Encyclopedia of Analytical Chemistry: Applications, Theory, and Instrumentation, 15 Volume Set PDF
Preview Encyclopedia of Analytical Chemistry: Applications, Theory, and Instrumentation, 15 Volume Set
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,Online2006–2011JohnWiley&Sons,Ltd. Thisarticleis2011GovernmentofCanadainCanadaand2011JohnWiley&Sons,Ltdintherestoftheworld. ThisarticlewaspublishedintheEncyclopediaofAnalyticalChemistryin2011byJohnWiley&Sons,Ltd. 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,Online2006–2011JohnWiley&Sons,Ltd. Thisarticleis2011GovernmentofCanadainCanadaand2011JohnWiley&Sons,Ltdintherestoftheworld. ThisarticlewaspublishedintheEncyclopediaofAnalyticalChemistryin2011byJohnWiley&Sons,Ltd. 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,Online2006–2011JohnWiley&Sons,Ltd. Thisarticleis2011GovernmentofCanadainCanadaand2011JohnWiley&Sons,Ltdintherestoftheworld. ThisarticlewaspublishedintheEncyclopediaofAnalyticalChemistryin2011byJohnWiley&Sons,Ltd. 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 EncyclopediaofAnalyticalChemistry,Online2006–2011JohnWiley&Sons,Ltd. Thisarticleis2011GovernmentofCanadainCanadaand2011JohnWiley&Sons,Ltdintherestoftheworld. ThisarticlewaspublishedintheEncyclopediaofAnalyticalChemistryin2011byJohnWiley&Sons,Ltd. DOI:10.1002/9780470027318.a0101m.pub2 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. EncyclopediaofAnalyticalChemistry,Online2006–2011JohnWiley&Sons,Ltd. Thisarticleis2011GovernmentofCanadainCanadaand2011JohnWiley&Sons,Ltdintherestoftheworld. ThisarticlewaspublishedintheEncyclopediaofAnalyticalChemistryin2011byJohnWiley&Sons,Ltd. DOI:10.1002/9780470027318.a0101m.pub2 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. EncyclopediaofAnalyticalChemistry,Online2006–2011JohnWiley&Sons,Ltd. Thisarticleis2011GovernmentofCanadainCanadaand2011JohnWiley&Sons,Ltdintherestoftheworld. ThisarticlewaspublishedintheEncyclopediaofAnalyticalChemistryin2011byJohnWiley&Sons,Ltd. DOI:10.1002/9780470027318.a0101m.pub2 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 EncyclopediaofAnalyticalChemistry,Online2006–2011JohnWiley&Sons,Ltd. Thisarticleis2011GovernmentofCanadainCanadaand2011JohnWiley&Sons,Ltdintherestoftheworld. ThisarticlewaspublishedintheEncyclopediaofAnalyticalChemistryin2011byJohnWiley&Sons,Ltd. DOI:10.1002/9780470027318.a0101m.pub2
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