Mini-review

IR microspectroscopy: potential applications

in cervical cancer screening

Michael J. Walsh a, Matthew J. German b,1, Maneesh Singh c, Hubert M. Pollock b,

Azzedine Hammiche b, Maria Kyrgiou c,2, Helen F. Stringfellow c,

Evangelos Paraskevaidis d, Pierre L. Martin-Hirsch c, Francis L. Martin a,*

a Biomedical Sciences Unit, Department of Biological Sciences, Lancaster University, Lancaster LA1 4YQ, UKb Department of Physics, Lancaster University, Lancaster, UK

c Sharoe Green Unit, Lancashire Teaching Hospitals, Sharoe Green Lane North, Fulwood, Preston, UKd Department of Obstetrics and Gynaecology, University of Ioannina, Ioannina, Greece

Received 15 January 2006; received in revised form 14 March 2006; accepted 16 March 2006

Abstract

Screening exfoliative cytology for early dysplastic cells reduces incidence and mortality from squamous carcinoma of the

cervix. In the developed world, screening programmes have adopted a 3–5 years recall system. In its absence, cervical cancer

would be the second most common female cancer in these regions; instead, it is currently eleventh. However, there exist a number

of limitations to the smear test even given the removal of contaminants using liquid-based cytology. It is prohibitively expensive,

labour-intensive and subject to inaccuracies that give rise to significant numbers of false negatives. There remains a need for novel

approaches to allow efficient and objective interrogation of exfoliative cytology. Methods that variously exploit infrared (IR)

microspectroscopy are one possibility. Using IR microspectroscopy, an integrated ‘biochemical-cell fingerprint’ of the lipid,

protein and carbohydrate composition of a biomolecular entity may be derived in the form of a spectrum via vibrational transitions

of individual chemical bonds. Powerful statistical approaches (e.g. principal component analysis) now facilitate the interrogation

of large amounts of spectroscopic data to allow the extraction of what may be small but extremely significant biomarker differences

between disease-free and pre-malignant or malignant samples. An increasing wealth of literature points to the ability of IR

microspectroscopy to allow the segregation of cells based on their disease status. We review the current evidence supporting its

diagnostic potential in cancer biology.

q 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cervical cancer; Cytological screening; IR microspectroscopy; Principal component analysis; False negatives; Papanicolaou smear

Cancer Letters 246 (2007) 1–11

www.elsevier.com/locate/canlet

0304-3835/$ - see front matter q 2006 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.canlet.2006.03.019

Abbreviations: ATR, attenuated total reflection-FT-IR; CaP, prostate cancer; CIN, cervical intra-epithelial neoplasia; FT-IR, Fourier transform

IR; HSIL, high-grade squamous intraepithelial lesion; HPV, human papillomavirus; IBD, inflammatory bowel disease; IR, infrared; LBC, liquid-

based cytology; LSIL, low-grade squamous intraepithelial lesion; PaP, Papanicolaou; PCA, principal component analysis; PCs, principal

components; PTMS, photothermal microspectroscopy; SDS, sodium dodecyl sulphate; SNOM, scanning near-field optical microscopy; SNR,

signal-to-noise ratio; SRS, synchrotron radiation.* Corresponding author. Tel.: C44 1524 594505; fax: C44 1524 593192.

E-mail address: f.martin@lancaster.ac.uk (F.L. Martin).1 Present address: School of Dental Sciences, University of Newcastle, Framlington Place, Newcastle NE2 4BW, UK.2 Present address: Department of Obstetrics and Gynaecology, Hammersmith and Queen Charlotte’s and Chelsea Hospitals, Du Cane Road,

London W12 0HS, UK.

M.J. Walsh et al. / Cancer Letters 246 (2007) 1–112

1. Introduction

The development of an objective and automated

approach to cancer screening to improve sensitivity and

reduce underreporting is imperative. Cancer pro-

gression from pre-malignancy to invasive and/or

metastatic growth may take many years. Because

earlier detection is associated with better prognosis, a

sensitive and specific test to identify atypical cells is

critical in cancer screening. Infrared (IR) microspectro-

scopy appears to be capable of identifying biochemical

biomarkers of dyskaryosis and has potential appli-

cations in screening to augment efficiency.

2. A current perspective of cervical cancer screening

Cytological screening for cervical cancer has

arguably met many of the prerequisites necessary for

a well-population programme. The disease is suffi-

ciently common to justify mass screening, is associated

with significant mortality, effective treatment is

Fig. 1. Chronology since the discovery of the smear test of the implementat

http://www.cancerscreening.nhs.uk/; accessed 13th December 2005).

available for pre-invasive or early invasive disease

and, detection and treatment of a presymptomatic state

results in benefits beyond those obtained through

treatment of early symptomatic disease [1]. A cervical

screening programme was introduced into the UK in

1988 and the Papanicolaou (Pap) smear has resulted in

reductions in cervical cancer incidence and mortality

(Fig. 1). Incidence has fallen by 26% since 1992, and

mortality as a consequence of this disease has dropped

from 7.1/100,000 in 1988 to 3.7/100,000 in 1997 [1].

The second most common female cancer worldwide,

long-term implementation of a UK screening pro-

gramme means that it is now the eleventh most

common female cancer in this region [2].

The aetiology of cervical cancer is strongly

associated with viral infection (i.e. human papilloma-

virus (HPV)); other risk factors include low socio-

economic class, multiple sexual partners, smoking and

poor diet. Specific oncogenic HPV genotypes may be

the main initiating and/or promoting entities in the

progression of clinically invasive squamous carcinoma

ion of screening for squamous carcinoma of the cervix (adapted from

M.J. Walsh et al. / Cancer Letters 246 (2007) 1–11 3

as these are present in O95% of cervical cancer [3].

Categorisation criteria of pre-invasive disease should

be well-defined to reduce inappropriate diagnosis.

Cervical intra-epithelial neoplasia (CIN) is a histologi-

cal description of pre-invasive disease and is cate-

gorised into three sub-categories known as CIN1, CIN2

and CIN3. In the US and now many other countries, the

Bethesda classification of cytological abnormalities is

used. Increasing CIN in exfoliative cytology is

categorised by grades of dyskaryosis and correlates

with severity of atypia. CIN1 is associated with low-

grade squamous intraepithelial lesion (LSIL) or mild

dyskaryosis that may regress; increasing dyskaryosis is

categorised as high-grade squamous intraepithelial

lesion (HSIL) and in these sub-categories (CIN2 or

CIN3) the cytology is severely atypical, unlikely to

regress and may progress to invasive disease [4].

Histologically confirmed, high-grade CIN2 or CIN3

pre-invasive disease is treated on diagnosis.

In modern practice, a brush is used to sample the

transformation zone of the cervix. Exfoliated cells are

either transferred onto a microscope slide or placed into

liquid medium known as liquid-based cytology (LBC)

solution [5]. Despite the successes of organised

screening programmes, the Pap smear may lack

sensitivity (percentage of true-positive cases detected)

and/or specificity (percentage of true-negative cases

that are negative) giving relatively high rates of false

positives or false negatives, respectively. A false

positive is a smear that is identified as dyskaryosis

but turns out disease-free. This lack of specificity,

although resolvable, may lead to unnecessary referrals

for colposcopy with consequent patient anxiety and

financial costs. Lack of sensitivity might result in an

apparently disease-free woman with occult disease

progressing to invasive carcinoma within the 3–5 years

routine recall period, i.e. a false negative. In an

extensive systematic review of cytology diagnostic

accuracy, the mean sensitivity was 59% with the

specificity being 69%; the accuracy within published

primary screening was less [6].

Sub-optimal sampling and/or mistakes due to human

subjectivity may contribute to errors. Responsible for

some two thirds of errors, sampling may be associated

with the provision of an unrepresentative or inadequate

sample. This aspect of smear accuracy can only be

achieved by better smear-taker training. Human error

accounts for the other third of smear mis-interpretation;

a result of poor management, lack of training or

tiredness on the part of the cytologist. The low

prevalence of cervical disease in regularly screened

populations makes the task of the screening

cytotechnologist difficult. Squamous atypia can be as

low as 5–7% [7] so the vast majority of smears are

negative. High vigilance is difficult to maintain under

these circumstances, and fatigue can contribute to

screening errors [8].

Cervical smears are often contaminated by red

and/or white blood cells, leukocytes, bacteria and

mucins. To reduce the number of inadequate smears,

the sampling process in many countries now incorpor-

ates LBC (Fig. 1). In LBC solution, exfoliated cells are

transferred in a liquid fixative, subsequently filtered or

centrifuged and applied as a thin film on a microscope

slide. However, LBC methodologies may also under- or

over-estimate disease severity. There appears to be

conflicting data in the published literature on the true

decrease of inadequate smears and any advantages, if

they exist in the diagnostic performance with LBC [9].

Other screening approaches currently being investi-

gated include HPV genotyping [10]. HPV testing

increases the screening sensitivity and its potential

role is being assessed by randomised controlled trials.

Organised cervical cancer screening programmes

are expensive as highlighted by the UK programme

(Fig. 1). With the proposed introduction of a

vaccination programme, one might presume that the

necessity for cervical cancer screening might ultimately

become redundant [11]. However, phasing out this

screening programme should be tempered by some

considerations. First, the current vaccination is against

select high-risk HPV genotypes and brings with it the

unpredictability regarding the long-term efficacy of any

viral vaccine [12]. Secondly, the aetiology of cervical

cancer is most probably multi-factorial; removal of one

dominant pre-disposing factor may result in the

emergence of another. It is now important to look to

cheaper, automated and more objective alternatives.

One such strategy has been to develop automated

screening using image interpretation software that has

the capacity of rapidly interrogating slides and high-

lighting areas of concern within a slide for the

cytoscreeners to focus on [8]; this approach may

facilitate more accurate and productive screening.

These recently developed computer-aided systems are

based on cellular visual image characteristics. It also

has the potential to increase job satisfaction and

performance [13].

IR microspectroscopy seems to possess the potential

to be applied as a screening tool [14,15]. It has been

applied to identify cancer biomarkers in de-waxed

tissue sections cut from paraffin blocks [16]. Although,

paraffin contributes to the acquired spectra, it is still

possible to successfully discriminate between benign

M.J. Walsh et al. / Cancer Letters 246 (2007) 1–114

lesions and invasive disease [17]. Thus, with appro-

priate ethical guidance, there is the possibility to

conduct retrospective validation studies employing

archived tissue and corresponding case notes. Data

obtained using IR microspectroscopy is now readily

interrogated for biomarker identification using

advanced computational techniques. However, this

technology needs to be validated in large prospective

studies before it might lead to an alternative to present

options for automated cytology screening.

3. Why IR microspectroscopy?

Spectroscopic methods applied to cancer studies

include Fourier Transform IR (FT-IR) microspectro-

scopy [18–24], attenuated total reflection-FT-IR

(ATR) microspectroscopy [16], Raman microspectro-

scopy [25–28] and photothermal microspectroscopy

(PTMS) [29]. The underlying principle is that an IR

beam is focused on a received sample (i.e. cells) that

absorbs this energy; it is how the signal is

subsequently detected that determines the method of

microspectroscopy. These methods permit the detec-

tion and measurement of cellular biomarkers includ-

ing DNA, RNA, lipids, phosphate and carbohydrates

[22,23,29]. Thus, acquired spectra of cells and/or

tissues may give a detailed ‘biochemical-cell finger-

print’ that varies dependent on clinical status

[19,22,23]. Other unusual spectral features such as

Mie-type scattering that may be associated with

varying opacity of intracellular molecules may be

exploited to distinguish between pyknotic and mitotic

nuclei but such phenomena remain to be fully

understood [30].

Microspectroscopy is not only applicable to cervical

cancer screening [19,31] but also, to any tissue type that

needs to be assessed for abnormalities [20]. Because of

the heterogeneous nature of exfoliative cytology, blood

cell lysis buffers may improve the spectral content

obtained from cervical smears using FT-IR microspec-

troscopy [21]. Although cell abnormalities are identifi-

able histologically, IR microspectroscopy potentially

allows for an objective and accurate assessment when

used in combination with computational tools that

facilitate the identification of variations in metabolites

that suggest pathological alterations [32]. The structural

alterations associated with the transformation of normal

cells into malignant and the intermediate continuum of

changes in this transition may be characterised using

this approach [19].

4. Biochemical-cell fingerprint

Cellular biomolecules absorb the mid-IR (lZ2–

20 mm) via vibrational transitions that are derived from

individual chemical bonds; this may yield richly-informa-

tive ‘fingerprint’ spectra relating to structure and confor-

mation [29]. Absorbance bands in vibrational spectra give

rise to peaks that correspond to different molecular bonds;

the main ones are amide I (z1650 cmK1), amide II

(z1550 cmK1), amide III (z1260 cmK1), asymmetric

phosphate (z1225 cmK1), symmetric phosphate

(z1080 cmK1) and glycogen (z1030 cmK1). In

vibrational spectra, different ratios and/or conformations

of biomolecules give rise to subtle changes in these peaks

(i.e. shape, shift and/or intensity) and indicate intracellular

alterations [16]. The amide I peak occurs due to the

n(CaO) protein amide bond while, the amide II peak is due

to d(N–H) and n(C–N) protein bonds [33]. A shift in the

centroids of amide I or II peaks are indicative of alterations

in the secondary structures of intracellular proteins [33].

Phosphate bands are due to nsðPOK2 Þ and nasðPOK

2 Þ and may

indicate phosphorylated proteins [14,29]. The major

spectral region of interest occurs between 900 and

1800 cmK1 [29]; however, wavenumbers 2800 to

3000 cmK1 may also contain a biomolecular signature of

interest, i.e. for pre-malignancy [15].

For IR microspectroscopy to be applied to cancer

diagnosis a robust biomarker(s) is required and candi-

dates exist. As an indirect indicator of cellular metabolic

turnover, one such potential biomarker is the glycogen/

phosphate ratio (ratio of absorbance intensities at 1030

and 1080 cmK1) [22]. When compared to a spectral

signature derived from normal cells, IR spectra pointing

to reduced glycogen levels and increased phosphate

levels are suggestive of a state of rapid cell proliferation;

this might indicate the presence of malignancy [20].

However, while cervical cancer glycogen levels decrease

and phosphate levels increase, melanoma may not exhibit

similar changes in glycogen levels [22]. Increases in

RNA/DNA ratio (ratio of absorbance intensities at 1121

and 1020 cmK1) is a biomarker for melanoma, colorectal

cancer and cervical cancer [20,22]. Such biomarkers will

require retrospective (on archival material) and prospec-

tive studies employing large cohorts before their

diagnostic potential can be validated.

5. Technologies employed in microspectroscopy

5.1. FT-IR microspectroscopy

In FT-IR microspectroscopy, an IR beam passes

through a received sample mounted on an IR

M.J. Walsh et al. / Cancer Letters 246 (2007) 1–11 5

transparent window, e.g. BaF2, ZnS. The spatial

resolution of FT-IR microspectroscopy is limited by

the diffraction limit [34]. The black body source of

radiation conventionally used for FT-IR microspectro-

scopy is relatively dim [35] and this may give rise to

weaker absorbance spectra with a low signal-to-noise

ratio (SNR). Samples that are opaque to IR radiation

may be difficult to analyze in transmission mode, i.e.

mitotic chromosomes [29,36,37]. Current techniques

often rely on the interrogation of a dried monolayer

sample; spectra of samples in their original aqueous

state often exhibit less intense amide II and phosphate

bands suggesting that dehydration affects the secondary

structure of proteins [38]. However, FT-IR microspec-

troscopy is a fast, objective and non-destructive

approach that has already been shown to discriminate

between cell states [16,19,20,22] and may be adaptable

to a high-throughput analysis, i.e. for imaging of tissue

microarrays [17,39].

Applied to exfoliative cervical cytology, FT-IR

microspectroscopy identified significant spectral differ-

ences between normal compared to invasive smears

[18]. Combining FT-IR microspectroscopy with princi-

pal component analysis (PCA), normal cervical epi-

thelium possessed strong glycogen and symmetric

phosphate peaks whereas invasive cells exhibited a

reduced glycogen band and increased symmetric and

asymmetric phosphate bands [40]. FT-IR microspectro-

scopy with chemometric analysis successfully charac-

terised normal, dysplastic and malignant cervical smears

[19]. It was also possible to discriminate between normal

and invasive cervical smears using FT-IR microspectro-

scopy combined with probabilistic neural networks [31].

Cervical cancer tissues, compared to normal, were

associated with decreases of between 23 and 49% in

glycogen/phosphate levels and, increases of between 38

and 150% in RNA/DNA ratios [31].

Several other cancers also appear to have pre-

malignant stages, i.e. polyps may progress to colon

cancer. Not only may FT-IR microspectroscopy success-

fully discriminate colon tissue as being normal or

abnormal but it may distinguish between polyps and

cancer using advanced computational techniques [20].

Colon cancer may also develop from crypts that appear

histologically normal; using FT-IR microspectroscopy,

altered spectra compared to normal were derived from

such crypts [41]. Inflammatory bowel diseases (IBD)

such as Crohn’s disease or ulcerative colitis may, but not

always, progress to colon cancer. When compared to

normal and cancerous, the spectral fingerprint of

IBD tissues has facilitated predictions of individual

tissues in which there was a greater likelihood of

progression [32]. Classification of acute lymphoblastic

leukaemia with and without blasts has been achieved with

FT-IR microspectroscopy spectra of lymphocytes and

subsequent cluster analysis, and this correlated with

clinical response to chemotherapy [42]. Age-related

structural changes (e.g. 8-hydroxypurine lesions) give

rise to a DNA phenotype in non-malignant prostate

tissues of older men (55–80 years) with features similar to

primary CaP [43]. In normal prostate, a metastatic CaP

DNA phenotype may occur that shares structural

similarities to DNA isolated from metastasizing tumours;

this exhibits a different conformation compared to a

primary CaP phenotype [44]. Prostate cancer (CaP) is

graded according to severity, i.e. by Gleason grade. For

80% of formalin-fixed, paraffin-embedded prostate

tissues interrogated using FT-IR microspectroscopy, an

IR spectral classification of CaP that correlated with the

Gleason grading system was found [23].

IR microspectroscopy may possess the potential to

identify whether CIN is likely to progress. CIN1 may

regress to normal so a spectral biomarker(s) associated

with progression could reduce unnecessary treatment

[45]. The use of different fixative techniques may also

lead to IR spectral changes. Formalin or ethanol

fixation appears to result in the least severe spectral

alterations [29,35]. It is likely that future FT-IR

microspectroscopy will involve sampling live tissue

or cells in situ. Discrimination of FT-IR spectra of

single, living cervical cancer HeLa cells in solution has

been achieved [46] despite spectral interference by

water, which also absorbs in the mid-IR range.

Development of these methodologies is important to

investigate the applicability of using FT-IR microspec-

troscopy in diagnosis of living tissue without biopsy,

i.e. skin cancer [47].

FT-IR microspectroscopy allows microspectro-

scopic tissue imaging with minimum sample prep-

aration and without the use of dyes [35]. A tissue image

or map is produced according to spatial fluctuations in

the intensity of a particular absorbance band, i.e.

phosphate levels at z1080 cmK1. Referenced to a

database, image analysis allows comparative diag-

noses, i.e. with prostate tissue arrays; based on

differences in the phosphate band, good discrimination

between disease-free and CaP can be obtained [17].

Clustering algorithms applied to spatially resolved

microspectroscopic data may increase the information

content of such IR images [48]. A phosphate-based

image derived using FT-IR microspectroscopy corre-

lated well with the histopathology of tumour cell

invasion in lung cancer; in invading cells, shifts in the

amide I and II peaks were also noted [49]. Coupled to a

M.J. Walsh et al. / Cancer Letters 246 (2007) 1–116

synchrotron radiation (SRS) source, FT-IR imaging

may be exploited to obtain sub-cellular resolution [50].

5.2. Synchrotron-radiation (SRS)-FT-IR

microspectroscopy

A synchrotron consists of a large storage ring

through which electrons pass in a magnetic field.

Synchrotron IR sources generate a highly collimated

beam of photons and may deliver a high brilliance light

source giving better SNR in FT-IR microspectroscopy.

With a higher brilliance than used in conventional FT-

IR microspectroscopy, it may be delivered through a

very small sampling aperture (e.g. 10 mm!10 mm) in

order to improve the spatial resolution up to the point at

which it becomes diffraction-limited [14,51]. Through

this smaller aperture, SRS potentially delivers a

stronger signal and a higher spatial resolution that

may be focused at the single-cell level. SRS-FT-IR

microspectroscopy was used to identify abnormal

characteristics in oral tumours [14] suggesting the

applicability of this method for post-operative screen-

ing for residual invasive cells. It may also be applied to

acquire spectra of single, living cells in media [51,52].

However, this valuable tool in spectroscopic analysis is

limited due to the costs and space requirements of

running a synchrotron facility.

5.3. Attenuated total reflection-FT-IR (ATR)

microspectroscopy

In ATR microspectroscopy, a sample is placed in

intimate contact with an IR-transparent element with a

high refractive index [33], generally either ZnSe, type

II diamond, or Ge. When the beam of radiation is

passed through this denser element onto the less dense

medium (i.e. the sample), it is partially reflected off

the sample surface. The fraction of the incident beam

that is reflected increases as the angle of incident

radiation becomes larger. Beyond a certain critical

angle that is a function of the refractive indices of

crystal and sample, total internal reflection occurs. In

this case, the beam acts as if it penetrates a small

distance beyond the reflecting surface and into the less

dense medium before reflection occurs. The penetrat-

ing radiation, whose intensity decays with distance, is

thus an ‘evanescent wave’ [53]. The depth of

penetration, which varies from a fraction of a

wavelength up to several, depends on the index of

refraction of the element and the angle of the incident

radiation with respect to the interface between sample

and element. It is also wavelength-dependent,

increasing with increasing wavelength. This has the

consequence that if the less dense material selectively

absorbs certain wavelength components of the

evanescent radiation, then attenuation of the reflected

beam occurs preferentially at the wavelength of

absorbance bands. This phenomenon is known as

attenuated total reflection; via an FT-IR spectrometer,

the spectral absorption characteristics of the sample

are derived. As a function of shifts in the amide I

band, ATR microspectroscopy has been used to track

changes in the secondary structure of proteins [54].

By employing fibre optic cables to act as the element,

ATR microspectroscopy may be potentially applied as

a non-invasive in vivo technique and has been used to

generate tissue maps differentiating between normal

and malignant tissue [55].

We conducted a pilot study using ATR microspec-

troscopy to determine whether it is possible to

successfully distinguish between normal, pre-malig-

nant and invasive cervical smears in LBC (Fig. 2(A)–

(D)). Of the exfoliative cervical cytology samples

examined, two were characterised as histologically

normal, two as low-grade (i.e. CIN1), two as high-grade

(i.e. CIN2/3) and two exhibited severe dyskaryosis (i.e.

probably invasive carcinoma) (Table 1; Fig. 3).

ThinPrep solution (Preserv Cyte Solution, Cytyc

Corp., Boxborough, MA, USA) containing exfoliative

cytology was centrifuged, the supernatant removed and

the resultant cell pellet washed in autoclaved water

prior to application of re-suspended cellular material, at

room temperature, to 1 cm!1 cm Low-E reflective

glass slides (Kevley Technologies, Chesterland, OH,

USA). These were allowed to air dry prior to being

placed in a dessicator until analysis. Spectra were

acquired using a Bruker Vector 22 FT-IR spectrometer

with Helios ATR attachment that contained a diamond

crystal (Bruker Optics Ltd, Coventry, UK). Data was

collected in ATR mode and spectra (8 cmK1 spectral

resolution, co-added for 32 scans) were converted into

absorbance using Bruker OPUS software. Sodium

dodecyl sulphate (SDS) was used to clean the ATR

crystal after every five spectral acquisitions, or prior to

the first spectral analysis of a sample. Each time the

crystal was cleaned a new background reading was also

taken prior to recommencing spectral analysis. Spectra

were baseline corrected using OPUS software and

normalised to amide I (z1650 cmK1) absorbance

band. A minimum of 10 spectra were acquired from

each individual sample (Fig. 2(A)–(C)).

IR microspectroscopy may give rise to a large

amount of data, which cannot be sufficiently analysed

using univariate analysis. This has led to the use of

9001100130015001700

0.0

0.5

1.0

1.5

2.0

2.5Normal vs. CIN1

Wavenumber /cm-1

Wavenumber /cm-1

Wavenumber /cm-1

Abs

orba

nce

/au

9001100130015001700

0.0

0.5

1.0

1.5

2.0

2.5Normal vs. CIN2/3

Abs

orba

nce

/au

9001100130015001700

0.0

0.5

1.0

1.5

2.0

2.5Normal vs. Severe dyskaryosis/

? invasive

Abs

orba

nce

/au

C

D

A B

PC1

PC 2

PC 3

Fig. 2. IR spectra (nZ10 from each individual donor) acquired from exfoliative cytology (LBC specimens). Those with a histological

characterisation as normal (nZ2 individuals) were compared to (A) mild dyskaryosis (CIN1; nZ2), (B) severe atypia (CIN2/3; nZ2) or (C) severe

dyskaryosis (? invasive carcinoma; nZ2). In PCA, each spectrum became a single point, or score, in n-dimensional space and the data was analysed

for clustering: (D) three-dimensional scores plots on PCs 1, 2 and 3 demonstrated segregation of spectra for different categories of exfoliative

cytology. Each symbol represents a single IR spectrum as a point in ‘hyperspace’. The & symbol represents participant 1 and the % symbol

represents participant 2 for each category of exfoliative cytology listed in Table 1.

Table 1

LBC specimens: background details of donors (nZ8)

LBC spe

cimen

Age (y) Smoking

status

No. of

children

HPV status

Normal-1 54 No 3 Negative

Normal-2 47 Yes 2 Negative

CIN1-1 28 No 1 High-risk

CIN1-2 28 Yes 2 High-risk

CIN2/3-1 31 No 2 High-risk

CIN2/3-2 41 No 2 High-risk

IC-1 65 No 3 Negative

IC-2 67 No 4 High-risk

CIN, cervical intraepithelial neoplasia; IC, invasive carcinoma

(severe dyskaryosis); HPV status (presence (high-risk) or absence

(negative) of oncogenic HPV genotypes) was ascertained using the

Hybrid Capture II assay.

M.J. Walsh et al. / Cancer Letters 246 (2007) 1–11 7

other analytical techniques such as PCA, linear

discriminant analysis [23], advanced neural networks

[20], probalistic neural networks [31] and chemometric

analysis [19]. Using Pirouette software (Infometrix,

Woodinville, WA, USA), PCA was performed on the

exfoliative cervical cytology spectra obtained

following ATR microspectroscopy (Fig. 2(D)). PCA

is a multivariate data analysis technique that allows

cluster analysis of spectroscopic data by plotting each

spectrum as a point in ‘hyperspace’, and using selected

principal components (PCs) as coordinates when the

data is viewed in a particular direction. Fig. 2(D) shows

a clear separation along PCs 1, 2 and 3 between spectra

derived from normal exfoliative cytology (nZ2)

compared to those derived from CIN1 (nZ2), CIN2/3

M.J. Walsh et al. / Cancer Letters 246 (2007) 1–118

(nZ2) or severe dyskaryosis (nZ2). Segregation of

spectra from cytology exhibiting severe dyskaryosis did

not appear to be influenced by HPV genotype

(Fig. 2(D), Table 1). Fig. 3(A) and (B) shows

representative examples of ThinPrep cytology samples

exhibiting normal histology or severe dyskaryosis,

respectively; IR microspectroscopy appears to possess

the potential to objectively discriminate between such

samples.

5.4. Photothermal microspectroscopy (PTMS)

A new spectroscopic technique, PTMS, exploits

near-field scanning probe microscopy so that the spatial

resolution is no longer subject to the diffraction limit.

PTMS has recently been applied to the microspectro-

scopic analysis of cells [29,34]. In PTMS, a narrow IR

beam is focussed on a received sample. This IR

radiation is absorbed by the sample and results in an

increase in temperature. Resultant temperature

increases are detected using a miniaturised scanning

Fig. 3. Representative histological ThinPrep LBC specimens from (A) a no

(probably invasive carcinoma). Scale bar, z50 mm.

thermal probe that acts as a thermometer. Thus,

characteristic vibrational spectra are obtained. The

advantage of PTMS is that it is non-destructive and

requires even less sample preparation than FT-IR

microspectroscopy [34]. Other microspectroscopic

approaches may be limited by the fact that some

intracellular material, i.e. M-phase chromatin, is too

dense for IR absorption [36,37]; PTMS overcomes this

as the resultant temperature increase is measured not

the IR passing through the sample [29]. PTMS also

allows for a higher-than-diffraction limit resolution

[34]. Using ultra-miniaturized probes, PTMS is capable

of mesospatial resolution unachievable using conven-

tional techniques.

5.5. Raman microspectroscopy

Whereas IR microspectroscopy measures the absor-

bance of light by a sample, Raman microspectroscopy

is a vibrational spectroscopic technique that measures

an inelastic light scattering process in which photons

rmal preparation, and (B) a preparation exhibiting severe dyskaryosis

M.J. Walsh et al. / Cancer Letters 246 (2007) 1–11 9

incident on a sample transfer energy to or from

molecular vibrational modes [56]. Because such

frequency shifts (i.e. energy transfers) are unique for

each molecule, resultant Raman spectra provide

detailed information that is inaccessible by absorption

measurements; these may concern the structure and

dynamics of materials and can be obtained by Raman

microspectroscopy [26,27]. The selection rules for

Raman and IR activity of vibrational modes are

different, and therefore the two techniques can be

considered complementary to a large extent [57]. One

advantage of the Raman scattering technique is that

operation is possible in aqueous solutions and as such,

this means that this method has potentially for in vivo

applications such as distinguishing between cancerous

and non-cancerous cells, or the interrogation of viable

cells [28]. For instance, this has facilitated investi-

gations into molecular interactions between potential

therapeutic regimes and intracellular targets [58].

Recent technical developments in scanning near-field

optical microscopy (SNOM) have allowed sub-wave-

length Raman mapping using near-field probes to be

achieved, although the very low Raman signal is

compounded by the low intensity of the sub-

wavelength excitation source (nW-mW) or the low

collection efficiency [59]. The efficiency of the near-

field probe to deliver excitation light to, or collect

Raman scattered light from the sample is probably the

key factor in determining the success of Raman SNOM

mapping. The method has been shown to differentiate

between normal and cancerous tissue [60–64]; how-

ever, the signal upon which it relies is up to six times

weaker than that using IR microspectroscopy [39].

6. Conclusion

IR microspectroscopy possesses the potential to be a

valuable tool in cancer diagnosis in the post-genomic

era where a more comprehensive understanding of

disease progression is being sought [65]. Of the

technologies described, sufficient data does not yet

exist to indicate which is the most applicable in a

screening or diagnostic setting. There is currently an

urgent need for automated, sensitive and objective

approaches applicable to well-population screening

programmes that would allow the identification of

small numbers of pre-malignant cells. Spectral bio-

markers that might be identified could also be

correlated with other molecular markers of suscepti-

bility [66]. To standardise this approach for cervical

cancer screening, validation studies examining

the spectral characteristics of disease progression

(normal through to CIN1, CIN2/3 and, finally, invasive

carcinoma) in parallel with conventional histological

approaches are required. It is our contention that were

IR microspectroscopic methods applied for screening

and/or diagnostic purposes, logistical and safety

considerations would suggest that tissue samples were

fixed as is conventionally done. Although fixation itself

would undoubtedly alter some of the spectral charac-

teristics of a living tissue, it would also probably lend

itself to a standardisation of this approach and allow for

the re-evaluation of archived samples. This would

facilitate the implementation of retrospective studies on

archived material where findings could then be

correlated with case notes. Also, IR microspectroscopy

has a wide range of other applications such as in post-

operative screening, tracking of cancer and predicting

the response of cancer to drugs. There are also other

disease states where spectral maps of chemical

functional groups may be correlated with pathological

alterations [67] while the characterization of cell

populations within complex tissue architecture is also

a possibility [68]. Future work must elucidate common

biomarkers for abnormalities and continuing advances

in computation analysis will increase the useful

extraction of information from spectral data. An

important aim is to standardise spectroscopic

approaches and to undertake larger studies to establish

a database of normal vs. abnormal cells.

Acknowledgements

This work is funded by Rosemere Cancer Foun-

dation (M.J.W., M.S. and P.L.M-H.) and EPSRC grant

GR/S75918/01 (M.J.G., A.H., H.M.P. and F.L.M.).

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