize the spectral properties of absorbance/reflection and fluorescence of

different dyes and fluorophores. Figure 1 shows how the entire region of the electromagnetic

spectrum has become relevant in the field of medical diagnosis and treatment with radiations

with different frequencies (wavelengths) having their own significance.

The application of spectroscopy in the various aspects of disease diagnosis and management can

be illustrated through the versatility of FTIR based research in the field of colon cancer. Colon

cancer has been a favored model system for study of FTIR applications mostly for its relevance

to disease management as it is one of the cancers with a high prevalence (third most common)

yet with a high survival rate upon successful diagnosis and timely intervention (8). The field

began with preliminary observations of the utility of FTIR for colon cancer diagnosis (9). The

studies have centered mainly around diagnostics (10-12). Additionally this model system has

been helpful in the development of data analysis systems (13,14). The model system has also

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been used to monitor the spread of the disease and stimulate the process in animal models to

evaluate its applicability (15,16). Various drugs and their mode of interaction for colon cancer

have used FTIR as an analytical technique (17-18). Similarly FTIR has been used for evaluating

the products that were developed for selective targeting into colon cancer tissues/cells (19-22)

and characterization of these compounds (23-24). In this manner the spectroscopic techniques
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become relevant to the entire field of biomedical research from diagnosis to

prevention/remediation of a disease.

Advances in various spectroscopic techniques

While spectroscopy has been in use in the field of biomedicine, recent advances have been

spectacular when considered in terms of the technical advancements made in the area of both

basic and translational research as well as clinical application. The development of FTIR based

diagnosis which is an emerging field is described in brief as a reflection of this trend. From the

days of its discovery as a technique suitable for distinction of normal and abnormal colonic

tissues (9), the colonic tissues have been extensively used as a model to evaluate the diagnostic

potential of FTIR spectroscopy (25). The advantages of using colonic tissues are that it has a

well defined architecture (Figure 2) that is altered during different kinds of abnormalities. With

the ability of pattern recognition for data analysis along with the infrared microspectrometer

(26), and the development of protocols to study consecutive sections for comparison between

normal and abnormal tissues (27) with their spectral data, this model gained further acceptance.

IR spectra of colonic tissues like cervical tissues under normal conditions contain defined

signatures from carbohydrates (glycogens) in the form of triad which is reduced or altered during

abnormal metabolism associated with disease or malignancies (28). These markers provided a

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base for the development of tissue diagnosis while monitoring changes in signals associated with

the decrease in glycogen. These findings determined the pathology which was verified in parallel

by evaluating consecutive sections by an expert pathologist (29). Later the signals from

glycogen/carbohydrates, nucleic acids, lipids and others were found to be equally efficient for

not only detection of cancer in biopsies but also to variety other abnormalities and malignancies
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from levels of cell to tissues (4,5,30), giving rise to the concept of common biomarkers. This

became important as possible utility of the instrumentation which was broadened and re

evaluated making investments at clinical levels a possibility. The development of focal plane

area (FPA) detectors, cluster analysis and other multivariate computational methods for

automated data analysis make the system more objective and further strengthened its

translational capabilities.

One important area where the spectroscopic techniques are relevant is during the continuous

monitoring of diseases in situ. While preliminary studies have been conducted using FTIR based

technique owing to its ease and speed, (31), further research of its application for evaluation

during endoscopies is required. This is important as currently only a visible range of light with or

without tissues staining is being used during colonoscopy, endoscopy and colposcopy.

Colonic tissue contains crypts which may be obtained in transverse or longitudinal sections

during the biopsy processing (Figure 3). While initial studies for detection of colonic

abnormalities using FTIR utilized only the transverse sections owing to their distinct morphology

under a microscope even without any staining (12) later studies also established the importance

of the longitudinal configuration of the crypts (32-34). The advances in computational analysis

further highlighted the importance and potential of the measurement along the crypt (35). Thus

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FTIR spectroscopy maybe used for evaluation of a tissue even though the biopsy may not be

suitable for other processes like immunofluorescence due to its configuration. The developments

of methods to overcome the effects of tissue processing and elimination of artifacts have further

added to the possibility of making the system more sensitive and useful (36).

Another technical requirement of this field is the ATR and FEWS systems (37) as these have a
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direct implication for development of probes suitable for endoscopy. In this regard, work has

been limited to skin and tissues of the kind that are on the surface as these have better

accessibility to surface scanning probes (38) or comparing the transmission system with the total

reflectance system (39). With advances it is likely that more research will be conducted if not

with human subjects but at least with animal models to advance the knowledge in this regards. A

schematic diagram of the FEWS system is shown in figure 3.

There have been studies highlighting the potential of FTIR spectroscopy to monitor patients

(31,40) by monitoring the changes in body fluids. While it is quite logical that these types of

studies are directly relevant to diagnosis of malignancy or monitoring the same in case of blood

related diseases(40), examination of body fluids as mentioned above may also be an easier way

to assess for diseases which are present in a different organ other than blood (41-43). Thus,

measurements on body fluids, especially blood samples through measurements of plasma and

WBC maybe an alternate approach which is minimally invasive for rapid diagnosis of solid

tumor since the whole body responds through the natural immune system to malignancy though

the effects are maximum at the site of origin in the initial stages. The field is expanding to

include diagnosis of pathologies in other human organs/tissues (44-47). For example, FTIR and

fluorescence microscopic studies on different types of gallstones from adults and children have

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shed light on their chemical composition and nucleation center. It was concluded that the inner

core of the stone had more of fatty acids and calcium carbonate, which could have initiated the

nucleation events for the stone formation. Bilirubin and cholesterol might be the risk factors in

children and adults respectively for developing gallstones disease (GSD) where stones are

formed in the gallbladder. (48) However, as seen from figure 3b the information provided by
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FTIR in terms of chemical composition is more detailed compared to fluorescence spectroscopy.

Obstacles in translating basic research in spectroscopy

One of the main hurdles in the application of spectroscopy in clinical settings has been the

transfer of observations under laboratory settings to clinically compatible methodologies that can

be carried out by less technically qualified personnel. While instruments like FACS which apply

fluorescence as their basic principle are now widely accepted as a means to identify abnormality

at the cellular levels, imparting a certain degree of objectivity to the approach, the diagnosis of

tissues is still largely subjective with immune-histochemistry being the main stay. This also is

the reason why expertise of the pathologist and his availability becomes a crucial factor in

deciding the status of a biopsy. To overcome these undesirable factors from contributing to the

complexity of the diagnosis, a reliable and rapid technology that is simpler to establish and

utilize under clinical conditions is required. Though techniques like CT scan, ultrasound and

PET are now routinely used to support the physician‟s observations, the continuous and regular

usage of these imaging techniques is largely unwarranted. Moreover, with the increasing costs of

health care these would become less affordable for the growing elderly population. As mentioned

before there are regions of the spectrum which are less invasive and harmless to the organs and

tissues and which remain under exploited in the field of biomedicine though their application has

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been well established in other fields like agriculture (49), food science(50), forensics(51),

pharmaceuticals (52,53) and allied industries (54). The mid IR and near IR regions of the spectra

is one such area which can provide further scope for incorporation into the field of biomedicine.

The major obstacle in this regard is the unambiguous establishment of a correlation between

tissue histology and the spectra obtained of the corresponding region. In the last two decades
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sufficient work has been carried out by several groups which unequivocally show that indeed the

different tissues and cells have unique fingerprints which is altered during disease conditions.

Yet most of these studies have been carried out on biopsies or samples and under ex vivo

conditions (55-59) making their direct application in situ less obvious. This has been the major

obstacle that has hindered the utilization of the potential benefits of IR spectroscopy on a large

scale.

Another important obstacle in the field of spectroscopy applications in biomedicine has been the

sample size. In most studies, sample sizes from human origin are small as most are obtained

during biopsies and rarely from excision of organs for pathological evaluation and are sparingly

available for any extra basic studies. Thus human samples are precious, and the quantity that is

expendable becomes small. This is one reason that the field of FTIR spectroscopy is not well

established due to lack of sufficient number of samples for obtaining statistically acceptable

numbers. This along with the fact that there has to yet be specifically designed experiments for

spectroscopy using animal models makes the progress of translation of any significant

observations slow. There have been attempts to circumvent this aspect by developing

computational models (39) which may help to partly alleviate this problem. While techniques

like CT Scans may be used to generate a 3D image of an organ or tissue in situ (60), continuous

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exposure to this region of radiation maybe harmful. Unfortunately though infrared is less

damaging, due to its minute energy and penetration powers into tissues, currently its usage for

3D imaging is limited, though penetration depth is expected to be significantly larger in the near

infrared (NIR) region compared to MIR

The unique potential of the spectroscopic techniques

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Most modern medical diagnostics depend on spectroscopic principles at some point of their

application, be it the fluorescence in immunofluorescence based techniques like FACS/immuno-

histochemistry or the routine methods like ELISA which depends on the absorbance of products

from enzymatic reactions, though not obvious. Except a few techniques like X-rays that are used

for monitoring structural features, the spectroscopic techniques are quantitative, lending a high

degree of sensitivity and precision to data analysis. This is the main stay and unique capability of

the spectroscopic techniques in general. On the other hand different clinical instruments may

base their diagnostic capabilities to a defined range of radiations that they are based on making

their features and potential unique. Most FTIR based techniques though not yet clinically applied

are reagent free and need little or no processing though they require a IR transparent matrix for

transmission mode acquisitions. FTIR based techniques also utilize the unique fingerprint as

their principle which is now widely recognized and thus a reference point is easily available

when comparing a new sample with an existing data set. Similarly, measurements carried out

using many diagnostic kits rely on a valid standard with well established spectroscopic properties

(either naturally or as a derivative there off). Many spectroscopic techniques have been

developed to overcome the barriers that exist in various technologies. While FTIR spectroscopy

based on vibrational characteristics of molecules is affected by water absorption, Raman

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spectroscopy based on the same physical phenomenon is not. However the requirement of lasers

for Raman spectroscopy makes it less likely to be used for in situ or in vivo applications as far as

human studies are concerned. The ability of FTIR spectroscopy is being expanded to include

different types of cancers but additionally different types of abnormalities are being screened

using the technique though at laboratory scale (61-68). The application has now been extended to
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body fluids and other tissues which are akin to a minimally invasive or non-invasive procedure

on the patient for diagnosis (69) and applicability and limitations of different approaches are

being studied with methods to increase signal from low abundant compounds that may have

diagnostic significance (70). The potential and prevalent application of FTIR spectroscopy and

its relevance is displayed in figure 4.

Future directions and prospects

It is interesting to note that though fluorescence spectroscopy was invented later than FTIR

spectroscopy, the former has been inculcated into many aspects of clinical research. This has

been made possible by the parallel discovery of several fluorophores which can be excited at

various wavelengths and conjugated to antibodies. This single technical breakthrough has

gradually expanded into a technology like FACS which is now clinically acceptable because of

its ability to bridge the gap between biological processes and physical phenomenon. In the case

of FTIR this has not been as spontaneous as the ability to be an independent methodology for

evaluation is still being questioned by conventional pathologists in spite of overwhelming

amount of literature in support of the validity of unique fingerprint. Additionally there are

several demerits in the application of fluorescence spectroscopy compared to infrared

spectroscopy. The comparison is presented in table 2. As can be seen that FACS is still

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considered at par with other clinical techniques owing to its unequivocal diagnostic capabilities,

which cover up its various demerits like long analysis time, complex staining protocols,

requirement for specific reagents etc. One possibility of improving the FTIR diagnosis is through

the utilization of several unique biomarkers to further classify the tissues as well as the

possibility of adding a substance (marker) with unique and differential fingerprint in the IR
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region as successfully done in FACS. While in the case of FACS and Immunofluorescence these

can be easily visualized using fluorophores with different emission/excitation properties

conjugated to antibodies targeting different markers, lending weight to the observation through

co-localization or simultaneous staining, in case of FTIR and Raman spectroscopy, such

biomarkers are obtained from the same spectrum. This is both an advantage and a disadvantage

as a single spectrum (or average of spectra) becomes the determinant of the diagnosis.

It is an inexpensive technique and is suitable for veterinary purposes in which case the possibility

of investment towards the health of the animal (71) is less as well as during times of epidemics

there is a need for a rapid diagnostics (72).

One area of research pertaining to spectroscopy with wide implications is the ability to use it for

in situ diagnosis. Though there have been pilot studies (73,74) in this regard yet a lot has to still

be attained. However modifications of this approach may find relevance in diverse fields as well

(75) and may lead the way for implementing similar principles for in situ diagnosis of ailments

based on spectral properties of surface tissues. The utility of the infrared diagnosis is at the

forefront of such research owing to the fact that all other radiations in the spectra are of high

energy and can cause extensive damage if used on live tissues. With the increasing incidents of

cancers of the gastrointestinal tract and the reproductive organs, which can have a positive

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impact on the life of patients if detected early and routinely, the ability of the mid IR

spectroscopy to detect changes in tissues at molecular level manifested that a spectral changes

can in future enable the development of probes that can be inserted into the organs. These may

then be used in tandem or simultaneously along with the routine colposcopy or

colonoscopy/endoscopy for further validation of the results. While the histopathological

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relevance of FTIR spectroscopy in clinical evaluation of biopsies has been more or less

established, such a study pertaining to real time and in situ measurements of normal and

malignant (abnormal) tissues is yet to be established. One approach would be to begin such kind

of studies with animal models. However presently most materials used as a matrix or scaffold for

IR spectroscopy are made up of materials like silver Halides, ZnSe or halides of other cations

(Ba, Ca) with low ductility and malleability. Most fiber optics systems used cables that contain

polymers that may have a dense signature in the IR region, leading to high background levels

unsuitable for their utilization in the probes. Moreover these measurements are likely to be of a

few micron depths like 1-2 microns, just scanning the surfaces of the lumen. Thus a higher

sensitivity as well as specificity of the biomarkers is desirable. With the possibility that most

such measurements would inevitably end up with signatures form the membranes comprising

mostly lipids (and proteins) the spectral range available for biomarker identification is further

limited to the higher wavenumber and the signatures from amide I-III (along with lipids). This

would exclude the region between 900-1300 cm1 where most biomarkers have been validated

for diagnosis till date, baring a few where the higher wavenumber region has been dealt with.

Thus further research in the utility of the higher wavenumber region and model compounds that

may stimulate the membrane architecture in normal and abnormal tissues would be required and

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would possibly be the direction in which research will progress towards making the translation a

possibility.

The area of FTIR spectroscopy aimed at biomedical applications would continue to depend on

allied disciplines like physics and mathematics (computer science) for continuous innovation to

increase the data analysis and interpretation. With several different approaches being undertaken
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over the last decade in this aspect (76-80) using models systems for constant validation of the

technique for biomedical applications.

The inexpensive characteristic of FTIR based diagnosis makes it a technique suitable for

utilization in veterinary applications (81-85) where both the cost as well as rapidity become

determinants. Additionally as the samples are from animals or animal models, it is easier to

maintain a better homogeneity in the sample while evaluation. This area would expand in the

future due to growing importance of agriculture as well as for techniques required for evaluation

of animal samples, the effects of climate change being a major parameter.

New evidence suggests that the FTIR spectra from body fluids may be highly specific for each

individual, making it relevant in forensic studies (86). Further studies for other organ deposts like

stones from gall bladder and bladder may also continue to classify the stones in a rapid manner

for quick initiation of the therapy (87). This may also be in tune from the perspective of

personalized medicine which is rapidly becoming a reality, helping to take advantage of the

unique potential of FTIR spectroscopy by establishing a basic pattern for a patient‟s sample and

monitoring it over time. The possibility of combining different spectroscopic techniques for

improving the diagnostics or analysis of samples is one area that could easily have a clinical

application due to increased sensitivity from two or more different readouts simultaneously (88)

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or undertake other types of spectroscopy like non-linear spectroscopy to determine structure or

composition (89). A schematic representation of FTIR spectroscopy in biodiagnostics as shown

in Figure 5 would predict this outcome for clinical settings. This can be easily forecast with the

entry of bench top type of diagnosis of biological samples being possible using single cell

systems (90).
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Conclusions

Spectroscopic techniques have been a vital part of biomedical research and application. Growing

demand for rapid and precise diagnosis further has brought the focus towards developing

instrumentation for exploiting this range of wavelengths of the electromagnetic spectrum. While

most of the spectra are utilized, in the recent past the mid IR and NIR regions has been gaining

importance. There are several technical constraints that need to be overcome before this region is

utilized for routine clinical purposes. The growing research in the utilization of mid IR

spectroscopy is a reflection of the importance of spectroscopy in biomedicine and allied

disciplines like forensics.

Acknowledgments

Many thanks are due to Plabani Sahu for her careful assistance with the figures.

Funding

This research work was supported by the Israel Science Foundation (ISF) and the Israel Cancer

Association (ICA).

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90. Hughes, C., Henderson, A., Kansiz, M., Dorling, K.M., Jimenez-Hernandez, M., Brown,

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Analyst.140:2080-5.
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Table I Comparison of Fluorescence spectroscopy and IR vibrational spectroscopy.

Feature Infrared Fluorescence

spectroscopy spectroscopy

Clinical Application Not yet use under Currently in use for

clinical or laboratory diagnosis of cell and

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setting for biomedical fluid abnormality and

applications in cell biology.

Flexibility, FPA, Microscopy Modified for cells and

modifications and system, ATR and cytokine/biochemicals

adaptation FEWS based, using bead based

transmission based. technologies

Reagent requirements None Mostly based on

antibodies for markers

and fluorochrome

conjugation to those

Data acquisition and Rapid data about Detection of

analysis several biochemicals maximum 15 markers

from each individual

cells. Needs

calibration and

compensation to be

made every time.

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Sample suitability Cells, tissues, fluids Single cell

(dried) suspensions and fluids

only

Requirement for Histochemical Not required or not

additional techniques complementation in applicable

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case of tissue samples

Cost Relatively cheap Expensive due to

when applied requirement of several

reagents

Sample Not destroyed or Modified during

modified after preparation and

evaluation. Still usually lost not

available for re- recovered after data

evaluation acquisition

Suitability for future High due to non Not applicable

in situ applications toxicity of radiation As the instrument

needs laser sources

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Figure 1. Schematic representation of the various regions of the electromagnetic spectrum

and their utilization in biomedicine.

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Figure 2. Hematoxylin eosin stained sections of a colonic biopsy showing the crypts in

longitudinal section (left) and cross section (right) with the spectrum in mid IR region

between 800-1800 cm1 in the center as a representation of the data output using FTIR

microscopy. The red circles indicate the measurement areas usually used for diagnosis of

colonic abnormalities (malignancies) and usually represent a diameter of 100 microns.

The data are obtained from a consecutive unstained section.

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Figure 3. (a) Schematic diagram of a FEWS system and its application in FTIR based

diagnosis of abnormalities on surfaces amenable to probes. The system of FEWS

operates on the ATR principle. As depicted in figure 3, the system consists of a source of

light and a modulator that generates IR beam. The IR beam can be passed through a fiber

optic system as represented inside the dashed trapezoid with a probe whose surface

would be in contact with the organ. This is the possible way of using the fiber-optic

system. On the top of the figure is a IR transparent medium on which a traditional sample

is mounted and the measurements made using the same system to establish the

biomarkers. The transmitted light is detected by the detector and the signals processed to

obtain the spectra. (b) Comparison of information obtained from green stones of adult

gall bladder using two spectroscopic techniques. Adapted from reference 48.

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Figure 4. Application of FTIR spectroscopy in biomedicine.

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Figure 5. Schematic representation of the application of FTIR spectroscopy in clinical

diagnostics.
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