Table Of ContentBIOMEDICAL SPECTROSCOPY: INTRODUCTION 1
Medicine, known as ‘‘the art of science of healing
Biomedical Spectroscopy:
disease’’, is more art than science to some, where the
Introduction patient is treated as a whole and not as the site in
which a particular disease has chosen to manifest itself.
However, to many others medicine is a life science that
Henry H. Mantsch
rests firmly upon a foundation of biological sciences,
National Research Council, Winnipeg, Canada which in turn, trace their origin to the basic physical
sciences of chemistry, physics and mathematics. Sadly, in
spite of our success to combat disease and illness, there
are still health care problems, for which there is little
1 Introduction 1
we can do. Even if the best of contemporary medicine
2 Overview of Contributions 1 were universally available, regardless of financial barriers,
3 Apologia and Outlook 3 cancer would continue to kill, rheumatoid arthritis would
Abbreviations and Acronyms 3 still cripple, and Alzheimer’s disease would continue to
render many elderly people helpless. Although modern
medicine appears powerless to reduce the impact of
these diseases, largely due to our inability to treat them
successfully, the paucity of effective diagnostic techniques
1 INTRODUCTION plays a significant role in the continued occurrence of
these diseases. Early diagnosis is an important component
Biomedical Spectroscopy, fortuitously the first section in of preventive medicine, and spectroscopic methods
this comprehensive encyclopedia, is one of the youngest play an increasingly important role in biodiagnostics.
branches of analytical chemistry. Throughout much of Medical biospectroscopy uses the entire arsenal of
their history, medicine and spectroscopy have evolved electromagnetic radiation, from the high-energy end
quite separately, yet in the new millennium the two (gamma rays) down to the low-energy end (radio waves),
solitudes are closing the gap that has separated them to probe individual biomolecules and indeed the whole
for so long as they cross-pollinate each other increasingly. human body.
Although spectroscopy, by its very nature, has always had
an interdisciplinary focus, the recent marriage between
spectroscopy and medicine is only now beginning to bear 2 OVERVIEW OF CONTRIBUTIONS
fruit.
It is unfortunate that the word ‘‘chemical’’ has acquired The Biomedical Spectroscopy section consists of 15 arti-
such a bad reputation in the lay press and yet the same cles, among which are five dedicated to optical, infrared
public is increasingly captivated by ‘‘natural biochemi- (IR), and magnetic resonance (MR) spectroscopy. From
cals’’. So what are natural biochemicals, and what is a the most ancient times, medical practitioners performed
natural substance? To answer this question we have to go physical examinations of their patients using their eyes as
back almost 20 billion years to when the universe arose optical spectroscopes. Indeed, analytical methods based
with a cataclysmic explosion that hurled hot, energy-rich on optical spectroscopy were used early in medical diag-
subatomic particles throughout space. Gradually, as the nosis, and even today many tests performed in the clinical
universe evolved, the various chemical elements were chemistry laboratory still rely on visible spectroscopy to
created, including those in all living organisms on earth. monitor chemical or enzymatic color reactions. Bioan-
Hence, we humans are literally made of stardust. The alytical methods based on IR spectroscopy or on MR
unique molecules from which living organisms are con- spectroscopy are of more recent vintage, as are several
structed are called biomolecules and were selected during optical approaches such as optical coherence tomography
the course of evolution for their fitness to perform spe- or photodynamic therapy.
cific functions. It is therefore quite legitimate to ask what Biodiagnostic methods fall into two large categories:
the purpose or the specific biological function of a given (a) biomedical spectroscopy and (b) biomedical imag-
biomolecule in a living organism may be. When examined ing. The former is based on the interaction of selected
separately these biomolecules conform to all the physical electromagnetic waves with individual or collections of
and chemical laws that describe the behavior of inanimate biomolecules. The resulting ‘‘molecular spectra’’, repre-
matter, and yet living things possess unique properties not sented as plots of intensity versus electromagnetic energy,
displayed by collections of inanimate molecules, thus pre- provide answers to the questions ‘‘what?’’ (qualitative
senting a distinctive challenge to the analytical chemist bioanalytical chemistry) and ‘‘how much?’’ (quanti-
and to the biospectroscopist alike. tative bioanalytical chemistry). The second category,
Encyclopedia of Analytical Chemistry
R.A. Meyers (Ed.) Copyright John Wiley & Sons Ltd
2 BIOMEDICAL SPECTROSCOPY
biomedical imaging, goes on to answer the question photodynamic therapy, is reviewed by Ro¨ der (Photody-
‘‘where is what?’’, by localizing and mapping the spec- namic Therapy). Photodynamic therapy, known as PDT
troscopic information. Both biomedical spectroscopy and to its practitioners, uses photosensitizers that are non-
biomolecular imaging can be performed either ex vivo, on toxic in the dark but become toxic after photoactivation
extracted biofluids or excised tissue, when the biomaterial by light. The evolution of photodynamic treatment over
is brought to the spectrometer, or in vivo, in which case three generations of photosensitizers is illustrated by a
the electromagnetic radiation is delivered to the target number of clinical applications with particular emphasis
area of the patient via optical fibers, endoscopes, catheters on skin diseases and cancer.
or even through space (e.g. radio waves in MR imaging). A second group of contributions deals with IR spec-
In an ideal world, all diagnostic procedures would be troscopy, a more recent tool in medical research and
noninvasive, but we live within the constraints of the real practice. Both IR spectroscopy and the complementary
world. technique of Raman spectroscopy, derive information
The individual articles in the Biomedical Spectroscopy from the vibrations of chemical bonds in the biomolecules
section are largely self-contained, each covering a particu- of interest and are therefore referred to as vibrational
lar area of expertise of the contributing author(s). A first spectroscopies. Jackson and Mantsch (Infrared Spec-
group of contributions deals with optical spectroscopy. troscopy, Ex Vivo Tissue Analysis by) introduce the
The term ‘‘optical spectroscopy’’ is not synonymous with reader to the basics of ex vivo tissue analysis by mid-IR
visible spectroscopy, which at times can be confusing. spectroscopy, focusing on two facets of such an analysis.
Optical spectroscopy involves transitions between elec- First, there is an experimental aspect related to potential
tronic energy levels, and thus extends beyond the violet pitfalls with spectroscopic measurements on samples as
into the ultraviolet and beyond the red into the near-IR. complex as human tissue. The second aspect concerns the
Ramanujam (Fluorescence Spectroscopy In Vivo) pro- interpretation of spectra, meaning the proper translation
vides a comprehensive account of optical fluorescence of spectroscopic information into diagnostic, medically
and absorption spectroscopy, introducing the technique, relevant information using such tools as chemometrics
the types of chromophores and fluorophores, and the and nonsubjective multivariate statistical classification
spectrometers and fluorimeters used in clinical settings. methods. Every type of human tissue, although struc-
Both absorption and fluorescence spectroscopies have turally highly complex, has a unique vibrational pattern
been explored extensively as diagnostic tools, in particu- (fingerprint) in the mid-IR region, which is different in
lar for cancer (precancer) screening in epithelial surface healthy and in diseased tissue. This has led to the cre-
layers of various organ sites (cervix, bladder, gastro- ation of a new field, IR histopathology. In vivo tissue
intestinal tract, trachelial tube, and oral cavity). The analysis by IR spectroscopy is addressed by Sowa et al.
contribution by Ramjiawan et al. (Fluorescence Imaging) (Near-infrared Spectroscopy, In Vivo Tissue Analysis
is dedicated to optical fluorescence imaging. Fluores- by). Compared to both visible light and mid-IR radiation,
cence, a zero-background technique, has a much higher near-IR light can traverse a greater distance into tissue,
sensitivity and specificity than absorption spectroscopy therefore the spectral range of near-IR is ideally suited
or imaging. In particular immunofluorescence imaging, a for in vivo tissue spectroscopy and imaging. The pene-
technique based on the interaction of labeled antibodies tration depth of near-IR light is of the order of several
with specific antigens, shows great diagnostic potential, centimeters, whereas only the top 10–20 µm of tissue can
even if it is not yet a common sight in hospitals. Heise be explored by mid-IR light. The clinically relevant tis-
(Glucose, In Vivo Assay of) tackles glucose, the Holy sue chromophores oxy- and deoxyhemoglobin, oxy- and
Grail of all analytes assessed in biological fluids. It is the deoxymyoglobin, oxidized and reduced cytochrome c,
dream of many diabetics, and of their attending physi- as well as water, provide vital information related to
cians, to do away with the daily finger pricking and to oxygen delivery, storage, utilization and tissue hydra-
depend on a simple, noninvasive optical wand for deter- tion/dehydration. The article by Shaw and Mantsch
mining their blood glucose levels. As the reader will find (Infrared Spectroscopy in Clinical and Diagnostic Anal-
out, great progress has been made towards this goal, ysis) offers an overview of the clinical analyses that have
but we are not quite there yet. Fercher (Optical Coher- been carried out by IR spectroscopic methods on such
ence Tomography) provides an up-to-date synopsis of the common biological fluids as serum, whole blood and urine,
novel field of optical coherence spectroscopy and tomog- as well as on less-common body fluids such as amniotic
raphy, addressing a number of applications in medicine fluid, synovial fluid, cerebrospinal fluid, and saliva. The
that range from ex vivo biopsy studies in dermatology, term ‘‘IR clinical chemistry’’ was coined as an analytical
urology, and gynecology to in vivo imaging in ophthal- technique that does not require chemical or biochemical
mology, dentistry, and gastroenterology. Another type reagents for the quantitative determination of analytes;
of optical spectroscopy suitable for medical applications, instead, the analysis relies on chemometric algorithms. A
BIOMEDICAL SPECTROSCOPY: INTRODUCTION 3
digression into the realm of microbiology, utilizing mid- stellar performance of fMRI in the field of neuroscience
IR spectroscopy as the investigative tool, is recounted has filled in many blanks on the human brain map, and
by Naumann (Infrared Spectroscopy in Microbiology). fMRI is now a premier method for the study of brain
This methodology offers an alternative analytical tool for function. Finally, Yuan (Magnetic Resonance Angiogra-
the detection, enumeration, classification and identifica- phy) exploits the emerging use of MR in angiography, an
tion of pathogenic bacteria in a clinical setting. Finally, area until recently reserved for X-ray radiologists. The
Ozaki and Noda (Two-dimensional Vibrational Corre- name ‘‘angiography’’ applies to any imaging modality
lation Spectroscopy in Biomedical Sciences) introduce that can visualize blood vessels and blood flow. Poten-
the reader to a new modality for extracting addi- tial clinical applications are discussed, the challenge being
tional information from IR spectra. Two-dimensional coronary MR angiography because the three-dimensional
correlation spectroscopy already has revolutionized MR structure of the coronary tree changes shape during each
spectroscopy and is expected to benefit IR biomedical heart contraction. A concern sometimes voiced with in
spectroscopy as well. vivo imaging is the potentially harmful electromagnetic
A third group of contributions explores nuclear mag- radiation. Although this may apply to X-ray imaging,
netic resonance (NMR) in medicine. There is now a molecular spectroscopic imaging uses low-energy elec-
tendency to drop the ‘‘N’’ in NMR, because the word tromagnetic fields and very few adverse effects have been
‘‘nuclear’’ is unpopular. Even though optical and IR recorded.
spectroscopy have been around much longer, MR spec-
troscopy enjoys remarkable success today. Winter and
Bansal (Magnetic Resonance, General Medical) pro-
3 APOLOGIA AND OUTLOOK
vide an authoritative overview of multinuclear NMR in
medicine. As MR spectroscopy involves the absorption
While I want to thank all the contributing authors for
of certain radiofrequencies by individual nuclei in a mag-
their effort and dedication, as editor of the Biomedical
netic field, it must be performed in a magnet. The higher
Spectroscopy section I also apologize to my colleagues in
the magnetic field, the greater the detection sensitivity,
the biospectroscopic community whose work could not be
a fact which has fuelled the race for ever higher-field
included. I had to be selective and no doubt this selectivity
magnets. Smith and Blandford (Magnetic Resonance
reflects my own bias and preoccupations with certain
in Medicine, High Resolution Ex Vivo) introduce the
aspects of the exploding field of biomedical spectroscopy.
reader to the intricacies of ex vivo high-resolution MR
As to the future of biomedical spectroscopy, I am
spectroscopy in medicine. The applications they highlight
optimistic that in the process of growing and maturing
range from the study of inborn errors of metabolism to
it will embrace other traditional areas of analytical
the diagnosis of neurological disorders and many types
chemistry. When pressed to be more specific, I would
of cancer. The last three contributions describe various
like to refer the reader to a statement by Alan Kay, one
MR imaging modalities. Richards (Multinuclear Mag-
of the founders of Silicon Valley in California, ‘‘the best
netic Resonance Spectroscopic Imaging) discusses MR
way to predict the future is to invent it’’. So, good luck
spectroscopic imaging, a procedure for generating spa-
and ‘‘happy hunting grounds’’ to future generations of
tially resolved maps and images of selected biomolecules
bioanalytical chemists.
in tissue (ex vivo), or in the body (in vivo). This molecular-
level imaging, also known as chemical shift imaging, is
similar to chemical group imaging by IR spectroscopy.
ABBREVIATIONS AND ACRONYMS
Both imaging modalities allow the noninvasive mapping
of chemicals in the body, although IR imaging is limited
to external body parts such as skin, whereas the whole fMRI Functional Magnetic Resonance Imaging
body is accessible to MR imaging. Richter (Magnetic Res- IR Infrared
onance Imaging, Functional) familiarizes the reader with MR Magnetic Resonance
functional magnetic resonance imaging (fMRI), perhaps NMR Nuclear Magnetic Resonance
the most powerful imaging technique available today. The
FLUORESCENCE IMAGING 1
of burns and transplant tissue. In addition, monitoring
Fluorescence Imaging
of vascular parameters during cardiac surgery presents
exciting opportunities. For example, low oxygen levels (e.g.
Bram Ramjiawan, Michael Jackson, and Henry during bypass surgery) in the heart can result in alterations
Mantsch in microvascular permeability. As ICG is largely bound
Institute for Biodiagnostics, Winnipeg, Canada to serum albumin, it should not be seen in extravascular
spaces in normal hearts. However, increased permeability
will allow albumin to diffuse into the extravascular spaces,
and diffuse fluorescence across the surface of the heart
1 Introduction 1 will be seen. In principle, the infusion of polymers
2 Chromophores 2 (e.g. dextrans) of various molecular weights labeled with
2.1 Endogenous Chromophores 2 dyes that fluoresce at various wavelengths will allow
2.2 Exogenous Chromophores 3 the assessment of the porosity of capillary beds in such
systems.
3 Immunofluorescence Imaging 5
Immunofluorescence techniques have the potential to
3.1 Choice of Antibody 5
provide unmatched sensitivity and specificity. The unique
3.2 Choice of Fluorophore 6
nature of antibody–antigen interactions ensures specific
4 Choice of Detection Systems 7 delivery of the fluorophore to the site of interest. The
4.1 Charge-coupled Device Cameras 7 specific interaction of labeled antibodies with antigens
4.2 Filter Selection 7
means that the fluorophore persists in the body for
4.3 Typical Experimental Set-up 7
a prolonged period of time (days). Following a single
5 Immunofluorescence Imaging of Tumors 8 injection of labeled antibody, repeated measurements on
5.1 Qualitative Analysis of Images 8 the same site over the course of hours or days allow
5.2 Quantitative Analysis of Images 9 kinetic information to be readily obtained. In principle,
5.3 Statistical Analysis of Images 11 this means that the effect of therapeutic intervention, i.e.
6 Summary and Future Prospects 11 radiation therapy, chemotherapy, etc., can be monitored.
Abbreviations and Acronyms 13
Related Articles 13
1 INTRODUCTION
References 14
Owing to their high sensitivity, fluorescence techniques
have found a valuable place in biology. The sensitivity
A number of fluorescence imaging techniques show diag- of fluorescence methods is due in large part to the fact
nostic promise. Imaging endogenous fluorescence has that they are ‘‘zero background’’ techniques. In other
been proposed as a method for cancer diagnosis. Unfor- words, in the absence of the chromophore of interest,
tunately, tissue autofluorescence is relatively weak and no signal (other than random noise) can be detected.
poor contrast between malignant and normal tissue is Any signal that is detected must therefore arise from the
seen. Contrast may be enhanced with the addition of chromophore of interest.
fluorescent materials that are selectively accumulated by Fluorescence methods with potential for use in
malignant cells, such as fluoroscein or porphyrin deriva- medicine utilize both intrinsic and extrinsic chro-
tives. The limited penetration of light at the emission mophores. The number of intrinsic chromophores (i.e.
maxima of these materials restricts the use of fluores- chromophores that occur naturally in tissues) is limited;
cence techniques utilizing these chromophores to superfi- the most important are listed in Table 1. A number
cial phenomena. However, many potential applications of literature reports suggest that tissue nicotinamide
still exist. For example, monitoring fluorescence dur- adenine dinucleotide (reduced form) (NADH) fluores-
ing surgery may allow resection margins to be clearly cence may prove useful in the detection of malignancies,
delineated. the rationale being that changes in the metabolic sta-
Other exogenous chromophores that may have may tus of malignantly transformed cells are reflected in
have diagnostic utility include indocyanine green (ICG). changes in fluorescence signals arising from NADH..1;2/
Techniques based upon visualization of the distribution Unfortunately, intrinsic fluorescence is typically low in
.3/
of ICG fluorescence (i.e. choroidal angiography) are most tissues and applications to date are limited.
already prominent in ophthalmology. ICG fluorescence The range of extrinsic chromophores of use (i.e.
imaging may also find a useful niche in monitoring chromophores which are added to samples in some
Encyclopedia of Analytical Chemistry
R.A. Meyers (Ed.) Copyright John Wiley & Sons Ltd
2 BIOMEDICAL SPECTROSCOPY
Table 1 Selected chromophores and their excitation and 2 CHROMOPHORES
emission properties
2.1 Endogenous Chromophores
Chromophore Excitation Emission
maximum (nm) maximum (nm)
Although a number of endogenous fluorescent chro-
Tryptophan 275 350 mophores exist in the body (see Table 1), their use in
Collagen 340 395 in vivo fluorescence studies can be problematic. The most
Elastin 460 520
common problems are as follows:
NADH 350 460
Porphyrins 400 610, 675
1. The low level of fluorescence typically seen in tissues.
ICG 790 815
Cy3 550 565 2. Overlap of excitation and emission wavelengths for
Cy5 690 710
absorption. Many endogenous chromophores may be
Cy7 750 777
excited at similar wavelengths. For example, collagen
fluorescence may be stimulated by illumination at
340 nm, very close to the excitation wavelength of
manner) is obviously much wider, and fluorescent
NADH (350 nm). Overlap of excitation and emis-
chromophores are available covering a wide spectral
.4;5/ sion wavelengths can be even more problematic. For
range [from the visible to the near-infrared (NIR)].
instance, whereas tryptophan and NADH fluores-
Typically, extrinsic chromophores are introduced into
cence is characterized by well separated excitation
samples either as free agents or attached to carrier
wavelengths (275 and 350 nm, respectively), the emis-
materials such as antibodies to specific materials present
.6– 8/ sion maximum for tryptophan fluorescence is seen at
in the sample.
350 nm. Hence, tryptophan fluorescence at 350 nm
Detection of fluorescent materials in samples may be
may be masked owing to absorption of the emitted
achieved using either spectroscopic or imaging technolo-
radiation by NADH.
gies. Spectroscopic techniques may be used to analyze
small regions of tissue and provide information relat- 3. Ultraviolet (UV) or visible excitation and emission
ing to the average concentration of fluorescent materials maxima. As discussed above, chromophores that
within the sample. Fluorescence spectroscopy may be per- exhibit excitation and emission maxima in the
formed in vitro, e.g. using homogenized tissues, or in vivo, UV or visible spectral regions limit studies to the
.1;9/
e.g. with the use of fiber-optic bundles. The advantage characterization of superficial structures, owing to
to the spectroscopic approach is that fluorescence across limited penetration of light through tissues at these
the entire spectral range of interest is acquired, allowing wavelengths. In addition, illumination of tissues with
information on multiple chromophores to be obtained UV light is obviously undesirable.
with one measurement. However, the obvious disadvan-
tage to this approach is that only a small volume of tissue Despite these limitations, imaging fluorescence arising
can be analyzed, and the resulting signal is an average from endogenous chromophores has been demonstrated
signal from that tissue volume. Spatial information is not to have clinical potential, particularly in the diagnosis
obtained. of malignancies. For example, it has been demonstrated
Spatial information may be obtained by fluorescence that malignant tissue shows enhanced accumulation of
imaging, in which the distribution of fluorescence intensity endogenous porphyrins, which exhibit fluorescence in the
.7;10/
is measured as a function of position within the sample. red part of the visible spectrum. It has therefore
Fluorescence imaging is typically achieved using imag- been suggested that changes in fluorescence arising from
ing arrays such as charge-coupled device (CCD) cameras endogenous porphyrins may be useful in tumor detection.
equipped with appropriate filters. While spatial informa- Differences in NADH fluorescence are also apparent
tion may be obtained in this manner, fluorescence imaging between normal and malignant cells and tissues. NADH
has typically been performed at a single frequency. Thus, exhibits relatively strong fluorescence at 350 nm, whereas
C
spectral information is lost. Multiple imaging frequen- its oxidized counterpart, NAD , exhibits only weak flu-
C
cies (to detect multiple chromophores) require changing orescence. The ratio of NADH to NAD is decreased
between multiple filters, a tedious procedure. The devel- in malignant tissues, thus leading to a reduced fluores-
opment of the liquid-crystal tunable filter (LCTF) has cence signal in malignant tissues. It should be stressed
removed this limitation. The transmission characteristics that factors that result in metabolic disturbances may
C
of LCTFs may be varied under computer control, allowing also potentially affect NADH/NAD ratios and pro-
the user to vary the imaging wavelength without chang- duce altered tissue fluorescence signatures. The specificity
ing filters. With such filters, fluorescence spectroscopic of NADH fluorescence methods for malignancies is at
imaging becomes feasible. present unclear.
FLUORESCENCE IMAGING 3
2.2 Exogenous Chromophores Accumulation of porphyrins, including hematopor-
phyrin derivative and protoporphyrin IX, have been used
Exogenous chromophores are those introduced into
to diagnose a number of forms of cancer in humans.
samples by some mechanism either to allow the detection
In vivo fluorescence imaging of skin showed higher
of specific materials (e.g. when attached to antibodies), to
fluorescence in basal cell carcinomas lesions than the
probe environmental parameter (e.g. pH) or to provide
surrounding normal tissue. Clear demarcation of lesion
contrast (e.g. angiography). As discussed above, the
borders was seen in images. Malignant tumors of the
number of potential exogenous fluorescent chromophores
breast, head and neck region and urinary bladder have
is large, allowing greater flexibility in the choice of the
been visualized using a fluorescence imaging system using
spectral range to be employed for excitation and emission
100-ns long optical pulses at 390 nm delivered via an
monitoring. In fact, in many instances chromophores can
endoscope system which was also coupled to the imaging
be chemically tailored to meet excitation and emission
system. Tumor detection was achieved based on differ-
criteria.
ential fluorescence between normal and malignant tissue,
The chromophore of choice depends upon a number
related to the selective uptake of tumor-marking agents
of factors, including the nature of the experiment, exci-
such as hematoporphyrin derivative and levulinic acid,
tation and emission wavelengths and chemical structure.
and natural chromophore differences between various
Chemical structure is important if the fluorophore is to
tissues. A clear demarcation from normal surrounding
be attached to a carrier material: the fluorophore must
tissue was found in measurements of superficial bladder
contain a reactive group or be capable of derivatization
carcinoma and in vitro investigations of resected breast
or produce an active group which will allow chemical
.16/
cancer.
linkage to the carrier material. In many respects, the lim-
While the use of porphyrins as chromophores in
itations in chromophore selection imposed by the nature
fluorescence imaging is appealing, it is not without
of the experiment and excitation and emission wave-
.6;7;10;14/
drawbacks. The most important drawbacks are
lengths are more severe. The applications of fluorescent
the time required to achieve significant differential
dyes that show excitation and emission wavelength max-
accumulation of the porphyrin in malignant tissue and
ima in the UV or visible regions of the spectrum are
potential side effects such as photosensitization.
restricted owing to the limited penetration of UV and
visible light into tissues. Thus only fluorescence signals
from superficial chromophores can be recorded in these 2.2.2 Visible Fluorophores: Fluorescein
spectral regions. Dyes which fluoresce in the far-red or
In contrast to porphyrin derivatives, toxic side effects
NIR regions have greater potential diagnostic use, owing
associated with fluorescein use are minimal. Fluorescein
to the enhanced penetration of light through tissues in
has been used for many years in studies of the vascular
these spectral regions.
beds within the eye. Illumination of the retina with blue
light produces green fluorescence in the vessels of the eye
2.2.1 Ultraviolet/Visible Fluorophores: Porphyrin
following injection of a bolus of fluorescein. Acquisition
Derivatives
of images (typically with the use of photographic film)
Any fluorescent material that is selectively accumulated of the distribution of fluorescence produces angiograms,
by tumor cells can in principle be used as a diagnostic images of the vessels within the eye. While fluorescein
marker. In this respect, many therapeutic agents have angiography is slowly being superseded by ICG angio-
potential as diagnostic agents. Porphyrin derivatives used graphy, other applications of fluorescein fluorescence are
in photodynamic therapy are particularly well suited in being pursued.
.6;10 – 15/
this respect. Already used as therapeutic agents Malignant tissues show an enhanced accumulation of
(with toxicity data, etc., available) these agents are fluorescein compared with normal tissues. At neutral pH
selectively accumulated by malignant cells and exhibit fluorescein exists as a mixture of charged and uncharged
fluorescence. Illumination with light at around 400 nm species. Accumulation of fluorescein by cells results from
allows a fluorescence signal to be imaged in tissues passive diffusion of the neutral form of the dye across the
and cells at 600–650 nm, depending upon the nature of cell membrane. In malignant tissues, the extracellular pH
the porphyrin derivative. The utility of this approach is often lower than that seen in normal tissue. This lower
has been demonstrated by imaging the fluorescence pH shifts the dissociation curve of fluorescein, resulting
arising from a hematoporphyrin derivative accumulated in a higher concentration of the neutral form of the dye in
in colon adenocarcinoma cells implanted in the leg of a the extracellular fluid. This in turn results in an increased
rat. Illumination at 337 nm 24 h following administration passive diffusion of the dye across the cell membrane.
of a low dose of the porphyrins resulted in detectable As tumor cells are generally able to maintain a neutral
fluorescence and visualization of the tumor. intracellular pH, the equilibrium within the cell is shifted
4 BIOMEDICAL SPECTROSCOPY
in favor of dissociation, reducing the concentration of the can be obtained. By studying the time course of fluores-
neutral form of the dye and reducing outward diffusion. cence changes in images, hemodynamic information can
.17/
The dye therefore accumulates within malignant cells. be obtained. Static images and hemodynamic information
Preferential uptake of fluorescein by malignant tissues can be used to evaluate vision-related problems such as
.18;23/
has been known for more than 50 years, However, age-related macular degeneration.
the dye exhibits green fluorescence when excited with Monitoring tissue vascularity is important in many
blue light. In other words, the excitation and emission other clinical settings, and ICG fluorescence imaging
maxima are both in the visible portion of the spectrum. may play an important role in these areas. One obvious
Penetration of visible light through tissues is poor application of ICG fluorescence imaging is monitoring
and fluorescein spectroscopy and imaging are limited vascularization of tumors. Typically tumors have high
to accessible surfaces. Fluorescein imaging is therefore metabolic rates, and so have requirements for high rates
most useful if endoscopic techniques are used to study of blood flow. As the tumor develops, this high blood
organs such as the lungs or the gastrointestinal tract.
flow requirement is met by neovascularization, that is,
Endoscopic detection of fluorescein fluorescence has been
the formation of new blood vessels. Thus many tumors
demonstrated for gastric cancer. Even more promising is
are extremely well vascularized. Imaging the distribution
the finding that dysplasia could be detected based upon
of blood vessels within tumors is therefore of potential
fluorescein fluorescence in 22 of 23 specimens of cheek
diagnostic utility. This technique has been demonstrated
.9/
pouch from carcinogen-treated Syrian hamsters.
to be effective for studying vascular bends in melanoma
Of course it should be noted that conditions other
of the retina. However, it should be stressed that only a
than malignancies might induce a fall in extracellular
limited subset of tumors will be amenable to study in this
pH, e.g. inflammatory conditions. If such conditions are
manner. An absolute requirement for such a technique is a
also associated with maintenance of intracellular pH, i.e.
clear differentiation between the vascular structures of the
the formation of a pH gradient across the cell, then
tumor and surrounding tissues. If this clear differentiation
they may show fluorescein accumulation and enhanced
is not present, then diffuse fluorescence from ICG in
fluorescence may result. The specificity for fluorescein for
the vasculature of the surrounding tissue will mask
malignant tissues has yet to be determined.
fluorescence of ICG in vessels within the tumor. In other
words, the zero background advantage of fluorescence
2.2.3 Near-infrared Fluorophores: Indocyanine Green techniques will be lost, reducing sensitivity and specificity.
In addition to providing contrast for studying vas-
If information from deeper, non-surface structures is
culature, ICG has also been used in the investigation
required, fluorophores that exhibit excitation and emis-
.25/
of burns. In contrast to the above studies, studies
sion maxima in the far-red or NIR spectral regions,
involving burns have focused upon the degree of ICG
in which tissues are relatively transparent to light, are
fluorescence recorded at various times after a burn was
required.
induced was correlated with clinical outcome of burns
The most widely used NIR fluorescent dye is ICG (also
of various depths. Burns were produced in anesthetized
known as Cardio-Green or Fox Green). ICG exhibits
pigs by placing a heated brass block on the skin. Burn
absorption and emission maxima at 780 and 810 nm,
depth was varied by varying the time of contact between
respectively, wavelengths at which light can readily
the block and the skin. Following creation of the burn,
traverse several millimeters of tissues. Originally used
a bolus of ICG was injected into the circulatory system
to monitor cardiac and hepatic function (based upon the
at various time points, the skin illuminated at 780 nm
transit time of a bolus of the fluorescent dye through
the circulatory system), ICG has recently become more and fluorescence images acquired using a CCD camera.
widely utilized as a contrast agent in studies of the Comparison of fluorescence in burns and adjacent nor-
vasculature..18 – 21/ mal tissue revealed interesting differences that could be
The most common application of ICG fluorescence in related not only to the stage of the burn (i.e. time after
vascular studies is without doubt in choroidal angiogra- the burn was created) but also to burn depth. Fluores-
phy, i.e. imaging the choroidal vessels of the eye, vessels cence images from deep burns (i.e. those produced by
that provide 70% of the nutrient flow to the retina. A prolonged contact between heated metal block and the
discussion of this active field of clinical use is beyond the skin) showed a decreased fluorescence intensity at the
scope of this article and the interested reader is referred burn site compared with superficial burns. This difference
.18;22 – 24/
to a number of papers on this subject. It is suffi- in fluorescence intensity was related to a decreased ICG
cient to say here that by coupling a CCD camera to the content in deeper vessels in the deep burn. In superficial
fundus camera normally used to view the retina in a clini- burns, such vessels remain largely intact, allowing ICG
cal setting, fluorescence images of the choroid vasculature flow and a strong ICG fluorescence signal. In deep burns
FLUORESCENCE IMAGING 5
these vessels are damaged and become occluded, reducing during and after use. Specialized precautions, equipment
blood and ICG flow and reducing the fluorescence signal. and training are required for their use. Immunofluores-
In addition to discriminating between burns of different cence techniques eliminate the need for radionuclides
thickness, ICG fluorescence imaging was also able to and the associated specialized equipment, expertise and
discriminate between fresh and older (24 h) burns. A health and safety risks while maintaining sensitivity.
significant (twofold) elevation of fluorescence intensity Success in immunofluorescence imaging depends upon
was seen in images of fresh burns compared with images of the correct choice of antibody, fluorophore and detection
burns recorded after 24 h. This difference was attributed system.
to an increase in capillary permeability in fresh burns,
leading to an increased efflux of ICG from capillaries and
3.1 Choice of Antibody
accumulation in the extravascular space.
Finally, and perhaps most importantly, ICG fluores-
3.1.1 General Properties of Antibodies
cence imaging was able to discriminate between burns
which healed within 21 days and those which did not heal To be detectable by immunofluorescence techniques, a
within 24 h. At all time points, ICG fluorescence images substance must be immunogenic when introduced into a
showed a higher fluorescence intensity in burns which host animal, i.e. capable of inducing an immune response,
healed within 21 days compared with those which did and this immune response must result in the production
not heal. Presumably this difference may be explained of antibodies. Such immunogenic substances are termed
at least in part by a greater blood flow (leading to a antigens. Compounds that are immunogenic have certain
greater ICG fluorescence signal) in burns which would general characteristics that include the following: they
heal. This greater blood flow in burns that would heal are foreign to the immunized organism, generally of high
would have two beneficial effects, namely delivery of an molecular weight and chemically complex. Proteins are
adequate supply of nutrients to the regenerating tissues good examples of immunogenic compounds. When a
and removal of toxic waste products. protein is used as an immunogen, it induces an immune
response that results in the production of antibodies that
exhibit a remarkable specificity towards that protein.
Although the ideal condition for eliciting an immune
3 IMMUNOFLUORESCENCE IMAGING
response involves having foreign substances of high
molecular weight that are chemically complex, situations
The examples discussed above share a common drawback: exist in which an immune response can be mounted
a lack of specificity. In addition, toxicity may pose diffi- against simple compounds of low molecular weight.
culties. These problems can be alleviated with the use of In these situations, the small compound is rendered
more effective targeting strategies that do not require the immunogenic by chemical linkage to a high molecular
use of potentially toxic compounds. The most promising weight substance such as a protein.
of these alternative approaches is immunofluorescence Following incubation of the antigen in a host animal,
imaging. Immunofluorescence techniques combine the antibodies to the antigen may be isolated. Antibodies
sensitivity of fluorescence measurement methods with the are globular glycoproteins produced by B-lymphocytes in
specificity of immunochemistry, allowing highly specific response to the presence of foreign substances. Figure 1
detection of low concentrations of materials. Specificity shows the generalized structure of an antibody molecule.
is achieved with the use of an antibody specific to the At the molecular level, antibody molecules are made
material that the investigator wishes to detect, e.g. a up of four polypeptide chains, two identical light chains
cell surface antigen expressed uniquely by cancer cells. (25 000 Da) and two identical heavy chains (50 000 Da).
Exposure of cells to such an antibody labeled with a The structure of the antibody molecule is stabilized by
fluorescent dye results in accumulation of the labeled a number of disulfide bridges. There are two antigen-
antibody only on the surface of cancer cells and not on binding sites on each antibody molecule, each having
the surface of normal cells. Detection of a fluorescence both a constant and a variable region. This variable
signal from samples therefore confirms the presence of portion can adopt an apparently infinite variety of subtly
.26 – 30/
malignant cells. Specificity is provided by the unique different forms that allow it to bind specifically to a vast
.38/
nature of antibody–antigen interactions, while low limits variety of antigens. Interaction of an antibody with
of detection are possible owing to the inherent sensitivity an antigen is governed by noncovalent forces, including
of fluorescence techniques. hydrophobic, electrostatic and van der Waals forces and
Immunofluorescence techniques are of course related hydrogen bonding.
.31 – 37/
to radioimmunoassays. However, radiolabeled The nature of the heavy chain components of the
materials pose a human and environmental risk before, constant region of the antibody molecule determines the
6 BIOMEDICAL SPECTROSCOPY
Light chain to determine which colony produces the monoclonal
antibody with the desired properties. This colony is
then used to produce an essentially limitless supply of
monoclonal antibody with the desired properties.
Variable Fab fragment
Molecular homogeneity and an abundant supply
region
have revolutionized immunoassays. Monoclonal anti-
Constant bodies are not without disadvantages. Lower affinity
region
and the special techniques required for production
are the major disadvantages of monoclonal antibod-
Fc fragment ies compared with polyclonal antibodies. However, low
Heavy chain
affinities can be overcome by the careful selection of
high-affinity antibody-producing hybrids, and currently
many monoclonal antibodies have affinities in the range
10 12 1 .40/
10 –10 L mol .
3.1.3 Antibody Fragments
Figure 1 Generalized structure of an antibody molecule. In addition to the use of entire antibody molecules,
fragments of antibodies may also be used in immunoflu-
orescence experiments. Obviously, this must involve the
physical properties of the antibody, and antibodies are
use of the Fab fragment (i.e. the variable, high-affinity
grouped into five classes based upon these properties.
binding region) rather than the Fc fragment (i.e. the
The five functional classes of immunoglobulin (Ig) are
constant, non-binding region) of the antibody. The main
IgA, IgM, IgG, IgE and IgD. The biological properties
advantage in the use of antibody fragments rather than
of each of these classes are unique. For example, IgE is
the entire antibody lies in the potential for increased
the major class of Ig involved in allergy and binds with
delivery of antibody fragments to tumor sites in in vivo
high affinity to mast cells; IgG is the only class of Ig that
studies.
crosses the placenta, providing immunity to the fetus; IgA
In vivo immunofluorescence studies are limited by the
is the major antibody that is found in saliva and tears; IgM
rate of antibody delivery to the binding site. In part, the
can activate other components of the immune system to
rate of delivery is determined by the molecular weight of
.39/
rupture bacteria and other cells.
the antibody. The use of a relatively small fragment of
an antibody (MW 30 000–40 000) rather than the entire
3.1.2 Monoclonal Versus Polyclonal Antibodies antibody increases the rate of diffusion across capillary
.41/
walls and into the body of the tumor. This increased
Normal B-lymphocytes are each capable of producing an
rate of delivery of labelled material to the site of interest
antibody to a specific antigen determinant (the region of
obviously improves the changes of the fluorescent label
the antigen recognized by the antibody) when activated.
being detected.
As antigens usually have multiple antigenic determinants,
a mixture of antibodies is therefore produced in the serum
3.2 Choice of Fluorophore
of inoculated animals. Each activated B-lymphocyte
forms a clonal population of cells in the spleen, which The following characteristics should be taken into
produce the same antibody as the parent cell. Thus, a consideration in selecting the appropriate fluorophore:
polyclonal population of cells is found in the spleen,
1. Coupling properties. Coupling of a fluorophore label
secreting a wide variety of antibodies. Even relatively
to immunological reagents (antibodies or their frag-
simple antigens normally lead the generation of a mixture
.39/ ments) is an absolute prerequisite for immunofluo-
of antibodies with different specificities and affinities.
rescence imaging. The fluorophore must therefore
This problem of a heterogeneous antibody population
contain a reactive chemical group or be capable of
can be eliminated with the production of monoclonal
derivatization to introduce a reactive group such as
antibodies. By fusing the clonal B-lymphocytes from the
an isothiocyanate or ester group. Importantly, cou-
spleen with immortal myeloma cells, a group of immortal,
pling should not affect the affinity or specificity of
antibody-producing hybrids are produced. If individual
labeled antibodies or result in diminished signals.
hybrid cells are then harvested and grown in culture,
each individual cell gives rise to a colony of clonal cells, 2. Sensitivity. The sensitivity of immunoassays is highly
and each cell in the colony secretes the same (mono- dependent upon the properties of the fluorophore
clonal) antibody. The hybrid colonies are then screened used. To be useful a fluorescent dye must have a high
COOH
COOH
