516 IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 16, NO. 3, MAY/JUNE 2010

Multiphoton Microscopy of Live Tissues WithUltraviolet Autofluorescence

Chunqiang Li, Costas Pitsillides, Judith M. Runnels, Daniel Cote, and Charles P. Lin

(Invited Paper)

Abstract—Current research on multiphoton autofluorescencemicroscopy is primarily focused on imaging the signal from re-duced nicotinamide adenine dinucleatide (NADH) in tissue. NADHlevels in cells are useful reporters of metabolic information, as wellas early indicators in precancer and cancer diagnosis. While NADHis typically imaged in the 400–500 nm spectral window, the aminoacid tryptophan is the major source of tissue fluorescence in theUltraviolet range. Here, we briefly review current progress in multi-photon autofluorescence imaging of live tissues and cells, and reportour recent findings of in vivo mouse skin imaging based on mul-tiphoton excited tryptophan autofluorescence. This new methodenables noninvasive imaging of skin tissue at video-rate and al-lows for the visualization and identification of cellular componentsin the epidermis, dermis, and muscle layers. It is also possible toimage through small blood vessels in the mouse skin and observecirculating leukocytes in situ.

Index Terms—In vivo imaging, multiphoton-excited fluores-cence microscopy (MPM), tryptophan autofluorescence, video-rateimaging.

I. INTRODUCTION

F LUORESCENCE microscopy is the major optical imagingmodality in biomedical research. Multiphoton-excited flu-

orescence microscopy (MPM), which uses pulsed near infrared(NIR) lasers to excite fluorophores, has revolutionized the tradi-tional single-photon excitation method [1]. This nonlinear opti-cal excitation approach provides deeper tissue penetration, andresults in less photobleaching and photodamage [2]. There havebeen successful applications of in vivo fluorescence microscopyin multiple areas of medical research, such as neuroscience,immunology, and cancer research [2]–[4]. Most of these appli-

Manuscript received July 15, 2009; revised August 17, 2009; acceptedAugust 22, 2009. Date of publication October 6, 2009; date of current ver-sion June 4, 2010. This work was supported in part by the National Institutes ofHealth under Grant CA111519-01, and in part by Johnson and Johnson, Inc.

C. Li and C. P. Lin are with the Advanced Microscopy Program, Well-man Center for Photomedicine, Harvard Medical School, Massachusetts Gen-eral Hospital, Boston, MA 02114 USA, and also with the Center for SystemsBiology, Massachusetts General Hospital, Boston, MA 02114 USA (e-mail:li.chunqiang@mgh.harvard.edu; lin@helix.mgh.harvard.edu).

C. Pitsillides is with the Advanced Microscopy Program, Wellman Center forPhotomedicine, Harvard Medical School, Boston, MA 02114 USA. He is alsowith the Department of Biomedical Engineering, Boston University, Boston,MA 02215 USA (e-mail: pitsillides@helix.mgh.harvard.edu).

J. M. Runnels is with the Advanced Microscopy Program, Wellman Centerfor Photomedicine, Harvard Medical School, Boston, MA 02114 USA. She isalso with the Dana-Farber Cancer Institute, Harvard Medical School, Boston,MA 02115 USA (e-mail: runnels.judith@mgh.harvard.edu).

D. Cote is with the Centre de Recherche Universite Laval Robert-Giffard,Departement de Physique, Universite Laval, Quebec, QC G1J 2G3, Canada. Heis also with the Wellman Center for Photomedicine, Boston, MA 02114 USA(e-mail: daniel.cote@crulrg.ulaval.ca).

Color versions of one or more of the figures in this paper are available onlineat http://ieeexplore.ieee.org.

Digital Object Identifier 10.1109/JSTQE.2009.2031619

cations require the aid of exogenous fluorophores as contrastagents, such as membrane or cytoplasmic dyes, and fluorescentproteins. However, there are existing limitations to the use of allthe above agents [5]. Cells often need to be labeled with dyes invitro before being injected into animals, dyes can diffuse out ofcells over time and their concentration can decrease with suc-cessive cell divisions. It is often difficult to create geneticallyengineered animal models to express fluorescent proteins in thetargeted cells or tissues.

The major intracellular endogenous fluorophores are reducednicotinamide adenine dinucleatide (NADH) and dinucleatidephosphate (NADPH), riboflavins, and tryptophan [6]. The pyri-dolamine crosslinks in elastin and collagen are the primary ex-tracellular fluorophores. The indoleamine derivatives of tryp-tophan, e.g., serotonin (5-hydroxytryptamine, or 5-HT), havesimilar excitation and emission spectra as tryptophan [7]. Sero-tonin is a neurotransmitter that has various functions, includingcontrol of appetite, mood, and anger [8]. In skin epidermis, thecellular skeleton protein keratin, also emit fluorescence in thevisible spectral region [9]. Recently, it has been reported thatmelanin exhibits fluorescent emission in both visible and NIRregion [10], [11].

All these endogenous fluorophores have much smaller multi-photon excitation cross sections than exogenous ones [6], whichlimits their applications. Therefore, utilizing tissue autofluores-cence for in vivo imaging is still in its early stage. Among thesefluorophores, NADH and flavin adenine dinucleotide (FAD, re-duced form FADH2) have been given the most attention due totheir roles as metabolic enzymes cofactors [12]. NADH and FADimaging has been used to observe changes in cellular metabolicstatus and tissue morphological features.

In tissue, the ultraviolet (UV) autofluorescence is dominatedby tryptophan as the other two UV fluorescent amino acids, tyro-sine and phenylalanine, are often quenched by tryptophan [13].Tryptophan has a one-photon absorption peak at 280 nm, and itsemission spectrum is between 300 and 400 nm [14]. The averagecontent of tryptophan in about 2000 proteins is 1.4% [15]. There-fore, imaging tryptophan fluorescence in vivo can provide a newmodality to visualize cellular and subcellular protein contents.Using multiphoton excitation on tryptophan has the advantagesof avoiding UV excitation and reducing tissue scattering. Bothtwo-photon and three-photon excitation spectra of tryptophanhave been measured [7], [16]. There have been reports of mul-tiphoton microscopy imaging with tryptophan fluorescence onproteins, cells, and muscle tissues ex vivo [17]–[20], but to ourknowledge no in vivo imaging using tryptophan fluorescencehas been demonstrated yet.

1077-260X/$26.00 © 2009 IEEE

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TABLE IENDOGENOUS FLUOPHORES AND SPECTRA

In Section II, we briefly review the current status of tissueautofluorescence imaging. In Sections III and IV, we presentour video-rate in vivo imaging approach and results with bothtryptophan and NADH autofluorescence. Second harmonic gen-eration (SHG) microscopy is another type of nonlinear opticalmicroscopy in which the signal comes from coherent scatteringby noncentrosymmetric molecules [21]. It has been used to im-age collagen and muscle cells and is often integrated togetherwith MPM to provide information on tissue structure. Although,we do not review works on SHG microscopy, we will cite thereports related to tissue autofluorescence.

Imaging at high speed (30 frames/s) is needed to visualizecell trafficking events such as leukocyte rolling on vascularendothelial walls. At the same time, it is extremely helpful tominimize motion artifacts such as heartbeats and breathing eventhough the event under investigation may not require high imag-ing speed. Our group has developed video-rate multimodalitylaser scanning microscopy for in vivo cell tracking [22]. Wehave incorporated different lasers and detectors into this setupto achieve both NADH and tryptophan fluorescence imaging.

II. ENDOGENOUS FLUORESCENCE AND THEIR APPLICATIONS

IN IMAGING

The major endogenous fluorophores and their multiphotonexcitation spectra are listed in Table I. As most current fem-tosecond lasers are based on the titanium-doped sapphire as thegain media, the wavelength tuning range of such lasers is 690–1000 nm. Therefore, there is little data at excitation wavelengthshorter than 690 nm for most fluorophores.

A. NADH and FAD

Metabolism describes a set of chemical processes living cellsuse to harvest energy and synthesize proteins and other macro-molecules. A network of enzymes, proteins, and cofactors are

involved in these chemical reactions and the whole process isregulated by precise feedback mechanisms. Most of the en-ergy in eukaryotic cells is generated in the mitochondria viachemical reactions that convert energy substrates, e.g., glucose,into adenosine triphosphate (ATP). Therefore, examining themitochondrial functions is critical to understand physiologicalconditions. NADH (oxidized form NAD+ ) is a redox coenzymein the metabolic process that carries electron from one reactionto the next. NADH has a one-photon absorption below 380 nmwith emission between 400—and 500 nm. Using fluorescencespectroscopy to monitor NADH concentration, in order to studymitochondrial functions has a long history that started more50 years ago (reviewed in [12]). FAD, derived from riboflavin,is also a redox cofactor and is in equilibrium with NADH.Ratiometric redox fluorometry by measuring both NADH andFAD fluorescence provides a relative measurement of cellularmetabolism and overcomes problems such as tissue scatteringand absorption [27]. Redox ratio is defined as the ratio of thefluorescence intensity of FAD and NADH [28].

Fluorescence imaging of NADH and FAD provide extra mi-croscopic information of cellular metabolic status such as theirdistribution and concentration in cell mitochondria and cyto-plasm. Therefore, MPM with NADH and FAD autofluorescencehave been used to image skin and other epithelial tissues, e.g.,mucosal tissue and bronchus [29]–[31]. The different layers inskin epidermis such as stratum corneum, stratum granulosum,spinosum, and basal layer can be discerned using this method.Within the dermis, scattered cellular structures have been ob-served but the cell types are not readily identifiable [32]. Af-ter UV and visible irradiation, morphological and biochemicalchanges in the skin were assessed by this method with spectralanalysis of fluorescent contributions from NADH, FAD, ker-atin, lipids, lipofuscin, and collagen [33]. It has also been usedto follow the wound-healing process in guinea pig skin [34].NADH and FAD fluorescence has been used to study cellu-lar metabolism, apoptosis, and pyruvate response in pancreaticislet cells [35], [36]. Tissue structure and cellular organizationhave also been extracted from NADH and FAD autofluorescentimages [37], [38].

Cancer cells are thought to have increased cellular metabolicactivity; thus, the most frequent application of NADH and FADfluorescence imaging is in cancer studies. In normal epithelialtissue, more mature cells in the superficial layer of the strati-fied squamous epithelium have significantly higher redox ratiothan less differentiated basal cells, which indicates an increasein metabolic activity in superficial cells. While in precancerousepithelia tissue, the redox ratio is not significantly different be-tween basal cells and superficial cells [27]. Redox ratio in thesurface epithelium of normal–high risk ovarian tissue is highlyvariable. Normal–low risk samples have the lowest redox ratio.In contrast, cancerous tissue has the highest redox ratio [39].Basal cell carcinomas have clumps of autofluorescent cells withlarge nuclei, which can be discriminated from normal dermal tis-sue [40]. Morphological features of human nonmelanoma skincancers (NMSCs) are distinct from the peripheral tissue sur-rounding the lesions [41]. Traditional histopathological criteriafor diagnosing NMSCs, e.g., multinucleated cells, are clearly

518 IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 16, NO. 3, MAY/JUNE 2010

visible in these multiphoton autofluorescence images. The au-thors in this paper used an excitation laser with wavelength at780 nm and the detection band was 450–530 nm. As the endoge-nous fluorophores have broad emission spectra, the detected sig-nal could have contributions from NADH, FAD, keratin, elastin,collagen, and melanin. Interaction of breast cancer cells with thetumor stroma in metastasis models has been studied with NADHand FAD fluorescence and lifetime imaging [42].

B. Elastin

Elastin is one of the proteins that make up connective tis-sues and is the major extracellular fluorophore. Skin aging hadbeen studied with elastin autofluorescence and collagen SHGimaging. In both human facial skin fixed with formalin andlive forearm skins, areas of autofluorescence of elastic fibersincrease with age while collagen SHG signal decreases withage [43], [44]. In arterial walls, elastin fibrils exist in the in-tima layer and have been imaged in both normal arteries andatherosclerotic lesions [45], [46]. The potential application is tomonitor plaque progression by imaging elastin morphology.

C. Keratin

Keratins are a family of fibrous structural proteins that formthe hard and mineralized structures in skin epidermis. Two-photon excited fluorescence imaging with 760 and 860 nm hasdemonstrated keratin contributes to most of the fluorescence inthe spectral range of 400–600 nm from skin epidermis [9]. Sincekeratin excitation and emission spectra overlap with other fluo-rophores such as NADH and elastin, the absolute contributionof keratin to epidermal fluorescence is not yet known.

D. Melanin

Melanins, which serve predominantly as pigment in tissue,are heterogeneous polymers derived from amino acids. Melaninshave a monotonously decreasing absorption spectrum in the UVto NIR region. Although melanins are commonly viewed as non-or very weakly fluorescent, it has been shown that melaninshave fluorescence in the visible spectral region (400–700nm)under both one- and two-photon excitation [10], [47]. Recently,it was reported that melanins emit fluorescence in the NIR re-gion (820–920 nm) when excited at 785 nm [26]. Oxidationmay be the cause of melanin fluorescence as it has been shownthat nonoxidized melanins have very weak fluorescence com-pared with oxidized ones [48]. In skin tissue with melanoma,pigmented areas have much higher fluorescence intensity thannonpigmented areas with two-photon excitation at 800 nm [49].With one-photon excitation at 785 nm, cutaneous melanin inpigmented skin disorders has stronger fluorescence in the NIRregion (820–920m) than adjacent normal tissue [11]. These dis-coveries imply that melanin fluorescence imaging has potentialapplications in the diagnosis of melanoma.

E. Serotonin

Serotonin is a monoamine neurotransmitter synthesized fromtryptophan. Serotonin plays a role in a wide variety of neuro-

physiological functions including mood, anxiety, and appetitecontrol. Its role of the pathology in neural diseases, e.g., ag-ing, depression, and Alzheimer’s disease (AD), are under activestudy [8]. Similar to tryptophan, serotonin has one-photon ab-sorption below 300 nm with emission between 300 and 400 nm.Multiphoton excitation of serotonin with light from visible andNIR lasers avoids the photodamage caused by UV excitation [7].Quantitative measurements of serotonin concentration and dis-tribution in living neural cells have been measured with three-photon excited serotonin fluorescence [50]. The secretion pro-cess of serotonin and its uptake by mast cells have been studiedpreviously with MPM [25], [51].

III. MATERIALS AND METHODS FOR TRYPTOPHAN

FLUORESCENCE IMAGING

A. Laser Source

Fig. 1(a) shows the two-photon fluorescence excitation spec-trum of tryptophan taken from [16]. To perform two-photonimaging near 600 nm, a mode-locked Ti/sapphire laser (New-port, Spectra-Physics Maitai-HP, wavelength 700–1020 nm,100 fs pulse width, 80 MHz repetition rate) is used to pumpan optical parametric oscillator (Spectra-Physics OPAL, wave-length 1200 nm, 100 fs pulse width, 350 mW). The 1200 nmlaser pulses are focused into a β-barium borate crystal (BBO,CASIX USA, 2 mm thick) to achieve second harmonic genera-tion at 600 nm pulses with 60 mW power.

B. Video-Rate Laser Scanning Microscope

The laser beam exiting the β-BBO crystal is collimated anddeflected into a customer-built video-rate (30 frames/s) x–yscanner (polygon, galvanometer). The beam passes through adichroic beam splitter (Semrock, FF510-Di01) and is then fo-cused onto the sample with a 60×, N.A. = 1.2, water-immersionmicroscope objective lens (Olympus, UPlanAPO). The laserpower at the sample site is 10 mW. The fluorescence signal fromthe sample is epi-collected, deflected with the 510 nm long-pass dichroic mirror, and transmitted through a 330–380 nmbandpass filter (Semrock, FF01–357/44). For NADH fluores-cence imaging, the excitation light is provided directly by theTi/sapphire laser (730 nm) and the detection filter is changedto a 420–480 nm bandpass filter (Semrock, FF01–450/60). Theexcitation power of the sample is 20 mW. The fluorescent signalis detected by a photo multiplier tube (PMT) (Hamamatsu 3896)and the 2-D images in x–y plane are acquired by a frame grab-ber (Active Silicon, Snapper-8/24 PCI) installed on a Macintoshpersonal computer. The imaging speed is 30 frames/s and eachstatic image is an average of 30 frames. The schematic drawingof our microscope setup is shown in Fig. 1(b).

C. Animal Preparation

BALB/c mice (Jackson Laboratory) were imaged follow-ing administration of ketamine (100 mg/kg) and xylazine(15 mg/kg) anesthesia mixture. The mice were placed in atemperature-controlled tube and the ear skin was flattened on aglass slide using a 2% methocel gel. All animal experiments have

LI et al.: MULTIPHOTON MICROSCOPY OF LIVE TISSUES WITH ULTRAVIOLET AUTOFLUORESCENCE 519

Fig. 1. (a) Two-photon excitation (TPE) spectrum (solid line), one photon ab-sorption (OPA) spectrum at the corresponding two-photon wavelength (dashedline), and two-photon polarization ratio Ω (dotted line) of tryptophan at 0.01 Min aqueous phosphate buffer. (Reproduced with permission from [16].) (b) Di-agram of the video-rate multiphoton excited fluorescence microscope. OPO:optical parametric oscillator, PMT: photo multiplier tube.

been approved by the Subcommittee on Research Animal Careof Massachusetts General Hospital (Protocol # 2006N000058).

IV. RESULTS AND DISCUSSION

A. Epidermis

Gray scale MPM images of the BALB/c mouse ear epider-mis at different depths are shown in Fig. 2. Fig. 2(a) and (b)are NADH fluorescence images at 5 and 15 µm beneath theskin surface, respectively. Fig. 2(a) shows the layer of flat stra-tum spinosum cells. Columnar basal cells show up in Fig. 2(b)at a depth that is already near the epidermal–dermal junction.Fig. 2(c) and (d) are UV fluorescence images in the same loca-tions as in (a) and (b). The intracellular UV fluorescence signal

Fig. 2. Epidermis imaging. NADH fluorescence image of (a) mouse skinspinosum and (b) basal layer. Tryptophan fluorescence image of (c) spinosumand (d) basal layer (scale bar: 20 µm).

Fig. 3. Dermis imaging. (a) NADH fluorescence image of mouse skin dermis,and (b) tryptophan fluorescence image of the same location (scale bar: 20 µm).

in Fig. 2(c) and (d) comes primarily from the protein compo-nents of the cell cytoplasm and is much more evenly distributedthan NADH fluorescence, which has a punctuated pattern, corre-sponding to the distribution of mitochondria. It has been reportedthat keratin has a fluorescence emission spectrum in range of350 nm and above [9], therefore, the UV fluorescence signalin epidermis may be coming from both tryptophan and keratin.The relative contribution of these two fluorophores needs to befurther investigated.

B. Dermis

At 30 µm deep we are imaging into the dermis as shown inthe NADH and tryptophan fluorescence images, Fig. 3(a) and(b), respectively. In Fig. 3(a) a sebaceous gland is visible at thebottom left corner. Oxidized lipids are fluorescent in the visi-ble spectral region (400–600 nm), and are, therefore, detectedas “crosstalk” in our NADH fluorescence detection channel,

520 IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 16, NO. 3, MAY/JUNE 2010

Fig. 4. Muscle cells imaging. (a) NADH fluorescence image of mouse skinmuscle and (b) tryptophan fluorescence image of the same location (scale bar:20 µm).

which has a bandpass filter of 420–480 nm. The sebocytes,lipid-filled cells that produce sebum, in the sebaceous glandsare bright with dark nuclei, while in Fig. 3(c) sebocytes displaydarker cytoplasms. It has been demonstrated that coherent Anti-stokes Raman scattering (CARS) microscopy has the capabilityof imaging sebaceous glands in mouse skin by detecting theC–H vibrational bands in lipids [52]. In CARS images, the se-bocytes look bright with dark nuclei and the basal cells aroundthe gland have minimal contribution to the signal.

Dermal cells have been observed in previous skin imagingstudies with NADH fluorescence, however, the cell morphol-ogy was not well resolved [32]. In Fig. 3(a), we observe similarcellular structures (white arrow). In the tryptophan fluorescenceimage [Fig. 3(b)], the same cell can be distinguished (white ar-row) with dark nucleus and bright cytoplasm. The cell type hasnot been verified yet, but from their size (∼20 µm) and spinalshape they are most likely fibroblasts or mast cells. There area few more clearly visible dermal cells in Fig. 3(b), which aredifficult to identify in Fig. 3(a). As tryptophan is abundantly dis-tributed in the cytoplasm, the dermal cell bodies are much betterdefined in tryptophan fluorescence imaging rather than NADHimaging, in which NADH primarily locates in mitochondria.

In the dermis collagen makes up the extracellular matrix. Inour tryptophan fluorescence images shown in Fig. 3, we donot observe any fibrous structure similar to collagen. It hasbeen reported that collagen in human bone and tendon doesnot contain any tryptophan, as measured by the ion-exchangechromatographic method [53]. Our images indicate that mouseskin collagen contain little or no tryptophan.

C. Subdermal Muscle Cells

We also imaged muscle tissue with NADH and tryptophanfluorescence at a depth of 70 µm in the mouse skin as shownin Fig. 4(a) and (b), respectively. The NADH fluorescence im-age [Fig. 4(a)] reveals the A–Z bands of muscle cells with darknuclei. While the tryptophan fluorescence image [Fig. 4(b)] re-veals the overall cell delineation. Recently, muscle disease wasquantified with SHG microscopy images [54]. Parameters, suchas distribution of lengths of sarcomeres and muscle fiber cross-sectional areas, were measured. Both NADH and tryptophanfluorescence microscopy can provide the same resolution im-

Fig. 5. Tryptophan fluorescence image of leukocyte (white arrow) in bloodvessel (scale bar: 20 µm).

ages and can possibly be used to diagnose muscle disease suchas muscular dystrophies.

D. Effect of Hemoglobin on Tissue Imaging with MPM

Since the imaging depth of MPM in mouse skin is lim-ited to approximately 100 µm, hemoglobin absorption be-comes a significant issue in MPM imaging when imagingthrough a blood vessel and when its absorption coefficient ex-ceeds ∼100 cm−1 . At wavelengths used for most MPM studies(>700 nm), hemoglobin absorption coefficient is far less than100 cm−1 . At 600 nm, the wavelength used for two-photon ex-citation of tryptophan, the absorption coefficients of oxy- anddeoxy-hemoglobin are about 16 and 73 cm−1 , respectively. Theabsorption coefficients increase sharply below 600 nm, there-fore, the emission of both NADH and tryptophan are signifi-cantly attenuated by hemoglobin. The heme protein also con-tains tryptophan but its emission is quenched by nearby hememoieties [55], therefore, the blood vessels appear dark in tryp-tophan images (Fig. 5). While performing tryptophan fluores-cence imaging, we occasionally observed bright cells (whitearrow) inside the blood vessels, as shown in Fig. 5. Previous re-ports on fluorescence spectroscopy have shown that leukocytescontribute to tryptophan fluorescence under one-photon excita-tion [56], [57]. We performed in vitro tryptophan fluorescenceimaging on different populations of blood cells (leukocytes anderythrocytes, data not shown) and confirmed that leukocytes,but not erythrocytes (red blood cells) do not exhibit tryptophanfluorescence signal. Fig. 5 shows that it is possible to imageleukocytes in small vessels in the mouse ear vasculature.

E. Photodamage in MPM

Photodamage is an important concern especially for in vivobiomedical imaging. Although there have been several reportson this topic, the damaging mechanism in multiphoton mi-croscopy is not well understood yet, and the damage thresh-old is not fully characterized. It has been shown that heatingof the tissue is not the reason for photodamage in MPM, andtwo-photon absorption rather than one-photon absorption is theprocess that leads to damage [58], [59]. But another report hasstated that photodamage is a highly nonlinear process and isproportional to the integrated light intensity over space and

LI et al.: MULTIPHOTON MICROSCOPY OF LIVE TISSUES WITH ULTRAVIOLET AUTOFLUORESCENCE 521

time at a power of about 2.5 in two-photon microscopy usingexogenous calcium-indicator dye fluorescence [60]. As the ex-citation laser wavelength is in the NIR region, the endogenousabsorbers are probably cellular absorbers, such as the coen-zymes NADH and FAD as well as porphyrins. Excitation ofthese endogenous fluorophores will result in the formation ofreactive oxygen species (ROS) and indirect DNA damage. InNADH autofluorescence imaging excited with two-photon ab-sorption, reduction of DNA synthesis in rat basophilic leukemiacells occurred above a threshold dose of approximately 2 ×1053 photon2 cm−4 s−1 [61]. Using cell death as the criteria,the safety threshold for two-photon excitation with 780 nm de-creased from 22 to 7 mW as the laser pulse width shortened from2 ps to 240 fs while imaging Chinese hamster ovary cells [58].The damage threshold will increase with depth in scatteringtissue due to degradation of laser focus but the depth depen-dence of two-photon-induced damage in tissue has not beenthoroughly investigated. In our studies, we have not observedany obvious tissue damage in skin even after prolonged imagingat both NADH and tryptophan wavelengths, but further studiesare required to ensure the safety of this imaging modality.

V. CONCLUSION

We have demonstrated in vivo imaging of mouse skin withmultiphoton excited tryptophan autofluorescence. This newimaging modality enables the observation of epidermal and der-mal cells without the need for staining. It has been reported thatcervical epithelial cancers cells have much higher level of trypto-phan fluorescence than leukocytes [56]. This suggests potentialapplications in diagnosing epithelial cancers using tryptophanfluorescence imaging. Furthermore, we have used this techniqueto observe leukocytes trafficking in vivo, which is important inunderstanding immune response [62], [63]. Since all currentmultiphoton intravital microscopy requires labeling cells withcontrast agent, the ability to track leukocytes in vivo withoutexogenous labeling will significantly expand the applications ofMPM in immunology research.

ACKNOWLEDGMENT

The authors would like to thank Dr. N. Kollias andDr. P. Bargo from Johnson & Johnson, Inc. for insightfuldiscussions.

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Chunqiang Li received the Ph.D. degree in electrical engineering from thePrinceton University, Princeton, NJ, in 2006.

He is currently a Postdoctoral Fellow at the Advanced Microscopy Program,Wellman Center for Photomedicine, Harvard Medical School, MassachusettsGeneral Hospital, Boston, where he is engaged in research on nonlinear opticalmicroscopy with Prof. C. P. Lin. He is also associated with the Center forSystems Biology, Massachusetts General Hospital.

Costas Pitsillides received the Masters degree in mechanical engineering fromthe Massachusetts Institute of Technology, Cambridge, in 2002. He is currentlyworking toward the Ph.D. degree in biomedical engineering from Boston Uni-versity, Boston, MA.

He is at the Advanced Microscopy Program, Wellman Center for Pho-tomedicine, Harvard Medical School, Massachusetts General Hospital, Boston,where he is engaged in research on monitoring circulating cells by in vivo flowcytometry with Prof. C. P. Lin.

LI et al.: MULTIPHOTON MICROSCOPY OF LIVE TISSUES WITH ULTRAVIOLET AUTOFLUORESCENCE 523

Judith M. Runnels received the Ph.D. degree in genetics from Michigan StateUniversity, East Lansing, in 1981.

She was a Postdoctoral Fellow in the Laboratory of Paul Howard-Flanders,Yale University, New Haven, CT, where she studied bacterial mutation andrecombination. Subsequently, she joined Naomi Rosenberg’s group at TuftsMedical School, Boston, MA, where she developed murine B cell lines throughretroviral immortalization. She is currently a Member of C. Lin’s group at theAdvanced Microscopy Program, Wellman Center for Photomedicine, HarvardMedical School, Massachusetts General Hospital, Boston, and I. Ghobrial’sgroup in medical oncology at Dana-Farber Cancer Institute, Harvard MedicalSchool, Boston, where she is engaged in characterizing multiple myeloma andWaldenstrom’s macroglobulinemia by using advanced imaging techniques.

Daniel Cote received the Ph.D. degree in physics from the University of Toronto,Toronto, ON, Canada, in 2003.

From 2002 to 2004, he was a Postdoctoral Fellow at the Ontario CancerInstitute, University of Toronto, where he was engaged in research on tis-sue optics and polarimetry under the supervision of I. A. Vitkin and B. C.Wilson. From 2004 to 2006, he was a Postdoctoral Fellow at the WellmanCenter for Photomedicine, Harvard Medical School, Massachusetts GeneralHospital, Boston, where he was engaged in research on microscopy with Prof.C. Lin. He is currently an Assistant Professor at the Centre de Recherche Univer-site Laval Robert-Giffard, Departement de Physique, Universite Laval, Laval,Quebec, QC, where he is engaged in research on neuronal activity imaging withnonlinear microscopy and spectroscopy. He is also a Visiting Scientist at theWellman Center for Photomedicine.

Dr. Cote is the Natural Sciences and Engineering Research Council of CanadaResearch Chair in Biophotonics.

Charles P. Lin received the Ph.D. degree in physical chemistry from the Uni-versity of Chicago, Chicago, IL, in 1986.

He was a Postdoctoral Fellow in Prof. W. S. Warren’s group at PrincetonUniversity, Princeton, NJ, where he was engaged in research on coherent laserspectroscopy. In 1989, he joined Dr. C. Puliafito’s group at Boston, where he wasengaged in research on intraocular laser microsurgery and participated in theearly development of optical coherence tomography for ophthalmic imaging. Heis at the Advanced Microscopy Program, Wellman Center for Photomedicine,Harvard Medical School, Massachusetts General Hospital, Boston, since 1994,where he has been engaged in research on advanced optical techniques for invivo imaging and laser therapy. He is also associated with the Center for SystemsBiology, Massachusetts General Hospital.