EJH 2014 04.qxp Hrev master 30/12/14 14:41 Pagina 320 ... · E-mail: [email protected] Key words: Endogenous-fluorophores, energetic-metabolism, functionality-monitoring. Conflict

European Journal of Histochemistry 2014; volume 58:2461

[page 320] [European Journal of Histochemistry 2014; 58:2461]

Autofluorescence spectroscopyand imaging: a tool for biomedical research and diagnosisA.C. Croce, G. BottiroliHistochemistry and Cytometry Unit,Institute of Molecular Genetics of theNational Research Council, Departmentof Biology and Biotechnology “LazzaroSpallanzani”, University of Pavia, Italy

Abstract

Native fluorescence, or autofluorescence(AF), consists in the emission of light in theUV-visible, near-IR spectral range when biolog-ical substrates are excited with light at suit-able wavelength. This is a well-known phe-nomenon, and the strict relationship of manyendogenous fluorophores with morphofunc-tional properties of the living systems, influ-encing their AF emission features, offers anextremely powerful resource for directly moni-toring the biological substrate condition.Starting from the last century, the technologi-cal progresses in microscopy and spectrofluo-rometry were convoying attention of the scien-tific community to this phenomenon. In thefuture, the interest in the autofluorescencewill certainly continue. Current instrumenta-tion and analytical procedures will likely beovercome by the unceasing progress in newdevices for AF detection and data interpreta-tion, while a progress is expected in the searchand characterization of endogenous fluo-rophores and their roles as intrinsic biomark-ers.

Introduction

The native fluorescence, or autofluores-cence (AF) is a widespread phenomenon,because of the common presence of intrinsicbiomolecules acting as endogenous fluo-rophores in the organisms of the whole lifekingdom. The strict relationship of manyendogenous fluorophores with morpho-func-tional properties of the living systems, whichare influencing their AF emission features,affords an outstandingly powerful resource forthe direct monitoring of the biological sub-strate condition.

The attention of scientists to autofluores-cence was accompanying the technologicalprogresses in microscopy and spectrofluorom-etry starting from the 1900’s, and during the

years, several articles and books reviewed theadvancements in the investigation and use ofAF in biological research and biomedical diag-nosis.1-8 These AF applications are part of awider range of analytical procedures based onthe interaction between electromagnetic radi-ations and biological material, defined asOptical biopsy. This term is actually used todescribe the possibility to obtain diagnosticinformation in situ, in a non-invasive or mini-mally invasive manner, without removal of tis-sue specimens and in the absence of exoge-nous markes.8-16

Given the huge number of AF based studiesthat makes a complete report of the overallstudies almost impossible, in this review thehistory and technological progresses related toAF are briefly summarized, followed by thedescription of some selected topics of interestin biomedical research and diagnosis, impli-cating some experience of the authors.

Autofluorescence: a brief history

The emission of light from calcium fluorideand from the organic compounds quinine andchlorophyll in solution was reported early in1838 by David Brewster in Scotland, andnamed fluorescence by George Stokes inCambridge. As reviewed by Frederick H.Kasten,2 the scientific community offeredincreasing interest in fluorescence observa-tions in biological compounds in parallel withthe development of suitable instrumentation,leading to the set-up of the first fluorescencemicroscopes. Autofluorescence is reported, in1911, as the first form of fluorescenceobserved at the microscope by Stübel, a physi-ologist at Jena University who investigated AFof single cells such as bacteria and protozoa, ofanimal tissues as in the teeth, and of variousbiological substances. In the immediate nextyears, several researchers paid attention toashes, plant tissues and products, and the eyelens, and attempts were made to use AF to dis-criminate bacterial pathogens. In comparisonwith animal substrates, the plant endogenousfluorophores were found to give rise to muchmore appreciable emission signals, because oftheir more favorable spectral properties andquantum efficiency. As a consequence, AF wasconsidered a powerful tool to study plant mor-phology and physiology,17-19 and many fluo-rochromes naturally present in plants such asquinones, coumarins, cyanines, tetrapyrrolesand alkaloids were commercially extracted tobe used as exogenous markers.

The availability of these fluorochromes, inaddition to those provided by chemical synthe-sis or modification of natural substances to

make them fluorescent (e.g., the biogenicamines treated with formaldehyde),20 led to anincreasing development of highly selective andsensitive procedures for labeling subcellularcomponents in cytology and histology.4 In thisconcern, an increasing number of new prod-ucts is made available by a continuous searchand synthesis activity, as from the numerouscommercial catalogues of fluorochromes/fluo-rescent dyes.

In the application of exogenous and inducedfluorescence-based assays, AF was often per-ceived as a nuisance, its signal resulting in abackground which may hinder the specificdetection of the exogenous marker emission.Different strategies were proposed to addressthis problem, for instance avoiding AF by prop-er optical filtering or its removal. The latterchance is important, in particular, when AFresults in a wide wavelength range and/or highamplitude emission. AF background can bereduced by bleaching it through pre-irradia-tion, as successfully demonstrated to improvethe DNA measurements after Feulgen stainingin single cells, or the immunofluorescencelabelling of the arterial vessels.21,22 The highphotolability of vitamin A was also exploited toselectively eliminate its unwanted AF signalfrom Ito stellate cells during the in vivo analy-sis of NADH fluorescence in the liver.23 The sig-nal from highly emitting endogenous fluo-

Correspondence: Dr. Anna Cleta Croce, Instituteof Molecular Genetics of the National ResearchCouncil, Histochemistry and Cytometry Unit,Department of Biology and Biotechnology“Lazzaro Spallanzani”, University of Pavia, via A.Ferrata 9, 27100 Pavia, Italy.Tel. +39.0382.986428 - Fax: +39.0382.986430: E-mail: [email protected]

Key words: Endogenous-fluorophores, energetic-metabolism, functionality-monitoring.

Conflict of interests: the authors declare no con-flicts of interest.

Acknowledgments: the Authors are particularlygrateful to all Partners contributing to their orig-inal studies mentioned in this review article, andwish to thank Fondazione Cariplo, grant n. 2011-0439, for supporting their work.

Received for publication: 21 November 2014.Accepted for publication: 4 December 2014.

This work is licensed under a Creative CommonsAttribution NonCommercial 3.0 License (CC BY-NC 3.0).

©Copyright A.C. Croce and G. Bottiroli, 2014Licensee PAGEPress, ItalyEuropean Journal of Histochemistry 2014; 58:2461doi:10.4081/ejh.2014.2461

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[European Journal of Histochemistry 2014; 58:2461] [page 321]

rophores such as lipofuscins and elastin wasalso demonstrated to be reducible by chemicaltreatments before the staining procedures.24,25

The use of fixatives also deserved attention.Aldehyde derivatives, for example, are wellknown to undergo condensation reactions withamines and proteins generating fluorescentproducts.20,26 The consequent increase in theoverall AF emission can thus affect the assaysrequiring fixation, in particular when specificfluorochromized biological probes (i.e., anti-bodies, lectins, receptor probes) are used todemonstrate the occurrence of targets whichare present in very low amounts. In thesecases, in the attempt to maximize thesignal/background ratio, it can be suggested touse fluorophores with high quantum yield orwith excitation/emission ranges longer thanthe blue region (which is the predominant onefor most endogenous fluorophores). At pres-ent, the major chances to circumvent the AFadverse effects are actually offered by theimprovement in the emission properties ofexogenous fluorophores, in terms of quantumyield and of spectral position and narrowing tofavor a selective optical filtering.

While representing a complication whencells and tissue are labeled with exogenousfluorochromes, AF by itself encompasses agreat potential in the analysis of animalorgans and fluids for both research and diag-nostic purposes. Actually, the morphologicaland metabolic conditions of cells and tissuesare strictly influencing the nature, amount,physico-chemical state, intra-tissue distribu-tion and microenvironment of the endogenousfluorophores: therefore, the consequentchanges in the emission properties of thesebiomolecules make them to act as intrinsicbiomarkers. The whole AF emission signal isthus carrying comprehensive information suit-able for the direct and real-time characteriza-tion and monitoring of physiological or alteredmorpho-functional properties of cells and tis-sues, in the absence of exogenous marker per-turbations.

The first observations on the dependence ofAF variations on tissue functional changesconcerned porphyrins.27-29As reviewed byKasten,2 porphyrins were observed to be pres-ent in different sites, from the sebaceous plugsof human hair follicles to the Harderian glandof rodents, and their red fluorescence was con-sidered as diagnostically meaningful, since atthat time porphyrins were not otherwisedetectable at the microscope. It is worth recall-ing that a noticeable increase of porphyrin flu-orescence was remarked in relation with bothmetabolic disorders and the presence of neo-plasia,27,30 these observations being particular-ly predictive of the later development leadingto the currently applied Photo-DynamicDiagnosis and Photo-Dynamic Therapy (PDD,

PDT).31 In the same early times, a variety ofvitamins (e.g., vitamin A, riboflavin, thiamin)and other substrates (such as lipofuscins,structural proteins, ceroid pigments) wereidentified as natural fluorophores by HerwigHampler at the University of Vienna (Austria),who began the fluorescence microscopy appli-cations in human pathology.2,4,32,33

At the end of the mid-20th century, particularattention was given to the coenzymesNAD(P)H and flavins, and to the monitoring oftheir redox equilibria in isolated mitochon-dria, single cultured cells and intact tissuesunder different metabolic conditions. Thesepioneering investigations by Britton Chanceand his coworkers divided the period of spo-radic AF observations on various kinds of bio-logical substrates from the new way of system-

atic AF investigation in biomedicine, focusedon specific targets and functions. The develop-ment of instruments such as the firstmicrospectrofluorometers and flow cytofluo-rometers made these first studies possible,and the progresses in AF investigations wereaccompanied by the continuous advancementin the instrumental equipment and analyticalprocedures, allowing to transfer the results ofexperimental studies to the clinical applica-tion.34-42

In general, NAD(P)H and flavins are thealmost unique responsible for the AF signalrising from the cell cytoplasm:43 this allows toperform investigations on cell redox metabo-lism without the need of supplementary analy-ses to distinguish them from other fluo-rophores. Figure 1 shows examples of AF dis-

Review

Figure 1. Examples of autofluorescence distribution patterns recorded from cultured, liv-ing pig cells. The emission signal rising from the cytoplasm around darker nuclei ismainly ascribable to NAD(P)H, the contribution of globular proteins being almost neg-ligible under the conditions used for detection (exc 366 nm; em 420-600 nm). The signalis more diffusely distributed in stem cells (a) than in a mature one (b), where structuresascribable to the typical morphology of mitochondria can be easily recognized. Thesefindings are consistent with a prevalent engagement in anaerobic energetic metabolismin stem cells, and in aerobic – mitochondrial – one in the mature one. Intracytoplasmicbrightly fluorescing granules (c,d) are ascribable to lipofuscins, accumulated as undigest-ed material from autophagic processes contributing to the maintenance of the stemnesshomeostasis or in cell eldering. Scale bar. 20 m.


Table 1. Endogenous fluorophores recurrently exploited as intrinsic biomarkers in autofluorescence studies.

Endogenous Biological Autofluorescence Autofluorescence fluorophores constituents (exc) / (em) ranges photophysical fingerprints and possible correlated alterations

Aromatic amino acids: Functional proteins (240-280 nm) / (280-350 nm) Spectral shape and amplitude (near UV, blue region tail) Phe, Tyr, Trp Cytokeratins Intracellular fibrous proteins (280-325 nm) / (495-525 nm) Spectral shape and emission amplitudeCollagen/Elastin Extracellular fibrous proteins (330-340 nm) / (400-410 nm) Excitation light birifrangence effects (350-420 nm) / (420-510 nm) spectral shape and emission amplitude, depending on maturation degree in eldering and fibrosis NAD(P)H Coenzymes of key enzymes (330-380 nm) / (440, 462 nm, bound, free) Spectral shape, emission amplitude in redox reactionsFlavins Coenzymes of key enzymes (350-370;440-450 nm) / (480/540 nm) (NAD(P)Hbound/free, NAD(P)Htotal/oxidized in redox reactions flavins ratios, depending on aerobic/anaerobic energetic metabolism, antioxidant defense, inflammation, carcinogenesisFatty acids Accumulated lipids (330-350 nm) / (470-480 nm) Spectral shape, emission amplitude and photosensitivity, depending on altered lipid metabolism Vitamin A Retinols and carotenoids (370-380 nm) / (490-510 nm) Spectral shape, emission amplitude and photosensitivity, depending on multiple functions including antioxidant and vision roles, and altered retinol metabolism Protoporpyrin IX and Protein prostetic group (405 nm) / (630-700 nm) Spectral shape, emission amplitude and photosensitivity,porphyrin derivatives depending on heme and iron altered metabolism Lipofuscins/ Miscellaneous (proteins, (UV, 400-500 nm) / (480-700 nm) Spectral shape, emission amplitude depending onLipofuscin like- lipids, retinoids) eldering, oxidation degree, cell stemness degreelipopigments/ceroids


tribution patterns in living cultured cells underdifferent biological conditions.44

The continuously increasing interest ininvestigating tissues rather than single cellswas accompanied by a progressive attention toadditional fluorophores which may be founddepending on the morpho-functional proper-ties of the target substrates. The endogenousfluorophores mainly recurrent in biomedicalAF-based studies are summarized in Table 1,and their representative spectral profiles areshown in Figure 2. The AF studies were there-after focused on two main topics: i) monitor-ing of metabolic functions of cells and tissuesunder normal and experimental conditions, ii)in situ, real-time diagnosis of oncological andnon-oncological diseases. Actually, the bound-ary between these two subjects of investiga-tion cannot be clearly drawn, since a differentextent of early and subtle metabolic changesas well as structural alterations of cells and tis-sues are likely to occur, as the first signs of dis-orders, degenerating diseases or neoplasia.45.-47 Therefore, the ability of AF analysis to detectprimary changes in cell and tissue morpho-functional properties is a promising potentialfor diagnostic applications. In this concern, AFanalysis of cells and tissue properties fullybelongs to Optical biopsy,15,48 its applicationsbeing developed to address and support differ-ent diagnostic procedures in biomedicine.

Review

Figure 2. Typical spectral profiles of autofluorescence emission from single endoge-nous fluorophores. Spectra were recorded by microspectrofluorometry from pure com-pounds in solution, except for the fibrous proteins collagen and elastin and lipopig-ments, recorded respectively from connective tissue of hepatic portal selected areas ofa liver cryostatic tissue section, or from remnant material co llected after organic extrac-tion of liver tissue homogenates. Spectra were normalized to the maximum emissionpeak for presentation, except for the broad emission of lipopigments. Excitation light:366 nm.



Technologies and data processing

The advances in excitation sources, detec-tion devices (including fiber optic probes forboth excitation and AF light guide),49 and dataanalysis procedures promoted great progressesin the applications of Optical biopsy on tissuesites which can be either directly or endoscop-ically accessed, as recently reviewed by Alfanoand Pu.15 The improvement in techniques anddevices used for AF signal collection and analy-sis (such as spectroscopy, microspectroscopyand imaging) is continuously growing toimprove the sensitivity and specificity in thedetection of different fluorophores in singlecells and tissues. As to the in situ imaging ofendogenous fluorophores, the first applica-tions of multiphoton microscopy allowing sub-micron resolution were followed by continuousprogresses in the optical techniques to investi-gate the cell metabolism through the micro-scope.50,51 A careful choice of the Near Infra-Red (NIR) excitation wavelength and powerwas however recommended, to preserve thereliability of the results obtained for livingcells minimizing photobleaching and damages,and the undesired occurrence of photoinducedfluorescent granules containing lipofus-cins.52,53 For example, suitable NIR measuringconditions resulted in an efficient, non-inva-sive detection of NAD(P)H and flavins in ratio-metric redox fluorometry to assess in situmitochondrial metabolic states.54 The twocoenzymes NAD(P)H and flavins, along withlipofuscins and the second armonic generationfrom collagen fibers were also exploited asintracellular and extracellular exclusivesources of imaging contrast to monitor the dif-ferentiation of human mesenchymal stem cellsin culture.55

In addition to the steady-state techniques,time-resolved AF contributed to improve thestudy of the respiratory chain functions as wellas of different metabolic activities in cells andtissues under normal and altered conditions.These applications took advantage of the dif-ferent fluorescence lifetimes characterizingthe signal decay of NAD(P)H in its free andbound state, and of flavins, being respectivelyof about 0.4-0.5 ns, 2.0-2.5 ns and 6 ns.56-58 Inthis relationship, technical set-ups for directlifetime detection and monitoring of tissue AFwere proposed, aiming to improve routine bio-medical and bio-analytical online analyses.59

Recently, a phasor approach has also beendeveloped allowing an easier fluorescence life-time data processing and interpretation,through a non-invasive, label-free, fit-free life-time imaging microscopy technique, escapingthe problems of exponential analysis to assessthe presence of multiple fluorescing species. Agraphical global view is given as an image,

each pixel contributing as a point to the plot.The position of each point identifies a specificfluorophore depending on its typical decayproperties, and a picture is provided allowingan overall and direct interpretation of data interms of the fluorophores presented.60

As to the processing and diagnostic inter-pretation of AF data in single cells, the meremeasurement of the overall emission signalsallowed to detect and isolate granulocytes andin particular eosinophils by means of flow cyto-metric or more generally of microfluidic sys-tems, the analysis of the spectral shape detect-

ed under different excitation conditions evenleading to a complete discrimination of theleukocyte families.61-64 A multispectral imagingAF microscopy technique, based on the simplecollection of monochrome images in the blue,green and red regions under 365 nm excita-tion, was also demonstrated to distinguishbetween non-neoplastic and malignant lym-phoid tissues.65 In bulk tissues, complex multi-variate analysis procedures were proposed toprocess overall emission data, and assess theirpotential for a specific and sensitive discrimi-nation of normal from diseased areas.66-69

Review

Figure 3. Unfixed cryostatic tissue sections from human colon, non-neoplastic mucosa(a) and adenocarcinoma at different staging (b,c), after Hematoxylin and Eosin staining.Non-neoplastic tissue (a) shows the layer organization: mucosa (m) at the inner surface,muscolaris mucosa (mm), submucosa (sm) with blood vessels and muscolaris esterna(me). The histological organization of non-neoplastic mucosa is subversed by the risingof the neoplasia. The highly fluorescing connective tissue in submucosa (d, and AF signalplot profile inside) is strongly affected, only some remnants being appreciable in the neo-plastic mass (e). Excitation: 366 nm, emission: 420-640 nm. Scale bars: a-c), 650 µm; d),100 µm; e), 170 µm.



Nearly in the meantime, a device was devel-oped, based on a high sensitivity camera to beapplied at endoscopy to collect AF signals inthe green and red regions, and to managethem in a ratio imaging by a processing unit todisplay then a color image.70 The validity of thisapparatus as a support for the detection of lungand colon neoplasia resulted in the develop-ment of commercial devices, currently used inthe clinics for pre-screening and diagnosticpurposes through endoscopy. The first-genera-tion Xillix® Laser-Induced FluorescenceEndoscopy (LIFE) (Xillix TechnologiesCorporation, Richmond, BC, Canada) receivedan FDA-PMA approval in USA in 1996, and theEuropean CE Mark in 1997. A Xillix’s nextdevice, Onco-LIFE, incorporating fluorescenceand white-light endoscopy was then developedfor an improved guide in the localization oflung and gastrointestinal cancers. The differ-ences observed in AF emission between neo-plastic and non-neoplastic regions were cor-rectly foreseen to be ascribable to multiplealterations, in tissue metabolism, presence ofporphyrins, blood circulation and histologicalarchitecture. Actually, progresses in the knowl-edge on the nature of single endogenous fluo-rophores present, their AF properties and theirchanges depending on both tissue structureand biological alteration, confirmed the sug-gestions of Takehana,70 helping the progres-sion of optical techniques for diagnostic appli-cations.11

The role of tissue optical properties, interms of absorption, reflection and scatterwere also considered, as factors influencingthe propagation of both excitation light andthe resulting fluorescence within bulk tissues,thus affecting the overall signal collected atthe tissue surface. Monte Carlo simulationmodels were therefore developed, with particu-lar reference to the neoplastic lesions in mul-tilayered epithelia, to investigate and validatethe dependence of the overall AF signal collect-ed at the tissue surface on the contribution ofthe typical spectra of their biological compo-nents, their histological arrangement, localiza-tion depth and optical properties.71-76 In thecolon, for example, the structural alterationinduced by the rising of the tumor mass affect-ing the presence and localization depth of sub-mucosa was demonstrated as the main respon-sible for the marked decrease of the whole AFsignal in neoplastic with respect to non-neo-plastic tissue (Figure 3).77-79 These findingsare explained considering the essential roleplayed by submucosa, the tissue layer with astrong fluorescence emission because of thepresence of collagen, and its involvement in AFexcitation/emission depending on its localiza-tion depth: submucosa AF signal accounts for agreat part of the whole AF emission detected atthe tissue surface in normal mucosa, while it

disappears following the submucosa derange-ment and loss induced by the rising of neopla-sia. Monte Carlo simulation modeling proce-dures were also applied in non-epithelial tis-sues, as in the case of AF imaging of NADHand flavoproteins to map brain activity in cor-tical areas.80

Considering the differences in the spectralprofiles of the endogenous fluorophores rele-vant for each kind of tissue, spectroscopy,spectrofluorometry and microspectrofluorome-try provided a powerful support to imaginginvestigations.81 In this view, following theassessment of typical excitation-emissionspectra of single endogenous fluorophores,excitation-emission matrices were defined, inthe attempt to better select the measurementconditions and improve data interpretation intissue spectroscopy.82 Analytical proceduressuch as spectral curve fitting or differential

imaging were developed to detect the natureand estimate the relative amount of the fluo-rophores contributing to the whole AF emis-sion of a cell or a tissue. An essential conditionfor the application of these procedures is obvi-ously the definition of the spectral parametersfor each single fluorophore considered in thebiological substrate under investigation.Spectral fitting analysis is thus based on theuse of functions representing typical emissionprofiles, as represented in Figure 4.83,84 It isworthwhile reminding that in the steady-stateanalysis of AF, the ability of the near-UV exci-tation to excite simultaneously the great partof endogenous fluorophores can be an accept-able compromise between a relatively simpleand cost effective instrumentation for bothspectroscopy and imaging studies, and theadvantage to collect comprehensive data onthe different fluorophores in the substrate

Review

Figure 4. Example of fitting analysis of an autofluorescence spectrum collected via fiberoptic probe from a rat liver under living conditions. Real measured spectrum and calcu-lated curve as the sum of the endogenous fluorophore contributions are shown, alongwith spectral functions representing each endogenous fluorophore. Before analysis, spec-tra are normalized to 100% at the peak maximum. Curve-fitting procedure is then per-formed to evaluate the relative contributions of each fluorophore to the overall emission.The procedure consists in an iterative non-linear analysis, based on the Marquardt–Levenberg algorithm through the finding of the true absolute minimum value of the sumof squared deviations of a combination of GMG (half-Gaussian Modified Gaussian) spec-tral functions, each of them representing the emission profile of a pure fluorophore. Thegoodness of fitting is verified trough the residual analysis and r2 coefficient of determi-nation, 0.898 in this case. Fitting parameters are reported as peak center wavelength ( )/ full width at half intensity maximum (FWHM): NAD(P)H free, 463 nm / 115 nm;NAD(P)H bound, 444 nm / 105 nm; vitamin A, 488 nm /102 nm; fatty acids, 470 nm /90 nm; flavins, 526 nm / 81 nm. Due to parameter variability because of heterogeneityin composition and fluorescing properties, the functions of lipopigments and proteinsare left free to adjust, respectively in the 530-600 nm range, and at <440 nm, to reachthe goodness of fitting analysis results. Excitation: 366 nm.



under investigation. The data collection from asingle measuring set can minimize or avoidthe artifacts which may likely derive from pho-tobleacing in sequential measurements of thesame area when changing excitation/emissionconditions, or from the sampling variabilityamong different measured sites when investi-gating metabolic equilibria. This could be thecase of NAD(P)H and flavins when studyingenergetic metabolism,34-44,53-56,80,84 or metabo-lism of biomolecules involved in altered lipidturnover and oxidative stress in fatty livers.85-87

NAD(P)H, flavins and energetic metabolism

Starting from the already reminded pioneer-ing works by Britton Chance,34-39 NAD(P)H andflavins are up to now the most extensivelyinvestigated endogenous fluorophores, finding

unceasing applications in biomedical researchand clinics as dynamic biomarkers of energeticmetabolism in single cells and in the monitor-ing of organ vitality and function. The proper-ties of NAD(P)H and flavins are brieflyreminded, considering their great role in AFbased Optical biopsy. The NAD(P)H is fluores-cent in the reduced state and flavins in the oxi-dized state, the AF emission properties beingstrictly dependent on the bound/freecondition.88-90 These coenzymes are the mainresponsible for the AF rising from cell cyto-plasm, their amount, and redox, bound-freestate being in close relationship with theirengagement in energetic metabolism, celloxidative defense, reductive biosynthesis, andsignal transduction.37,38,89,91-94

The flavin free derivatives, flavin mononu-cleotide (FMN) and flavin-adenin dinucleotide(FAD), exhibit very similar excitation/emis-sion properties in aqueous solution (pH 7),their absorption/excitation and emission max-ima being respectively at about 440-450 nm

and 525 nm.95,96 The fluorescence emissionfrom FAD, in turn, was reported to be stronglyaffected by the nature of the protein to whichthe prosthetic group is bound. Studies on iso-lated mitochondria indicated that, as to theflavoproteins engaged in energetic metabo-lism, -lipoamide dehydrogenase (FP5) andelectron transfer flavoprotein were the majorresponsible for the flavoprotein fluorescencesignal in the cell, contributing respectively forabout 50% and 25% of the total flavin signal.The remaining part was ascribed to the notbetter defined, dithionite reducible flavopro-teins. The emission of FP5 was found to occurin the 510-550 nm region, while that of FP3showed a maximum at about 480 nm.90,91

The fluorescence properties of NAD(P)Hdepend on the nicotinamide group, absorbingat about 365 nm and fluorescing in the 420-490nm region. The AF spectral shape does notshow appreciable differences between the twoderivatives NADH and NADPH, in general thebinding to enzyme molecules resulting in a

Review

Figure 5. Simplified scheme of the main metabolic pathways affecting the redox state of NAD(P)H and flavins, the coenzymes partici-pating to the reactions as reductive equivalent donor /acceptors. The star frames indicate the fluorescing reduced form of NAD(P)Hand oxidized form of flavins.



blue shift, an increase in the quantum yieldefficiency (the ratio between free and boundforms being of about 0.33), and a lengtheningof the fluorescence decay time.40,56,88,89 WhileNADPH is engaged as an electron donor forreductive biosynthesis and antioxidantdefense, NADH is mainly involved in reactionsleading to energy production. Glycolysis andthe citric acid Kreb’s cycle, taking place respec-tively in the cytoplasm or in the mitochondrialmatrix, produce the reduced form of NADH,while NADH reoxidation occurs mainlythrough oxidative phosphorylation, resultingin ATP and heat production. The NADH insidemitochondria cannot diffuse across theirmembrane, the transport of the reducingequivalents from the cytosol to mitochondriabeing carried out by shuttle systems, ensuringan equilibrium between cytoplasm and mito-chondria. Transhydrogenases, in turn, areenzymes able to transfer electrons fromNAD(P)H to NAD(P)+ in a reversible reaction,contributing to the cell resources to maintainredox homeostasis. A simplified representa-tion of the several interrelated metabolic path-ways involving changes in the redox state ofNAD(P)H and flavins is shown in Figure 5.Such complex situation can explain apparentlyconflicting results on the ability to inducedchanges in the redox state of NAD(P)H andflavins by drugs acting on mitochondria. Forinstance, blocking the respiratory chain bymeans of potassium cyanide is expected toproduce an increase in the NAD(P)H fluores-cence intensity and, conversely, a decrease inflavin emission. Yet, evidences inconsistentwith this assumption were reported,41 leadingto define bistable the reciprocal changes in theNAD(P)H and flavin reciprocal redox state, independence of the metabolic steady state ofthe cells undergoing metabolic alterations.89,97

Despite this complexity, these coenzymes weresuccessfully used as intrinsic biomarkers ofcell energetic metabolism, consistently withthe proposal of Huang,54 to represent the redox– fluorescent state of the cells by means of avery simplified equation:

FADH2(FP5) + NAD+ = FAD(FP5) + NADH + H+

where FP5 belongs to the dehydrogenase mul-tienzyme complex catalyzing the electrontransfer from pyruvate to NAD+, as a point ofelectron entry in the respiratory chain. In addi-tion to the huge amount of studies in singlecells, NAD(P)H and flavins were investigatedas dynamic biomarker of energetic metabolismin tissues and organs.

Studies on pancreas, demonstrated the AFsuitability to monitor the response to anoxia inthe assessment of progressive damage afterpancreatitis or transplantation.98 The particu-

Review

Figure 6. Unfixed cryostatic brain tissue sections from a glioblastoma bearing patient. a)Hematoxylin and Eosin staining evidences an altered stainability and cell density in neo-plastic (glioblastoma, gbl) tissue as compared to the surrounding non-neoplastic tissue(nnt). b) Autofluorescence imaging of a serial, unstained tissue section, shows a loweremission signal in neoplastic as compared to the non-neoplastic area; a quantitative rep-resentation of this difference is given by the amplitude distribution profile of the selectedarea indicated by the frame overimposed to the picture. Excitation: 366 nm; emission:420-640 nm; scale bars: 200 µm. c) Spectrofluorometry performed in vivo, via fiber opticprobe during surgical operation showed an even more marked lower autofluorescenceamplitude, and spectral profile changes in neoplastic tissue in comparison with the non-neoplastic tissues (white matter and cortex); spectra are shown as real values.



lar metabolic engagement of pancreatic isletcells and their implications in the developmentof diabetes inspired NAD(P)H and flavin basedAF studies aimed both at improving knowledgeon functional mechanisms and at facilitatingthe flow cytometric sorting and enrichment ofpure beta cell populations for transplantation.99-101 More generally, several experimental stud-ies on different organs such as heart, kidneyand liver were stimulated in order both toattain an effective real-time functional moni-toring and to support investigations on strate-gies aimed at assessing and facing possibleinjuries from ischemic conditions during sur-gery or transplantation.102-107

An in vivo multiparametric assessment oftissue and organ dysfunctions under patholog-ical states was also proposed, based on themeasurement of whole NAD(P)H AF emission,together with oxyhemoglobin (Hb-O2) andblood hemodynamics.42 In addition, it is toremind that the spectrofluorometric, real-timemonitoring of the tissue redox state allowed todetect and explain transient events, such as atemporary NAD(P)H oxidation accompanied byan increase in the NAD(P)Hbound/free ratioobserved soon after the induction of liverischemia. This phenomenon, opposite to anexpected increase in the NAD(P)H reducedstate after the block of its reoxidation throughthe respiratory chain in the absence of oxygen,was explained with a physiological compensa-tory vasodilatation attempt of liver microvascu-lature to respond to ischemia.108 Recently, thein vivo analysis of AF in a rat liver model oflipid accumulation made it possible to assessthe maintenance of energetic metabolismhomeostasis under oxidative stress. This effortwas not detectable ex vivo, while the combinedAF and biochemical analysis of tissue samplesconfirmed the expected alteration in mito-chondrial activity.87 These findings further con-firmed that changes in biological conditionscan affect the cell engagement in aerobic andanaerobic energetic metabolism. In thisrespect, an example is given by recent AF-based studies on the metabolic plasticity ofstem cells. This phenomenon entails the meta-bolic signatures of the stemness status, con-sisting in the switch from anaerobic to aerobicrespiration during differentiation, the oppositeoccurring during the reprogramming of somat-ic cells to pluripotency.109 The consequentchanges in AF emission at to both signalamplitude and spectral shape are mainlyascribable to alterations in the relative pres-ence of bound and free NAD(P)H, as demon-strated by numerous works performed usingdifferent optical systems on single cultured celland living tissues.44,55,110-113 The usefulness ofAF for a comprehensive analysis in the assess-ment of the actual stem cell differentiationdegree has been thus proved, the exclusive

Review

Figure 7. Unfixed cryostatic tissue section from a mouse mammary tumor.Autofluorescence image collected at t = 0 s of excitation-light irradiation from (a), andthe distribution pattern of the signal lost (b), detected through the differential analysisof images recorded from the same tissue area at t = 0 s and t= 10 s of continuous irradi-ation; excitation: 330-385 nm; emission <420 nm. The lost signal representing the mostphotolabile fluorescing species occurring in this tissue is ascribable to porphyrins. Thepresence of PpIX, in particular, is confirmed by the spectra recorded under 405 nm exci-tation from the same tissue area, showing its typical emission bands centered at 630nm and 700 nm. The band at about 670 nm is ascribable to pheophorbide or porphyrinoxidative products. The PpIX band amplitude is consistent with the low diffused signalin the vital mass and the much higher emission in necrosis, identified when the measuredarea has been retrieved after conventional Hematoxylin and Eosin staining (c), for adirect comparison with autofluorescence distribution in tumor living cell mass (vm) andnecrosis (n). Scale bar: 100 µm.



advantage to avoid undesired stimuli fromexogenous-markers being of particular valuewhen the very delicate metabolic equilibria ofstem cells are investigated for regenerativemedicine and zootechnology applications.

Apart from these recent studies, a preva-lence of anaerobic on the aerobic energeticmetabolism is known since a long time tocharacterize tumor cells, as early remarked byWarburg in 1956.114 This condition, likelyfavoring survival and invasiveness of tumorcells, results in general in low values in theNAD(P)Hbound/free ratio in neoplastic cells withrespect to the non-neoplastic ones. These AFresults have been justified by a favored pen-tose-phosphate pathway, reflecting a possibleimpairment of mitochondrial respiratoryactivity and involving fewer steps of interac-tion between the pyridinic coenzymes andenzymes as compared to the aerobic metabo-lism.89,115-118 This suggestion confirmed thestrict relationship between the AF propertiesof cells and their actual metabolic engage-ment, consistently with the finding that theNAD(P) Hbound/free ratio may vary among can-cerous cell lines, while remaining constant ina cell line under standard metabolic condi-tions.119

In solid tumors, the cell switch from theaerobic to the anaerobic energetic metabo-lism can contribute at a different extent tothe whole AF signal alteration, depending onthe tissue considered. In general, the effectsof energetic metabolism are of minor diag-nostic importance than tissue architecture inmultilayered epithelia. In fact, as alreadydescribed above, the growing of the neoplas-tic mass affects the integrity and localizationdepth of the highly fluorescing submucosa,resulting in a marked decrease of its contri-bution to the whole AF signal collected at thetissue surface.66,70,71,74,75,77-79 On the otherhand, in the central nervous system the sub-version of the energetic metabolism wasdemonstrated to contribute to the discrimina-tion between brain tumor mass (in glioblas-toma) and the non-neoplastic surroundingtissue.120,121 A role of the metabolic engage-ment was demonstrated by data from tissuesections, showing an alteration in theNAD(P)Hbound/free ratio accounting for both thedecrease in amplitude emission and the redshift of AF spectra in the tumor with respectto the normal tissue. In vivo measurementsperformed via fiber optic probe during sur-gery, in turn, showed much higher differ-ences between the neoplastic and non-neo-plastic tissue (Figure 6). These findings indi-cated also a role of the tissue optical proper-ties, since changes in blood supply and inarchitectural organization, leading to amarkedly increased cellularity in the tumormass, can alter absorption, reflectance and

the scatter phenomena, influencing themigration of light, and the yield of excitationand fluorescence collection from different tis-sue depths.9, 122 The dependence of AF signalson both structural and physiological proper-ties of brain areas, and their sensitivity tohemodynamic changes induced by sensoryactivation were further demonstrated byimaging studies of NADH and flavoproteins.80

More generally, the importance of the cellmetabolic engagement combined with tissueorganization and cellularity was supportedby different cases, concerning for exampleleukocytes becoming resident and accumu-lating in tissues with inflammation, orlymph node alterations in lymphoprolifera-tive disorders affecting AF signals;65 similar-ly, it was possible to discriminate neoplasticdegeneration or to detect in situ heart allo-graft rejection.123,124

Lipofuscins and lipofuscin-likefluorophores in aging, oxidative damage, and more

In addition to the diffuse fluorescence ofNAD(P)H and flavins, brightly emitting parti-cles ascribable to lipofuscin or lipofuscin-likelipopigments can be occasionally observed inthe cell cytoplasm.

Lipofuscin fluorescence covers the yellow-reddish region, the spectral shape and emis-sion amplitude being dependent on the vari-ability in composition (proteins, lipids,carotenoids), crosslinks and oxidation degreeof these heterogeneous compounds, and age-ing.125 In general, lipofuscins consist in undi-gested material remaining from phagocytosisand autophagy processes, accumulating as

Review

Figure 8. Normal (a,b) and fatty (c,d) liver from rat models. Autofluorescence imagescollected at t = 0 s of excitation light irradiation from unfixed, unstained cryostatic tissuesections (a,c), and topographical distribution of the whole signal lost (b,d), obtainedthrough the differential analysis of images recorded from the same tissue area at t = 0 sand t= 10 s of continuous irradiation. In the normal liver a signal loss occurs mainlyalong sinusoids, likely involving vitamin A accumulated in Ito cells. Fatty liver showsmarked signal decrease within vesicular structures likely corresponding to lipid droplets,while the bright light blue fluorescing connective component (*) observed at t = 0 s ofirradiation undergoes a much lesser decrease. Image levels are adjusted to optimize imageobservation. Excitation: 366 nm. Scale bars: a), 80 µm; c), 100 µm.



intracytoplasmic granules depending on physi-ological cell metabolic engagement, and theoccurrence of disorders and pathologies.115,126-130 The physiological intracellular accumulationof lipofuscins in ageing is commonly reportedin the liver (see below), or in central nervoussystem. In the latter case, an anomalousincrease in the presence of lipofuscins or lipo-fuscin-like lipopigments or ceroids has beendetected as a consequence of oxidative stress,as a response to pathological conditions (i.e.,the neuronal ceroid lipofuscinosis or Battendisease), or to toxic compounds.131-133 In thisconcern, the alteration of the postnatal devel-opment of the multilayered rat cerebellar cor-tex induced by cisplatin administration result-ed in an increase in the lipofuscin emissionsignal and alterations in NAD(P)H and flavincontributions.132 Actually, cerebellum duringdevelopment is known to be particularly vul-nerable to toxic compounds and radiation, andthese findings in response to the cisplatinoxidative damage confirmed that AF-basedstudies are especially appropriate to investi-gate the oxidative effects of chemical andphysical agent.134

The presence of lipofuscins is inversely cor-related with the differentiation degree in stemcells.44,55,113 This phenomenon has beenascribed to autophagic processes activelyengaged in the removal and oxidation of unde-sired subcellular structures, with particularreference to mitochondria. Regulating thenumber of mitochondria would preserve theprevailing anaerobic energetic metabolism ofstem cells, thus contributing to the stemnessmaintenance.135 The accumulation of lipofus-cin in stem cells should therefore be ascribableto the action of reactive oxygen species onautophagocytosed mitochondria, rather than tothe physiological ageing processes.

A particular case of brightly fluorescingintracellular granules in eosinophils wasreported by Grossi and Zaccheo136 already in1962. This AF emission was later ascribed atleast in part to FAD, and found to undergo afurther increase in amplitude and shift fromthe blue to the yellow region when the circulat-ing cells become resident in the tissues.61,137,138

The relatively high AF emission of eosinophilswas more recently exploited to isolate themuch less fluorescent neutrophils by flow cyto-metric cell sorting, to generate a nearly purequiescent cell population in an antibody-freeenvironment, an essential condition for subse-quent activation studies.139 Another particularcase of remarkable intracellular AF emittinggranules concerned the pigment cell compo-nents in the liver of some amphibians, likelyinvolving melanin in addition to lipofuscin-likeproducts. Alterations in both emission ampli-tude and spectral profile reflected variations inthe composition of these pigment constituents

Review

Figure 9. Autofluorescence emission spectra recorded at t = 0 and at t = 10 s of irradiationfrom unfixed, unstained cryostatic sections of rat livers: connective tissue of a portal area(a) and lipofuscin granules (b) from a normal liver parenchima, and lipid droplet (c)from a fatty liver. The changes induced in the whole emission profile by light irradiationare evidenced by the measured spectra normalized to the peak maximum. The responseto irradiation in terms of spectral shape and amplitude changes in the AF emission fromcollagen, lipofuscin-like lipopigments and vitamin A/fluorescing lipids is represented bythe curves of each single fluorophore spectral function. Excitation: 366 nm.



in relation with summer activity and winterhibernation cycles, suggesting an engagementto compensate changes in oxidative conditionsdepending on the seasonal liver plasticity.140,141

A strong yellow fluorescence rising after UVirradiation was also demonstrated to beinduced by peroxidation of melanins naturallypresent in different tissues, improving the pos-sibility to distinguish them from lipopig -ments,142 while studies on synthetic melaninsdemonstrated that the emission signaldepends on modifications in the compositionand polymerization degree of indolic compo-nents.143 These findings opened wide-rangingperspectives for a further development ofoptic-based diagnostic clinical applications, forexample in clinical dermatology as well as inthe environment monitoring through sentinelwild animals.

Finally, the importance of lipofuscins andretinoid derivatives accumulating in retinal-pigment-epithelial (RPE) cells as residues ofundigested outer segments of photoreceptorsis here only reminded, as compared with thehuge amount of work carried on about their AFcharacterization as to etiology, diagnosis andmonitoring of retinal diseases.144-146

Porphyrins and heme metabo-lism alteration

In general, the emission spectra from theabove described fluorophores cover the wholevisible range, the contributions in the redregion consisting in the low, longer wave-length tails of the emission spectra. However,distinct and well defined emission bandsbeyond 600 nm can be detected, that can beascribed to porphyrins and their derivatives.147

The porphyrin chemical structure is basedon a tetrapyrrolic ring, the most frequently nat-urally recurrent protoporphyrin IX (PpIX)being the ultimate precursor before iron inser-tion along the biosynthesis pathways of theheme group. This latter is an ubiquitous mole-cule, involved as a prosthetic group in proteinsand enzymes providing to functions rangingfrom transport of oxygen to catalysis and pig-mentation. Heme group is produced followinga series of reactions, and can induce a feed-back-inhibition of the key enzymes involved: -aminolevulinate synthase, -aminolevulinatedehydrase and ferrochelatase.

The red emission from iron-free, heme-based compounds was among the early report-ed observations of natural fluorescence.2 In1924, the occurrence of red fluorescence underultraviolet light illumination was described inan experimental rat sarcoma and correctlyattributed to the presence of endogenous por-phyrins.27 In 1929 the detection of porphyrin

fluorescence spectra in the tissues from apatient who died for congenital porphyria wasone of the first examples of AF microspectro-fluorometric assay.30 It cannot be neglectedthat the current PDD and PDT procedureswere initiated by these observations, followedby the early experiences on the first hemato-porphyrin derivatives, obtained from the bloodas iron free products to be administered asexogenous compounds.148 These derivativesshowed a preferential accumulation in tumortissues, where they were able to activate pho-tosensitizing phenomena under suitable lightirradiation.149-151 Following the first indicationsby Samuel Schwartz on the heterogeneouscomposition of porphyrin derivatives, mixturesenriched in their aggregated species which aremostly responsible for the selective localiza-tion in target tissues (e.g., Photofrin) or spe-cific chlorine derivatives (e.g., Foscan andVerteroporphyrin) were produced to improvePDD and PDT efficacy, resulting in prepara-tions approved for the clinical use.31,147,152-156

The complex chemical composition of por-phyrin preparations make their use in PDD andPDT difficult; in addition, porphyrins proved tobe less selective than expected for tumor cells,while exhibiting low tissue depth photoactiva-tion and inducing prolonged skin photosensitiv-ity. As a consequence, several other photosensi-tizing molecules were proposed. Few of them,however, received market approval, while suc-cessful parallel research demonstrated the use-fulness of -aminolevulinic acid (ALA) or itschemically modified derivatives, as pro-drugprecursors of PpIX.156-159

Although a contribution from bacteria couldnot be completely excluded, an increase in por-phobilinogen deaminase and a decrease of fer-rochelatase activities participating to hemesynthesis in tumor cells explained the selec-tive accumulation of PpIX in the neoplasticmass following the administration of ALA pre-cursors.160 The same enzymes are likelyresponsible for the systemic accumulation ofendogenous porphyrins in the absence of pro-drug administration in subjects bearingtumors or metabolic disorders. While theincreased presence of porphyrins in tumor tis-sue contributes to the above mentioned imag-ing ratio analysis for in situ detection oftumors,70 the increase of PpIX in the blood hasbeen proposed to support the differential diag-nosis of hidden tumors or altered metabo-lism.161-165 In this respect, AF imaging andmicrospectrofluorometric studies on a MMTV-neu (erbB-2) female C1 mouse, model of spon-taneously growing mammary tumor,166 showeda systemic marked increase of PpIX and por-phyrin derivatives along with their rising inthe neoplasia, in particular in the necroticmass, (Figure 7).167-169 The rising in PpIX fluo-rescence in the serum, however, could not be

completely explained by a reversed activity offerrochelatase removing iron from heme indeath tissue,168 and by the blood porphyrindrainage from the tumor. In fact, the ironscarcity and porphyrin increase observed inparticular in the spleen (the organ normallyengaged in heme catabolism and iron recy-cling) supported the role of a systemic subver-sion of metabolism in tumor bearing subjectsinduced by the presence of tumor cells requir-ing this element to face their metabolicrequirements.169,170

Miscellanea of endogenous fluorophores in liver disorders,and more

In mammalian livers, lipofuscins resultingfrom the ageing processes are of no real patho-logical importance, while already at the begin-ning of the 1940’s a ceroid material with fluo-rescence properties was observed to accumu-late in experimental cirrhotic rat livers, andascribed to degradation products of unsaturat-ed fatty acids.2,171,172

The nature of the lipofuscin-like lipopig-ments as peroxidized products of lipids andother intracellular macromolecules was sup-ported by later investigations, even leading totheir proposal as AF biomarkers of oxidativedamages induced in livers with lipid accumula-tion and oxidative stress.87,127,173,174 Actually, anincreasing number of endogenous fluo-rophores has been considered for comprehen-sive AF based diagnostic applications in hepa-tology, depending on the complex biochemicalcomposition characterizing the liver tissuedue to its engagement in several metabolicand detoxificating functions.84-87,175 In additionto NAD(P)H, flavins and lipofuscins, the over-all liver tissue AF emission can originate fromproteins, vitamin A and fatty acids likely play-ing a role as interconnected autofluorescentbiomarkers of liver disorders. Under normalconditions, vitamin A accumulates mainly instellate cells, the protein emission risingmainly from cytoskeletal and fibrous compo-nents, namely cytokeratins and collagen. Thislatter, in particular, is present in connectivesepta, i.e. the portal space, and in the delicateconnective matrix of perisinusoidal reticularfibers.23 The collagen AF contribution increas-es when stellate cells are activated by inflam-mation messages, becoming fibrogenic andlosing their role as vitamin A deposits duringdevelopment of liver fibrosis,46 the emissionamplitude and spectral shape being stronglydependent on the type, network density andageing of this extracellular protein.176,177 Theability of AF analysis to detect such alterations

Review



in tissue organization was demonstrated bymeans of different approaches, such as multi-photon analysis and intravital microscopy inrodent models of developing cirrhosis or toxin-induced fibrosis,178,179 and the role of collagenas early autofluorescent biomarker of develop-ing fibrosis was further supported by ex vivoAF characterization of human liverspecimens.180

Vitamin A was also proposed as an autofluo-rescent biomarker directly related to the con-tent of lipids within hepatocytes; this was con-sistent with previous findings of a direct rela-tionship between the changes in the amountand distribution of the vitamin A emission sig-nal and lipids, as demonstrated by in situ Nilered fluorochromization performed on the sametissue section after AF analysis.175,180

The indications on the presence of vitaminA and fatty acids were obtained exploiting thedifferent sensitivity to the light irradiationexhibited by the various endogenous fluo-rophores. Actually, photolability can be a fur-ther parameter improving the reliability of theinformation provided by both imaging andspectrofluorometry. In the normal liverparenchyma, continuous irradiation results inthe decrease of the blue emission from thecytoplasm of hepatocytes, mainly due to thephotobleacing of NAD(P)H, their anastomoz-ing plates being bordered by a more photore-sistant bright network ascribable to reticularfibers. In fatty livers the strong photolability ofvitamin A and fluorescing fatty acids helped todemonstrate their localization in lipid vesicles,highlighted by means of differential imagingand microspectrofluorometric analysis, simi-larly to the procedure already described for por-phyrins (Figures 8 and 9).

Autofluorescence properties can be exhibit-ed also by some lipid compounds, dependingon their molecular properties. Up to now, fewindications have been provided on AF emissionproperties and high photolability of some fattyacids such as arachidonic, linoleic and stearicacid, while other derivatives such as butirricand palmitic acid, triglycerids and phospho-lipids are in general poorly fluorescent.84,86,87,175

This apparent limitation in the AF detection oflipids, however, may give indications oninduced alterations in the fluorescent fattyacid pool. In fact, despite the complex liverlipidic composition and its dependence on sev-eral metabolic pathways, a rising in fluoresc-ing fatty acids was indicated by spectral fittinganalysis of AF signals recorded from livers sub-mitted to experimental warm ischemia. Thephenomenon was suggested to reflect analtered balance of acylation and deacylationrates, rather than of fatty acid -oxidation, theultimate steps of this energy production path-way requiring oxygen.86,181,182

As to diagnostic potential of AF in hepatol-

ogy, promising advancements concern also bilecomposition and its dependence on liver func-tionality. Bilirubin is excreted in the bile as thebreakdown product of systemic and intrahepat-ic heme degradation. Bile optical propertieswere first mainly investigated to set-up assaysfor its evaluation in the blood, with the aim toimprove diagnostic and therapeutic proce-dures to face hyperbilirubinemia.183-185 Morerecently the bichromophore nature of bilirubinhas been found to influence its AF amplitudeand spectral profile, characterized by two mainemission bands centered at about 517-523 nmand 570 nm. The phenomenon is due to anintramolecular energy transfer consequent toan exciton splitting phenomenon between thetwo chromophore groups composing the biliru-bin molecule, consisting in two non-completelysymmetric moieties each of them containingtwo pyrrole rings. The alterations in the mole-cule conformation depending on microenvi-ronment will thus affect the yield of bilirubinintramolecular energy transfer, reflected bydifferent extents of changes in its emissionproperties. The phenomenon, particularlyfavored by the near-UV excitation, is thus mak-ing bilirubin along with other bile components(proteins and biliary acids, fluorescing in theblue region) to act as sensitive intrinsic bio-markers of bile composition, and consequentlyof liver function.85,86

Being aware of the extensive applicationsbased on several endogenous fluorophores asintrinsic diagnostic biomarkers in biomedi-cine, only few more examples are brieflyreported. As to the extracellular structural pro-teins, apart the numerous studies on the diag-nostic importance of the collagen-rich submu-cosa affecting the whole AF signal recorded atthe surface of multilayered epithelial tis-sues,70,74,75,77-79,186 early spectroscopic character-ization of laser induced emission signals fromatherosclerotic aorta were followed by thedevelopment of multiphoton analytical proce-dures for elastin and collagen in situ analy-sis.187-190 In the case of collagen, as well as ofother supramolecular periodically organizedfibrous proteins with a non-centrosymmetricmolecular structure, it is also to underline thatmultiphoton analysis offers the possibility todetect the Second Harmonic Generation(SHG), that is the light emission at half theincident laser wavelength.190,191 These studiespaved the way to renewed histological analysisfor diagnostic applications through in situthree-dimensional visualization in intact tis-sues.192-194 It is worth mentioning also the pos-sibility to detect the accumulation of advancedglycation end-products in the skin, given bysimple and non-invasive spectroscopy as a sup-portive method in the diagnosis and control ofaging and of the glycemic complications in dia-betes.195,196

At last, the UV absorption properties ofmonoamine-aromatic neuromediators stimu-lated the development of different and sophis-ticate strategies exploiting their AF propertiesfor their detection and monitoring in livingcells and in the central nervous system.197,198

Three-photon excitation microscopy, for exam-ple, allowed to measure and monitor the distri-bution and content of neurotransmitter gran-ules within viable cells, providing a support tomore conventional extracellular secretionassays, e.g. carbon-fiber amperometry.199,200 Inparticular, the possibility provided by AF analy-sis to differentiated sequestered from unse-questred serotonin indicated an otherwisehardly detectable non-linear relationshipbetween the two fractions, a supplementaryfactor of particular value in differentiationand drug response studies of serotoninergiccells.201 In this respect, the AF spectroscopypotential for a direct monitoring of neurotrans-mitter was supported also by data obtained invivo, through a optic fiber probe positioned viastereotaxis, from the hippocampus region ofrats submitted to behavioral or pharmacologi-cal treatments affecting serotonin levels.202,203

Concluding remarks and futureperspectives

Only some selected topics deserving interestin the biomedicine field have been consideredin this review. This limited description, howev-er, may sufficiently illustrate the long-timeenduring and wide-ranging attention of thescientific community to AF of biological sub-strates. Endogenous fluorophores and theirrole as intrinsic biomarkers offer an excep-tionally powerful tool to characterize in realtime even subtle changes of interconnectedmorphological and metabolic properties ofcells and tissues under physiological or alteredconditions. In this concern, a very recentexample of a powerful AF application hasshown the possibility provided by two-photonexcitation for noninvasive, automated andquantitative assessment of mitochondrialorganization in intact, engineered epithelialtissues. Differences in the three-dimensionalmitochondrial organization reflect alterationsin metabolic activities and allow to discrimi-nate normal and precancerous conditions,playing a role as hopeful early cancer diagnos-tic tool.204

In the future, the interest in the AF phenom-enon will certainly continue. Current instru-mentation and analytical procedures will likelybe overcome by the unceasing progress in newdevices for AF detection and data interpretation;nevertheless, a progress is expected in thesearch, identification, photophysical characteri-

Review



zation and biological meaning of additional bio-molecules present in cells and tissues andexploitable as AF intrinsic biomarkers.

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EJH 2014 04.qxp Hrev master 30/12/14 14:41 Pagina 320 ... · E-mail: [email protected] Key words: Endogenous-fluorophores, energetic-metabolism, functionality-monitoring. Conflict