Encyclopedia of Analytical Chemistry: Applications, Theory, and Instrumentation, 15 Volume Set

9889 Pages·2000·99.95 MB·English

by  Robert A. Meyers

BIOMEDICALSPECTROSCOPY:INTRODUCTION 1
Biomedical Spectroscopy: Medicine, known as ‘‘the art of science of healing
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
HenryH.Mantsch
rests firmly upon a foundation of biological sciences,
NationalResearchCouncil,Winnipeg,Canada
which in turn, trace their origin to the basic physical
sciencesofchemistry,physicsandmathematics.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 wereuniversallyavailable,regardlessoffinancialbarriers,
3 Apologia and Outlook 3 cancerwouldcontinuetokill,rheumatoidarthritiswould
still cripple, and Alzheimer’s disease would continue to
Abbreviations and Acronyms 3
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,thepaucityofeffectivediagnostictechniques
1 INTRODUCTION
plays a significant role in the continued occurrence of
thesediseases.Earlydiagnosisisanimportantcomponent
BiomedicalSpectroscopy, fortuitously thefirstsectionin 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 (gammarays)downtothelow-energyend(radiowaves),
solitudes are closing the gap that has separated them to probe individual biomolecules and indeed the whole
forsolongastheycross-pollinateeachotherincreasingly. humanbody.
Althoughspectroscopy,byitsverynature,hasalwayshad
an interdisciplinary focus, the recent marriage between
spectroscopyandmedicineisonlynowbeginningtobear 2 OVERVIEWOFCONTRIBUTIONS
fruit.
Itisunfortunatethattheword‘‘chemical’’hasacquired 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),andmagneticresonance(MR)spectroscopy.From
cals’’. So what are natural biochemicals, and what is a the most ancient times, medical practitioners performed
naturalsubstance?Toanswerthisquestionwehavetogo physicalexaminationsoftheirpatientsusingtheireyesas
back almost 20billion years to when the universe arose optical spectroscopes. Indeed, analytical methods based
withacataclysmicexplosionthathurledhot,energy-rich onopticalspectroscopywereusedearlyinmedicaldiag-
subatomic particles throughout space. Gradually, as the nosis,andeventodaymanytestsperformedintheclinical
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
structedarecalledbiomoleculesandwereselectedduring opticalapproachessuchasopticalcoherencetomography
the course of evolution for their fitness to perform spe- orphotodynamictherapy.
cificfunctions.Itisthereforequitelegitimatetoaskwhat Biodiagnostic methods fall into two large categories:
thepurposeorthespecificbiologicalfunction ofagiven (a)biomedical spectroscopy and (b)biomedical imag-
biomoleculeinalivingorganismmaybe.Whenexamined ing. The former is based on the interaction of selected
separatelythesebiomoleculesconformtoallthephysical electromagnetic waves with individual or collections of
andchemicallawsthatdescribethebehaviorofinanimate biomolecules. The resulting ‘‘molecular spectra’’, repre-
matter,andyetlivingthingspossessuniquepropertiesnot sentedasplotsofintensityversuselectromagneticenergy,
displayedbycollectionsofinanimatemolecules,thuspre- provide answers to the questions ‘‘what?’’ (qualitative
senting a distinctive challenge to the analytical chemist bioanalytical chemistry) and ‘‘how much?’’ (quanti-
andtothebiospectroscopistalike. tative bioanalytical chemistry). The second category,
Encyclopedia of Analytical Chemistry,Online © 2006 John Wiley & Sons, Ltd.
This article is © 2006 John Wiley & Sons, Ltd.
This article was published in theEncyclopedia of Analytical Chemistry in 2006 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470027318.a0101
2 BIOMEDICALSPECTROSCOPY
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
troscopicinformation.Bothbiomedicalspectroscopyand to its practitioners, uses photosensitizers that are non-
biomolecularimagingcanbeperformedeitherexvivo,on toxic inthe darkbut become toxic after photoactivation
extractedbiofluidsorexcisedtissue,whenthebiomaterial 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
areaofthepatientviaopticalfibers,endoscopes,catheters onskindiseasesandcancer.
oreventhroughspace(e.g.radiowavesinMRimaging). 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,butwelivewithintheconstraintsofthereal practice. Both IR spectroscopy and the complementary
world. technique of Raman spectroscopy, derive information
TheindividualarticlesintheBiomedicalSpectroscopy fromthevibrationsofchemicalbondsinthebiomolecules
sectionarelargelyself-contained,eachcoveringaparticu- of interest and are therefore referred to as vibrational
larareaofexpertiseofthecontributingauthor(s).Afirst spectroscopies. Jackson and Mantsch (Infrared Spec-
group of contributions deals with optical spectroscopy. troscopy, Ex Vivo Tissue Analysis by) introduce the
Theterm‘‘opticalspectroscopy’’isnotsynonymouswith reader to the basics of ex vivo tissue analysis by mid-IR
visible spectroscopy, which at times can be confusing. spectroscopy,focusingontwofacetsofsuchananalysis.
Optical spectroscopy involves transitions between elec- First,thereisanexperimentalaspectrelatedtopotential
tronic energy levels, and thus extends beyond the violet pitfalls with spectroscopic measurements on samples as
intotheultravioletandbeyondtheredintothenear-IR. complexashumantissue.Thesecondaspectconcernsthe
Ramanujam (Fluorescence Spectroscopy In Vivo) pro- interpretationofspectra,meaningthepropertranslation
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
beenexploredextensivelyasdiagnostictools,inparticu- (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 etal.
contributionbyRamjiawanetal.(FluorescenceImaging) (Near-infrared Spectroscopy, In Vivo Tissue Analysis
is dedicated to optical fluorescence imaging. Fluores- by).Comparedtobothvisiblelightandmid-IRradiation,
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
orimaging.Inparticularimmunofluorescenceimaging,a for in vivo tissue spectroscopy and imaging. The pene-
techniquebasedontheinteractionoflabeledantibodies tration depth of near-IR light is of the order of several
with specific antigens, shows great diagnostic potential, centimeters,whereasonlythetop10–20µmoftissuecan
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 suechromophores oxy- and deoxyhemoglobin, oxy- and
Grailofallanalytesassessedinbiologicalfluids.Itisthe deoxymyoglobin, oxidized and reduced cytochromec,
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-
dependonasimple,noninvasiveopticalwandfordeter- tion/dehydration. The article by Shaw and Mantsch
miningtheirbloodglucoselevels.Asthereaderwillfind (Infrared Spectroscopy in Clinical and Diagnostic Anal-
out, great progress has been made towards this goal, ysis)offersanoverviewoftheclinicalanalysesthathave
but we are not quite there yet. Fercher (Optical Coher- been carried out by IR spectroscopic methods on such
enceTomography)providesanup-to-datesynopsisofthe commonbiologicalfluidsasserum,wholebloodandurine,
novelfieldofopticalcoherencespectroscopyandtomog- 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‘‘IRclinicalchemistry’’wascoinedasananalytical
urology, and gynecology to in vivo imaging in ophthal- techniquethatdoesnotrequirechemicalorbiochemical
mology, dentistry, and gastroenterology. Another type reagents for the quantitative determination of analytes;
ofopticalspectroscopysuitableformedicalapplications, instead,theanalysisreliesonchemometricalgorithms.A
Encyclopedia of Analytical Chemistry,Online © 2006 John Wiley & Sons, Ltd.
This article is © 2006 John Wiley & Sons, Ltd.
This article was published in theEncyclopedia of Analytical Chemistry in 2006 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470027318.a0101
BIOMEDICALSPECTROSCOPY:INTRODUCTION 3
digression into the realm of microbiology, utilizing mid- stellarperformanceoffMRIinthefieldofneuroscience
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
Thismethodologyoffersanalternativeanalyticaltoolfor function.Finally,Yuan(MagneticResonanceAngiogra-
the detection, enumeration, classification and identifica- phy)exploitstheemerginguseofMRinangiography,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- tialclinicalapplicationsarediscussed,thechallengebeing
tional information from IR spectra. Two-dimensional coronaryMRangiographybecausethethree-dimensional
correlation spectroscopy already has revolutionized MR structureofthecoronarytreechangesshapeduringeach
spectroscopy and is expected to benefit IR biomedical heart contraction. A concern sometimes voiced with in
spectroscopyaswell. 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 tromagneticfieldsandveryfewadverseeffectshavebeen
‘‘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 APOLOGIAANDOUTLOOK
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
ofcertainradiofrequenciesbyindividualnucleiinamag-
their effort and dedication, as editor of the Biomedical
neticfield,itmustbeperformedinamagnet.Thehigher
SpectroscopysectionIalsoapologizetomycolleaguesin
the magnetic field, the greater the detection sensitivity,
thebiospectroscopiccommunitywhoseworkcouldnotbe
a fact which has fuelled the race for ever higher-field
included.Ihadtobeselectiveandnodoubtthisselectivity
magnets. Smith and Blandford (Magnetic Resonance
reflects my own bias and preoccupations with certain
in Medicine, High Resolution Ex Vivo) introduce the
aspectsoftheexplodingfieldofbiomedicalspectroscopy.
reader to the intricacies of ex vivo high-resolution MR
As to the future of biomedical spectroscopy, I am
spectroscopyinmedicine.Theapplicationstheyhighlight
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
liketoreferthereadertoastatementbyAlanKay,one
MR imaging modalities. Richards (Multinuclear Mag-
ofthefoundersofSiliconValleyinCalifornia,‘‘thebest
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
tiallyresolvedmapsandimagesofselectedbiomolecules
bioanalyticalchemists.
intissue(exvivo),orinthebody(invivo).Thismolecular-
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
ofchemicalsinthebody, although IRimagingislimited
to external body parts such as skin, whereas the whole fMRI FunctionalMagneticResonanceImaging
bodyisaccessibletoMRimaging.Richter(MagneticRes- IR Infrared
onanceImaging,Functional)familiarizesthereaderwith MR MagneticResonance
functional magnetic resonanceimaging (fMRI),perhaps NMR NuclearMagneticResonance
themostpowerfulimagingtechniqueavailabletoday.The
Encyclopedia of Analytical Chemistry,Online © 2006 John Wiley & Sons, Ltd.
This article is © 2006 John Wiley & Sons, Ltd.
This article was published in theEncyclopedia of Analytical Chemistry in 2006 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470027318.a0101
Fluorescence Imaging suggest that tissue nicotinamide adenine dinucleotide
(reduced form) (NADH) fluorescence may prove useful
in detection of malignancies, the rationale being that
changes in metabolic status of malignantly transformed
Jihad-Rene´ Albani
cellsarereflectedinchangesinfluorescencesignalsarising
Laboratoire de Biophysique Mole´culaire, Universite´
fromNADH.(1–8)
deLillel,Villeneuved’Ascq,France
The range of extrinsic fluorophores of use (fluo-
rophoreswhichareaddedtosamples)isobviouslymuch
wider.Theycoverawidespectralrange(fromthevisible
1 Introduction 1 to the near-infrared (NIR)).(9–14) Extrinsic fluorophores
2 Fluorescent Chromophores or Fluorophores 1 are introduced into samples either as free agents or
2.1 Endogenous Fluorophores 1 attachedtocarriermaterialssuchasantibodiestospecific
materialspresentinthesample.(15–22)
2.2 Exogenous Fluorophores 2
Detection of fluorescent materials in samples may be
3 Immunofluorescence Imaging 4
achievedusingeitherspectroscopicorimagingtechnolo-
3.1 Choice of Antibody 5
gies. Spectroscopic techniques may be used to analyze
3.2 Choice of Fluorophore 6 smallregionsoftissueandprovideinformationrelatingto
4 Choice of Detection Systems 6 theaverageconcentrationoffluorescentmaterialswithin
4.1 Charge-coupled Device Cameras 7 thesample.Fluorescencespectroscopymaybeperformed
in vitro, e.g. using homogenized tissues, or in vivo, e.g.
4.2 Filter Selection 7
with the use of fiber-optic bundles.(1,23) The advantage
4.3 Typical Experimental Set-up 7
tothespectroscopicapproachisthatfluorescenceacross
5 Immunofluorescence Imaging Of Tumors 7
theentirespectralrangeofinterestisacquired,allowing
5.1 Qualitative Analysis of Images 7 informationonmultiplefluorophorestobeobtainedwith
5.2 Quantitative Analysis of Images 8 onemeasurement.However,theobviousdisadvantageto
thisapproachisthatonlyasmallvolumeoftissuecanbe
5.3 Statistical Analysis of Images 9
analyzedandtheresultingsignalisanaverageonefrom
6 Summary and Future Prospects 11
that tissue volume. Therefore, spatial information is not
Abbreviations and Acronyms 12
obtainedfromspectroscopicmeasurements.
Related Articles 12 Spatial information may be obtained by fluorescence
References 12 imaging in which distribution of fluorescence intensity
is measured as a function of position within the
sample.(24–26) Fluorescenceimagingistypicallyachieved
using imaging arrays such as charge-coupled device
Fluorescence imaging methodology is described. Princi- (CCD)camerasequippedwithappropriatefilters.While
plesaregiven,alongwithdifferentfluorophoresthatcanbe spatial information may be obtained in this manner,
used.Applicationssuchasimmunofluorescence,vascular, fluorescence imaging has typically been performed
burnsandtumorsstudiesaredetailed. at a single frequency. Thus, spectral information is
lost. Multiple imaging frequencies (to detect multiple
chromophores)requirechangingbetweenmultiplefilters,
1 INTRODUCTION a tedious procedure. Development of the liquid-crystal
tunable filter (LCTF) has removed this limitation. The
Owing to their high sensitivity, fluorescence techniques transmission characteristics of LCTFs may be varied
have found a valuable place in biology. Sensitivity of undercomputercontrol,allowingtheusertovaryimaging
fluorescencemethodsisdueinlargeparttothefactthat wavelength without changing filters. With such filters,
theyare‘zerobackground’techniques.Inotherwords,in fluorescencespectroscopicimagingbecomesfeasible.
theabsenceoffluorophore,nosignal(otherthanrandom
noise) can be detected. Any signal that is detected must
2 FLUORESCENTCHROMOPHORESOR
thereforearisefromthefluorophoreofinterest.
FLUOROPHORES
Fluorescence methods with potential for use in
medicineutilizebothintrinsicandextrinsicfluorophores.
2.1 EndogenousFluorophores
Number of intrinsic fluorophores, i.e. those occurring
naturally in tissues, is limited; the most important Although a number of endogenous fluorophores exist
are listed in Table1. A number of literature reports in the body (Table1), their use in in vivo fluorescence
EncyclopediaofAnalyticalChemistry,Online2006–2011JohnWiley&Sons,Ltd.
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DOI:10.1002/9780470027318.a0101m.pub2
2 BIOMEDICALSPECTROSCOPY
Table1 Selected fluorophores and their excitation and Differences in NADH fluorescence are also apparent
emissionproperties betweennormalandmalignantcellsandtissues.NADH
Fluorophore Excitation Emission exhibitsrelativelystrongfluorescenceat350nm,whereas
maximum(nm) maximum(nm) itsoxidizedcounterpart,NAD+,exhibitsaweakfluores-
cence.RatioofNADHtoNAD decreasesinmalignant
Tryptophan 275 350 +
tissues,leadingtoareducedfluorescencesignal.Itshould
Collagen 340 395
Elastin 460 520 be stressed that factors resulting in metabolic distur-
NADH 350 460 bances may also potentially affect NADH/NAD ratios
+
Porphyrins 400 610,675 andproducealteredtissuefluorescencesignatures.Speci-
ICG 790 815
ficityofNADHfluorescencemethodsformalignanciesis
Cy3 550 565
atpresentunclear.
Cy5 690 710
Cy7 750 777
2.2 ExogenousFluorophores
Exogenous fluorophores are those introduced into
studiescanbeproblematic.Themostcommonproblems
samples,toallowdetectionofspecificmaterials(e.g.when
areasfollows:
attached to antibodies), to probe environmental param-
The low level of fluorescence typically observed in eter(e.g.pH),ortoprovidecontrast(e.g.angiography).
• tissues. Asdiscussedabove,numberofpotentialexogenousfluo-
The very close excitation wavelengths. In fact, many rophoresislarge,allowinggreaterflexibilityinthechoice
• endogenousfluorophoresmaybeexcitedatsimilaror ofspectralrangetobeemployedforexcitationandemis-
closewavelengths.Forexample,collagenfluorescence sionmonitoring.Infact,inmanyinstances,fluorophores
may be excited at 340nm, very close to the exci- canbechemicallytailoredtomeetexcitationandemission
tation wavelength of NADH (350nm). In this case, criteria.
one should be sure, when performing fluorescence Fluorophoreofchoicedependsonanumberoffactors,
experiments,whichfluorophoreisexcited. suchasnatureoftheexperiment,excitationandemission
Overlap of excitation and emission wavelengths wavelengths, and chemical structure of the fluorophore.
• can be even more problematic. For instance, while Chemical structure is important if the fluorophore is to
tryptophan and NADH fluorescence is character- be attached to a carrier material: the fluorophore must
ized by well-separated excitation wavelengths (275 containareactivegroup,becapableofderivatization,or
and 350nm, respectively), emission maximum for produceanactivegroupthatwillallowchemicallinkage
tryptophan fluorescence is seen at 350nm. Hence, to the carrier material. In many respects, limitations in
tryptophan fluorescence at 350nm may be masked fluorophoreselectionimposedbytheexperimentnature
or disrupted owing to the absorption of the emitted andexcitationandemissionwavelengthsaremoresevere.
radiationbyNADH. Applications of fluorescent dyes that show excitation
Ultraviolet (UV) or visible excitation and emission and emission wavelength maxima in the UV or visible
• maxima.Asdiscussedabove,fluorophoresthatexhibit regionsofthespectrumarerestricted,owingtothelimited
excitation and emission maxima in the UV or visible penetrationofUVandvisiblelightintotissues.Thus,only
spectralregionslimitstudiestothecharacterizationof fluorescencesignalsfromsuperficialfluorophorescanbe
superficialstructures,owingtolimitedpenetrationof recorded in these spectral regions. Dyes that fluoresce
lightthroughtissuesatthesewavelengths.Inaddition, in the far-red or NIR regions have greater potential
illumination of tissues with UV light is obviously diagnostic use, owing to the enhanced penetration of
undesirable. lightthroughtissuesinthesespectralregions.
Despitetheselimitations,imagingfluorescencearising
2.2.1 Ultraviolet/VisibleFluorophores:Porphyrin
from endogenous fluorophores has been demonstrated
Derivatives
to have clinical potential, particularly in the diagnosis
of malignancies. For example, it has been demonstrated Any fluorescent material that is selectively accumulated
that malignant tissue shows enhanced accumulation of by tumor cells can in principle be used as a diagnostic
endogenousporphyrins,whichexhibitfluorescenceinthe marker. In this respect, many therapeutic agents have
red part of the visible spectrum.(16,27) It has, therefore, potentialasdiagnosticagents.Porphyrinderivativesused
beensuggestedthatchangesinfluorescencearisingfrom in photodynamic therapy are particularly well suited
endogenousporphyrinsmaybeusefulinthedetectionof in this respect.(15,27,35–46) Already used as therapeutic
tumor.(28–34) agents,porphyrinderivativesareselectivelyaccumulated
EncyclopediaofAnalyticalChemistry,Online2006–2011JohnWiley&Sons,Ltd.
Thisarticleis2011GovernmentofCanadainCanadaand2011JohnWiley&Sons,Ltdintherestoftheworld.
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DOI:10.1002/9780470027318.a0101m.pub2
FLUORESCENCEIMAGING 3
bymalignantcellsandexhibitfluorescence.Excitationat cells results from passive diffusion of the neutral form
around400nmallowsafluorescencesignaltobeimaged of the dye across cell membrane. In malignant tissues,
in tissues and cells at 600–650nm, depending on the extracellular pH is often lower than that seen in normal
porphyrin derivative nature. Utility of this approach tissue. This lower pH shifts the dissociation curve of
has been demonstrated by imaging the fluorescence fluorescein,resultinginahigherconcentrationofneutral
arising from a hematoporphyrin derivative accumulated formofthedyeinextracellularfluid.Thisinturnresults
in colon adenocarcinoma cells implanted in the leg of a in an increased passive diffusion of the dye across cell
rat. Excitation at 337nm 24h following administration membrane.Astumorcellsaregenerallyabletomaintain
of a low dose of the porphyrins resulted in detectable a neutral intracellular pH, equilibrium within the cell is
fluorescenceandtumorvisualization. shiftedinfavorofdissociation,reducingconcentrationof
Accumulation of porphyrins, including hematopor- neutral form of the dye and reducing outward diffusion.
phyrin derivative and protoporphyrin IX, has been used Thedye,therefore,accumulateswithinmalignantcells.(54)
to diagnose a number of cancer forms in humans. In Preferentialuptakeoffluoresceinbymalignanttissues
Vivo, fluorescence imaging of skin showed higher fluo- has been known for more than 50years. However, the
rescenceinbasalcellcarcinomaslesionsthansurrounding dye exhibits green fluorescence when excited with blue
normal tissue. Clear demarcation of lesion borders light.Inotherwords,excitationandemissionmaximaare
was seen in images. Malignant tumors of the breast, both in the visible portion of the spectrum. Penetration
head and neck region, and urinary bladder have been of visible light through tissues is poor, and fluorescein
visualized with a fluorescence imaging system, using spectroscopy and imaging are limited to accessible
100-ns long optical pulses at 390nm delivered via an surfaces.Fluoresceinimagingis,therefore,mostusefulif
endoscope system coupled to the imaging one. Tumor endoscopic techniques are used to study organs such as
detection was achieved based on the differential fluo- lungs or gastrointestinal tract. Endoscopic detection of
rescence between normal and malignant tissue, related fluorescein emission has been demonstrated for gastric
to the selective uptake of tumor-marking agents, such cancer.Evenmorepromisingisthefindingthatdysplasia
as hematoporphyrin derivative and levulinic acid, and couldbedetectedbasedonfluoresceinfluorescencein22
naturalchromophoredifferencesbetweenvarioustissues. of23specimensofcheekpouchfromcarcinogen-treated
Acleardemarcationfromnormalsurroundingtissuewas Syrianhamsters.(23)
foundinmeasurementsofsuperficialbladdercarcinoma
Of course, it should be noted that conditions other
andinvitroinvestigationsofresectedbreastcancer.(47)
than malignancies might induce a fall in extracellular
While the use of porphyrins as chromophores in
pH, e.g. inflammatory conditions. If such conditions are
fluorescence imaging is appealing, it is not without
alsoassociatedwithmaintenanceofintracellularpH,i.e.
drawbacks.(15,16,27,38) Themost important drawbacks are
formationofapHgradientacrossthecell,theymayshow
the time required to achieve significant differential
fluoresceinaccumulationandenhancedfluorescencemay
accumulation of the porphyrin in malignant tissue and
result.Specificityforfluoresceinformalignanttissueshas
potentialsideeffectssuchasphotosensitization.
yettobedetermined.
2.2.2 VisibleFluorophores:Fluorescein
2.2.3 Near-InfraredFluorophores:IndocyanineGreen
In contrast to porphyrin derivatives, toxic side effects
associated with fluorescein use are minimal. Fluorescein If information from deeper, nonsurface structures is
hasbeenusedformanyyearsinstudiesofvascularbeds required, fluorophores that exhibit excitation and emis-
within the eye. Illumination of retina with blue light sion maxima in the far-red or NIR spectral regions
produces green fluorescence in the vessels of the eye in which tissues are relatively transparent to light are
following injection of a bolus of fluorescein. Acquisition required.
of images (typically with the use of photographic ThemostwidelyusedNIRfluorescentdyeisICG(also
film) of the distribution of fluorescence produces known as Cardio-Green or Fox Green). ICG exhibits
angiograms, images of the vessels within the eye. While absorption and emission maxima at 780 and 810nm,
fluorescein angiography is slowly being superseded by respectively, wavelengths at which light can readily
indocyaninegreen(ICG)angiography,otherapplications traverse several millimeters of tissues. Originally used
offluoresceinfluorescencearebeingpursued. to monitor cardiac and hepatic function (based on the
Malignant tissues show an enhanced accumulation transit time of a bolus of the fluorescent dye through
of fluorescein compared with normal tissues.(48–53) At the circulatory system), ICG has recently become more
neutral pH, fluorescein exists as a mixture of charged widely utilized as a contrast agent in studies of the
and uncharged species. Accumulation of fluorescein by vasculature.(55–72)
EncyclopediaofAnalyticalChemistry,Online2006–2011JohnWiley&Sons,Ltd.
Thisarticleis2011GovernmentofCanadainCanadaand2011JohnWiley&Sons,Ltdintherestoftheworld.
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DOI:10.1002/9780470027318.a0101m.pub2
4 BIOMEDICALSPECTROSCOPY
The most common application of ICG fluorescence alsotoburndepth.Fluorescenceimagesfromdeepburns
in vascular studies is without doubt in choroidal (i.e.thoseproducedbyprolongedcontactbetweenheated
angiography,i.e.imagingthechoroidalvesselsoftheeye, metalblockandtheskin)showeddecreasedfluorescence
vesselsthatprovide70%ofthenutrientflowtotheretina. intensityattheburnsitecomparedwithsuperficialburns.
Adiscussionofthisactivefieldofclinicaluseisbeyondthe Thisdifferenceinfluorescenceintensitywasrelatedtoa
scopeofthisarticleandinterestedreaderisreferredtoa decreasedICGcontentindeepervesselsinthedeepburn.
numberofpapersonthissubject.(58,61,73–75)Itissufficient In superficial burns, such vessels remain largely intact,
tosayherethatbycouplingaCCDcameratothefundus allowingICGflowandastrongICGfluorescencesignal.
camera normally used to view the retina in a clinical In deep burns, these vessels are damaged and become
setting, fluorescence images of the choroid vasculature occluded, reducing blood and ICG flow and decreasing
canbeobtained.Bystudyingtimecourseoffluorescence fluorescencesignal.
changes in images, hemodynamic information can be Inadditiontodiscriminatingbetweenburnsofdifferent
obtained. Static images and hemodynamic information thickness, ICG fluorescence imaging was also able to
can be used to evaluate vision-related problems such as discriminate between fresh and older (24h) burns. A
age-relatedmaculardegeneration.(58,74) significant (twofold) elevation of fluorescence intensity
Monitoring tissue vascularity is important in many wasseeninimagesoffreshburnscomparedwithimagesof
other clinical settings, and ICG fluorescence imaging burnsrecordedafter24h.Thisdifferencewasattributed
may play an important role in these areas. One obvious to an increase in capillary permeability in fresh burns,
application of ICG fluorescence imaging is monitoring leadingtoanincreasedeffluxofICGfromcapillariesand
vascularization of tumors.(76–81) Typically, tumors have accumulationintheextravascularspace.
high metabolic rates and so have requirements for high Finally, and perhaps most importantly, ICG fluores-
rates of blood flow. As the tumor develops, this high cenceimagingwasabletodiscriminatebetweenburnsthat
bloodflowrequirementismetbyneovascularization,that healed within 21days and those that did not heal within
is, formation of new blood vessels. Thus many tumors 24h.Atalltimepoints,ICGfluorescenceimagesshowed
areextremelywellvascularized.Imagingthedistribution ahigherfluorescenceintensityinburnsthathealedwithin
of blood vessels within tumors is therefore of potential 21days compared with those that did not heal. Presum-
diagnosticutility.Thistechniquehasbeendemonstrated ablythisdifferencemaybeexplainedatleastinpartbya
to be effective for studying vascular bends in melanoma greaterbloodflow(leadingtoagreaterICGfluorescence
of the retina. However, it should be stressed that only a signal)inburnsthatwouldheal.Thisgreaterbloodflowin
limitedsubsetoftumorswillbeamenabletostudyinthis burnsthatwouldhealwouldhavetwobeneficialeffects,
manner.Anabsoluterequirementforsuchatechniqueis namely,deliveryofanadequatesupplyofnutrientstothe
acleardifferentiationbetweenvascularstructuresofthe regeneratingtissuesandremovaloftoxicwasteproducts.
tumorandsurroundingtissues.Ifthiscleardifferentiation
is not present, diffuse fluorescence from ICG in the
vasculature of surrounding tissue will mask fluorescence
3 IMMUNOFLUORESCENCEIMAGING
of ICG in vessels within the tumor. In other words, the
zero background advantage of fluorescence techniques
willbelost,reducingsensitivityandspecificity. Examples discussed above share a common drawback:
In addition to providing contrast for studying vascu- a lack of specificity. In addition, toxicity may pose
lature, ICG has also been used in the investigation of difficulties. These problems can be alleviated with
burns.(82–93) These studies have focused on the intensity the use of more effective targeting strategies that do
of ICG fluorescence recorded at various times after a not require the use of potentially toxic compounds.
burnwasinduced.Thisintensitywasthencorrelatedwith The most promising of these alternative approaches is
clinical outcome of burns of various depths. Burns were immunofluorescence imaging, which combine sensitivity
produced in anesthetized pigs by placing a heated brass of fluorescence measurement methods with specificity
blockontheskin.Burndepthwasvariedbyvaryingthe of immunochemistry. This combination allows highly
timeofcontactbetweentheblockandtheskin.Following specific detection of low concentrations of materials.
creationoftheburn,abolusofICGwasinjectedintothe Specificityisachievedwiththeuseofanantibodyspecific
circulatory system at various time points, the skin was to the material that investigator wishes to detect, e.g.
illuminated at 780nm, and fluorescence images were a cell surface antigen expressed uniquely by cancer
acquired using a CCD camera. Comparison of fluo- cells. Exposure of cells to such an antibody labeled
rescence in burns and adjacent normal tissue revealed withafluorescentdyeresultsinaccumulationoflabeled
interesting differences that could be related not only to antibody only on the surface of cancer cells and not on
thestageoftheburn(i.e.timeafterburnwascreated)but the surface of normal cells. Therefore, detection of a
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FLUORESCENCEIMAGING 5
fluorescence signal from samples confirms the presence Light chain
of malignant cells.(94–112) Specificity is provided by the
unique nature of antibody–antigen interactions, while
lowlimitsofdetectionarepossibleowingtotheinherent
sensitivityoffluorescencetechniques. Variable Fab fragment
region
Immunofluorescence techniques are of course related
to radioimmunoassays.(113–124) However, radiolabeled Constant
region
materials pose a human and environmental risk before,
during,andafteruse.Specializedprecautions,equipment,
F fragment
and training are required for their use. Immunofluores- c
Heavy chain
cencetechniqueseliminatetheneedforradionuclidesand
associated specialized equipment, expertise, and health
andsafetyriskswhilemaintainingsensitivity.
Success in immunofluorescence imaging depends on H H
O O
thecorrectchoiceofantibody,fluorophore,anddetection O O
C C
system.
Figure1 Generalizedstructureofanantibodymolecule.
3.1 ChoiceofAntibody
an apparently infinite variety of subtly different forms
3.1.1 GeneralPropertiesofAntibodies that allow it to bind specifically to a vast variety of
antigens.(125–133) Interaction of an antibody with an
To be detectable by immunofluorescence techniques, a
antigen is governed by noncovalent forces, including
substancemustbeimmunogenicwhenintroducedintoa
hydrophobic, electrostatic, and van der Waals forces,
hostanimal,i.e.capableofinducinganimmuneresponse,
andhydrogenbonding.
which results in antibodies production. Such immuno-
Natureoftheheavychaincomponentsoftheconstant
genic substances are termed antigens. Compounds that
region of the antibody molecule determines physical
are immunogenic have characteristics that include the
properties of the antibody. On the basis of these
following: they are foreign to the immunized organism,
properties, antibodies are grouped into five classes. The
generally of high molecular weight and chemically
five functional classes of immunoglobulin (Ig) are IgA,
complex. Proteins are good examples of immunogenic
IgM,IgG,IgE,andIgD.Biologicalpropertiesofeachof
compounds.Whenaproteinisusedasanimmunogen,it
these classes are unique. For example, IgE is the major
induces an immune response that results in the produc-
classofIginvolvedinallergyandbindswithhighaffinity
tionofantibodies,whichexhibitaremarkablespecificity
to mast cells; IgG is the only class of Ig that crosses
towardthatprotein.
theplacenta,providingimmunitytothefetus;IgAisthe
Although ideal condition for eliciting an immune
major antibody that is found in saliva and tears; IgM
response involves having foreign substances of high
can activate other components of the immune system to
molecularweightthatarechemicallycomplex,situations
rupturebacteriaandothercells.(134–138)
exist in which an immune response can be mounted
against simple compounds of low molecular weight. In
these situations, small compound is rendered immuno- 3.1.2 MonoclonalversusPolyclonalAntibodies
genic by chemical linkage to a high-molecular-weight
substancesuchasaprotein. Whenactivated,eachnormalB-lymphocytesiscapableof
Following incubation of antigen in host animal, producing an antibody to a specific antigen determinant
antibodies bound to the antigen may be isolated. (theregionoftheantigenrecognizedbytheantibody).As
Antibodies are globular glycoproteins produced by B- antigens usually have multiple antigenic determinants, a
lymphocytes in response to the presence of foreign mixture of antibodies is therefore produced in serum of
substances. Figure1 shows the generalized structure inoculated animals. Each activated B-lymphocyte forms
of an antibody molecule. At the molecular level, aclonalpopulationofcellsinthespleen,whichproduce
antibody molecules are made up of four polypeptide the same antibody as the parent cell. Thus, a polyclonal
chains, two identical light chains (25 000 Da) and populationofcellsisfoundinthespleen,secretingawide
two identical heavy chains (50 000 Da). Structure of variety of antibodies. Even relatively simple antigens
the antibody molecule is stabilized by a number of normally lead the generation of a mixture of antibodies
disulfidebridges.Therearetwoantigen-bindingsiteson with different specificities and affinities.(135,139–143) This
each antibody molecule, each having both a constant problemofaheterogeneousantibodypopulationcanbe
and a variable region. This variable portion adopts eliminatedwiththeproductionofmonoclonalantibodies.
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6 BIOMEDICALSPECTROSCOPY
By fusing the clonal B-lymphocytes from the spleen or ester group. Importantly, coupling should not affect
with immortal myeloma cells, a group of immortal, affinity or specificity of labeled antibodies or result in
antibody-producing hybrids are produced. If individual diminishedsignals.
hybrid cells are then harvested and grown in culture,
each individual cell gives rise to a colony of clonal Sensitivity. Sensitivity of immunoassays is highly depen-
cells, where each cell secretes the same (monoclonal) dentonpropertiesofthefluorophoreused.Tobeuseful,
antibody.Hybridcoloniesarethenscreenedtodetermine afluorescentdyemusthaveahighquantumyield.
which one produces the monoclonal antibody with the
desiredproperties.Thiscolonyisthenusedtoproducean Stability.Whenlinkedtoantibodies,fluorophoresshould
essentially limitless supply of monoclonal antibody with be nonreactive to materials within the sample of
thedesiredproperties. interest. Labeled materials should also be stable for
Molecular homogeneity and an abundant supply have extended periods of time. Stable signal levels both
revolutionized immunoassays. Monoclonal antibodies from day to day and from experiment to experiment
are not without disadvantages. Lower affinity and are required. Stable fluorophores decrease the need for
specialtechniquesrequiredforproductionarethemajor frequent standardization. It should be noted that many
disadvantages of monoclonal antibodies compared with fluorophoresaresensitivetofactorssuchastemperature
polyclonal antibodies. However, low affinities can be andpH.
overcome by careful selection of high-affinity antibody-
producing hybrids, and currently, many monoclonal Availability.Inorderforalabeltobeacceptedinresearch
antibodies have affinities in the range 10−10–10−12 L and in clinical setting, it should be readily available;
mol−1.(144) conditions for labeling the antibody should be mild
and easily optimized. Also, labeling should be highly
reproducible.
3.1.3 AntibodyFragments
In immunofluorescence experiments, fragments of anti- Excitation and Emission Maxima. Excitation and emis-
bodies may also be used in addition to entire antibody sionmaximaofthefluorophoremustalsobeconsidered.
molecules.(145–159)Obviously,thismustinvolvetheuseof For example, absorption maxima should not be in
the F fragment (i.e. the variable high-affinity binding spectral regions in which endogenous materials absorb
ab
region) rather than the F fragment (i.e. the constant strongly and emission maxima should not be in spec-
c
nonbinding region) of the antibody. The main advan- tral regions where endogenous chromophores exhibit
tage in the use of antibody fragments rather than entire significantemissionorabsorption.Inaddition,choiceof
antibody lies in the potential for increased delivery of absorption and emission maxima, and so fluorophore,
antibodyfragmentstotumorsitesininvivostudies. depend on whether the experiment is conducted in
InVivoimmunofluorescencestudiesarelimitedbythe vitro or in vivo. For example, dyes such as fluorescein
rateofantibodydeliverytobindingsite.Inpart,delivery isothiocyanate(FITC)arecommonlyusedinimmunoflu-
rate is determined by molecular weight of the antibody. orescencestudiesinvitro.However,suchdyesareexcited
Use of a relatively small fragment of an antibody (MW byUVorvisiblelightandfluoresceinthevisible.Unfor-
30000–40000)ratherthanentireantibodyincreasesrate tunately, visible light has limited penetration through
ofdiffusionacrosscapillarywallsandintotumorbody.(160) tissues. This limited penetration obviously reduces dyes
This increased rate of labeled material delivery to the utility for in vivo studies. In contrast, cyanine fluo-
site of interest obviously improves the changes of the rochromes haveemissionmaximainthefar-redspectral
fluorescentlabelbeingdetected. region.Bloodandtissuearerelativelytransparentatsuch
wavelengths, leading to enhanced transmission of light
3.2 ChoiceofFluorophore through tissues. Cyanine dyes such as Cy5 are therefore
moresuitedtoinvivoapplications.
The following characteristics should be taken into
considerationinselectingtheappropriatefluorophore:
Coupling Properties. Coupling of a fluorophore label to
4 CHOICEOFDETECTIONSYSTEMS
immunological reagents (antibodies or their fragments)
is an absolute prerequisite for immunofluorescence
imaging. Fluorophore must therefore contain a reactive ForinvivodetectionoffluorescenceusingvisibleorNIR
chemical group or be capable of derivatization to fluorophores, CCD camera systems equipped with the
introduce a reactive group such as an isothiocyanate appropriatefiltersarethedetectorsystemsofchoice.
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FLUORESCENCEIMAGING 7
4.1 Charge-coupledDeviceCameras
CCD camera To computer
Detection system of choice is the CCD camera. CCD
cameras are available with two-dimensional arrays of
detectors that allow two-dimensional images to be
acquired at wavelengths in the visible and NIR regions.
Advantages of such cameras include high quantum Band-pass filter
efficiency, low noise characteristics, flexibility, and
ruggedness. CCD cameras are available in many forms, Laser
Liquid-crystal
with many types of sensing element to choose from. diodes tunable filter
However, a two-dimensional array of silicon detectors
will provide adequate response over the spectral range Band-pass
200–1000nm and allow detection of most common filter
chromophores.
Figure2 Typical pictorial representation of experimental
set-up.
4.2 FilterSelection
If fluorescence detection at a particular wavelength is wavelength, intensity may still be sufficient to result
required, all other wavelengths must be prevented from in transmission of some light through the LCTF. To
reaching imaging array. This wavelength selection can eliminate this possibility, a band-pass filter should be
be achieved either with the use of fixed-wavelength inserted between the source and the sample to remove
filters such as band-pass or interference filters or by wavelengthsthatarenotofinterest.
using LCTFs. Band-pass and interference filters allow As an additional precaution, when high-intensity
light at predetermined wavelengths to be transmitted sources such as laser diodes are used, a band-pass filter
to the sensing element. If multiple wavelengths are of should be placed in front of the LCTF to eliminate
interest (i.e.if multiple antibodies labeled with different scatteredlightatthefrequencyofthelaserdiode.
fluorophores are used), a filter is required for each
wavelength.Requirementformultiplefiltersisremoved
ifLCTFsareused.Astheirnameimplies,LCTFsutilize 5 IMMUNOFLUORESCENCEIMAGINGOF
liquid-crystal technology and have tunable transmission TUMORS
characteristics. Transmission window of the filter is set
andchangedelectronically.Thus,imagescanbeacquired
5.1 QualitativeAnalysisofImages
at multiple wavelengths with no hardware adjustments.
In addition to time saving, LCTFs have the additional The experimental set-up described above was used to
advantage that as no moving parts are involved in studyinteractionofafragmentofanantitumorantibody
wavelength selection, registration of images at different labeled with Cy5 with tumor cells implanted in athymic
wavelengthsisexcellent. mice.(160) Antibody fragment was part of a human
monoclonal antibody to a cell surface antigen expressed
by a wide variety of human cancer cells. Athymic
4.3 TypicalExperimentalSet-up
mouse has two major advantages in such experiments.
A typical set-up for an immunofluorescence imaging First,athymicmouseishairless.Thisalleviatespotential
experimentisshowninFigure2.ACCDcameraequipped problems due to scattering of light by hair, which would
withazoomlensforfocusingismountedabovethesample not only reduce the amount of excitation light reaching
area. A computer-controlled LCTF for wavelength the area of interest but also result in blurring at the
selection is attached to the zoom lens. An illumination fluorescence site. Second, athymic mouse is immune
source is positioned so as to provide even illumination compromised. As such, it provides an excellent host for
over the sample area. The source may be a broad- human tumor cells, which will not be rejected following
band (white light) source or may be monochromatic implantation.Abilitytomonitorbindingoftheantibody
radiation provided by a laser diode. If laser diodes are fragmenttohumantumorcellsisimportantiftheantibody
thesourceofchoice,precautionsmustbetakenowingto is of human origin. This raises an important, if obvious,
the intense nature of the radiation produced. Although issue in immunofluorescence experiments: the choice of
laser diodes are essentially monochromatic, side lobes model system requires thought. It is crucial that antigen
(i.e. lower intensity emission on either side of the main to which antibody is raised is expressed in system under
emissionline)maybeevident.Althoughsuchsidelobes investigation. For example, the use of a carcinogen-
are much weaker than emission at the main emission treated animal as a model system may be inappropriate
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DOI:10.1002/9780470027318.a0101m.pub2