three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis...

Three-Dimensional Investigation and Scoring of ExtracellularMatrix Remodeling During Lung Fibrosis UsingMultiphoton MicroscopyANA-MARIA PENA,1,2,3 AURELIE FABRE,4,5,6 DELPHINE DEBARRE,1,2,3 JOELLE MARCHAL-SOMME,4,5,6

BRUNO CRESTANI,4,5,6 JEAN-LOUIS MARTIN,1,2,3 EMMANUEL BEAUREPAIRE,1,2,3

AND MARIE-CLAIRE SCHANNE-KLEIN1,2,3*1Laboratory for Optics and Biosciences, Ecole Polytechnique, 91128 Palaiseau, France2CNRS, 91128 Palaiseau, France3INSERM, U696, 91128 Palaiseau, France4INSERM, U700, Paris, France5Universite Paris 7, Faculte de medecine X. Bichat, 75018 Paris, France6AP-HP, Hopital Bichat-Claude Bernard, services de pneumologie et laboratoire d’anatomie-pathologique, Paris, France

KEY WORDS collagen; second harmonic generation; lung morphology; three dimensionalimaging

ABSTRACT The organization of collagen during fibrotic processes is poorly characterizedbecause of the lack of appropriate methodologies. Here we show that multimodal multiphoton mi-croscopy provides novel insights into lung fibrosis. We characterize normal and fibrotic pulmonarytissue in the bleomycin model, and show that second-harmonic generation by fibrillar collagenreveals the micrometer-scale three-dimensional spatial distribution of the fibrosis. We find thatcombined two-photon excited fluorescence and second-harmonic imaging of unstained lung tissueallows separating the inflammatory and fibrotic steps in this pathology, underlining characteristicfeatures of fibroblastic foci in human Idiopathic Pulmonary Fibrosis samples. Finally, we proposephenomenological scores of lung fibrosis and we show that they unambiguously sort out controland treated mice, with a better sensitivity and reproducibility in the subpleural region. Theseresults should be readily generalized to other organs, as an accurate method to assess extracellularmatrix remodeling during fibrosis.Microsc. Res. Tech. 70:162–170, 2007. VVC 2006 Wiley-Liss, Inc.

INTRODUCTION

An emerging application of multiphoton microscopyis the observation of unstained intact tissue based onendogenous sources of nonlinear signals (Diaspro,2002; Zipfel et al., 2003). In particular, intrinsic secondharmonic generation (SHG) by fibrillar collagen(Boulesteix et al., 2006; Brown et al., 2003; Cox et al.,2003; Freund et al., 1986; Konig and Riemann, 2003;Pena et al., 2005a; Stoller et al., 2003; Wang et al.,2002; Zoumi et al., 2002, 2004) permits to probe theextracellular matrix macromolecular organization, andcan be detected in combination with cellular andextracellular 2-Photon Excited Fluorescence (2PEF)endogenous signals. The understanding of fibroticpathologies should therefore benefit from the specific-ity and three-dimensional (3D) micrometer-scale reso-lution of SHG microscopy in intact tissues. In thiswork, we focus on idiopathic pulmonary fibrosis (IPF),which is a chronic interstitial lung disease with poorresponse to available medical therapies and potentiallyfatal prognosis. Most patients die of respiratory failurewithin 3–8 years of the onset of symptoms (ATS/ERS,2000). At this time, the pathogenesis of IPF has notbeen elucidated. The disease is thought to be due to al-veolar epithelial injury, leading to focal activation andproliferation of fibroblasts with mild inflammation,followed by extracellular matrix accumulation, and

destruction of the lung architecture (Selman et al.,2004). Studies in the past years have focused on theinflammatory component of the disease. More recentlythe contribution of fibrogenesis per se in the IPF patho-genesis has been reevaluated (Chapman, 2004). Thedifficulty in distinguishing the fibrotic response fromthe associated inflammatory response may, in part,explain our poor understanding of the underlying proc-esses and the absence of efficient treatments. Indeedthe first snag we are striking is the absence of a reli-able and reproducible method to measure the extent oflung fibrosis with conventional techniques.

In this study, we quantitatively assess the extent oflung fibrosis using multiphoton microscopy, and wereport a novel description of its 3D spatial distribution ina commonly used physiopathological animal modelobtained by intratracheal administration of bleomycin.

*Correspondence to: Marie-Claire Schanne-Klein, LOB, Ecole Polytechnique,Route de Saclay, 91128 Palaiseau cedex, France.E-mail: [email protected].

Received 26 June 2006; accepted in revised form 12 October 2006

Contract grant sponsors: Fondation pour la Recherche Medicale, College desProfesseurs de Pneumologie, l’Association Francaise pour la Recherche Thera-peutique, and Legs Poix, Chancellerie des Universites de Paris

This article includes Supplementary Material available at http://www.interscience.wiley.com/jpages/1059-910X/suppmat.

DOI 10.1002/jemt.20400

Published online 19 December 2006 in Wiley InterScience (www.interscience.wiley.com).

VVC 2006 WILEY-LISS, INC.

MICROSCOPY RESEARCH AND TECHNIQUE 70:162–170 (2007)

We first characterize multiphoton microscopy of unla-beled lung tissue and assign the various endogenous non-linear signals by comparing with conventional imagingtechniques. We show that multimodal SHG/2PEFmicros-copy allows one to visualize the fibrosis 3D architecture,as well as the accompanying inflammation in unstainedtissue. We then propose phenomenological scores of fibro-sis, and assess their reproducibility and sensitivity in thebleomycin model. Finally, we demonstrate the applicabil-ity of multiphoton microscopy to human lung samples.

MATERIALS AND METHODSBleomycin Animal Model

Experiments were performed on adult male C57Bl/6mice, 6–7 weeks old, weighing 20–24 g (Janvier, LeGenest Saint Isle, France), according to institutionalguidelines that comply with national and internationalregulations. The mice had free access to water and foodad libitum prior to and during pharmacological treat-ments. On day 0, the mice were anesthetized intra-muscularly with 100 lL containing ketamine hydro-chloride (45 mg/kg) and xylazine 2% (9 mg/kg), andthen were administered a unique dose of intratrachealbleomycin hydrochloride (bleomycin, Bellon, Aventis,Paris, France), 80 lg in 50 lL of 0.9% sterile saline. Na-ıve mice were used as controls.

At day 14 after instillation, animals were euthanizedwith intraperitoneal ketamine (60 mg/kg) and xylazine(8 mg/kg). In a first set of experiments, fresh lung tissuereadily dissected from the animal, without preparationwas directly used for SHG imaging or snapped frozenfor cryosections. In a second set of experiments, thelungs were fixed by inflation with a buffered 4% parafor-maldehyde (PFA) solution, following either sectioning ofthe aorta, or washing the lungs four times with 0.25 mLaliquots of 0.9% saline via the trachea. The lungs werefixed for 24 h, and were either used for SHG or embed-ded in paraffin, and 5 lm sections were cut and somekept unstained for SHG assessment. Other sectionswere stained with hematoxylin–phloxine–safran (HPS),picrosirius, Masson’s Trichrome, or orcein.

Frozen tissue was used for immunofluorescent label-ing of macrophages. Cryosections were incubated for1 h at room temperature with hamster antimouseCD11c antibody (6.25 lg/mL, BD Biosciences) andrevealed using goat antihamster IgG coupled to Alexa-fluor 568 (15 lg/mL, Molecular Probes, Invitrogen).

Human Lung Samples

We studied lung tissue samples from patients withIPF, obtained by open lung biopsy (n ¼ 2). IPF wasdiagnosed according to the ATS-ERS consensus criteria(ATS/ERS, 2000). Control lung tissue (n ¼ 2) wasobtained on resected specimens, remote from the soli-tary lesion, following surgical removal of a primarylung tumor. Formalin-fixed paraffin-embedded lungtissue was stained with HPS and Masson’s Trichrome,or left unstained for multiphoton microscopy.

Multiphoton Laser Scanning Microscopy

Combined SHG/2PEF imaging was performed on acustom-built laser scanning microscope incorporating afemtosecond Titanium-sapphire laser (Mira, Coherent),galvanometer mirrors (GSI Lumonics), and photon-

counting photomultiplier modules (Electron Tubes).SHG and 2PEF signals were separated, taking advant-age of their spectral differences: SHG signals are radi-ated at exactly half the excitation wavelength (430 nmfor 860 nm excitation), whereas 2PEF signals are red-shifted (>430 nm for 860 nm excitation), as exemplifiedin Figure 2d. Appropriate filters were used to rejectthe excitation (E700sp, Chroma) and to select the2PEF signal (bandpass filters or colored filters) or theSHG signal (interferential filter HQ430/20, Chroma, at860 nm excitation).

We used a 203, 0.95-NA objective lens (Olympus) inorder to combine a large field of view and a good spatialresolution. In the experiments reported here, the objec-tive back aperture was slightly underfilled, resultingin 0.8 effective excitation N.A., and typically 0.45 lm(lateral) 3 2 lm (axial) resolution near the sample sur-face. Pixel acquisition rate was 100 kHz. Samples wereobserved in an upright geometry (see Fig. 1e) anddetection channels were implemented both in thereflected and transmitted directions, to allow for simul-taneous detection of various multiphoton signals. Mul-tiphoton signals from thick tissues were all detected inthe backward direction, using a dichroic mirror(450DCLP, Omega, at 860 nm excitation) to dispatch2PEF and SHG on two different detectors. Fluo-rescence is isotropically emitted and convenientlydetected in the backward direction. As for SHG, whichis a coherent process, its emission pattern stronglydepends on the organization of the harmonophores inthe focal volume (Mertz and Moreaux, 2001). The ratioof backward-to-forward SHG was reported to varydepending on tissue (Han et al., 2005; Williams et al.,2005). Furthermore, when imaging thick tissues, scat-tering redirects a fraction of the forward-directed lighttowards the objective and contributes to some extentto the backward-detected signal. In our experimentalconditions we observed no significant differencebetween control and fibrotic samples in that respectand we conveniently detected backward SHG signalscomparable to intrinsic fluorescence signals. To visual-ize collagen fibers independent of their direction in thefocal plane and to minimize polarization effects, weinserted a quarter-waveplate on the laser path, result-ing in a nearly-circular incident polarization.

We also performedThird-Harmonic generation (THG)microscopy using a femtosecond optical parametric os-cillator (OPA, APE) providing 200 fs pulses at 1180 nm.TH signals were usually generated using 100 mW inci-dent power and selected with an interferential filter.THG imageswere recorded in the backward direction bytaking advantage of the scattering by the tissue as previ-ously described (Debarre et al., 2006).

Image analysis was performed using ImageJ (W.Rasband, National Institute of Health) and Amira(Mercury Computer Systems).

Spectra of the multiphoton endogenous signals wererecorded using a tunable interference filter (S-60,Schott), as previously described (Pena et al., 2005b).Absorption and fluorescence spectra of chemicals usedto stain the histological sections were recorded on aCARY 500 spectrophotometer (Varian) and an F-4500spectrofluorimeter (Hitachi). 2PEF spectra were re-corded using a custom-built 2-photon spectrofluorime-ter as previously described (Pena et al., 2005b).

Microscopy Research and Technique DOI 10.1002/jemt

163MULTIPHOTON MICROSCOPY OF LUNG FIBROSIS

Fig. 2. Assignment of the multiphoton endogenous signals in lungtissue. (a1) SHG/2PEF image and (a2) transmitted-light image of afixed histological section stained with picrosirius, showing peripherallung and an interlobar scissure (scale bar: 50 lm, same code colors as inFig. 1). SHG signal in the multiphoton image (white arrow) clearlycolocalizes with the picrosirius staining (black arrow) in the conven-tional histological image; (b1) SHG/2PEF image and (b2) transmitted-light image of a fixed histological section stained with orcein (scale bar:50 lm). Part of the 2PEF signal in the multiphoton image correspondsto elastic fibers in a bronchovascular axis as illustrated by orcein stain-ing (arrows). (b3) absorption (black), one photon-excited fluorescence(430 nm excitation, blue) and 2PEF (800 nm excitation, red) spectra ofan orcein solution; (c1) 2PEF image with 1100 nm excitation of a frozen

histological section showing CD11c expression by alveolar macrophages,corresponding to the circular structures observed in Figure 1c; immuno-labeling is confirmed by (c2) in situ 2PEF spectra recorded from macro-phages (black solid line) and from interalveolar structures (blackdashed line), which exhibit nonspecific labeling. The small spectral com-ponent on the blue side corresponds to intrinsic fluorescence frommacrophages as evidenced by the 2PEF spectrum recorded in an unla-beled section (red solid line); (d) In situ spectra of multiphoton signalsin fresh lung tissue: 720 nm excitation of NAD(P)H fluorescence (blueline), 860 nm excitation of macrophages (red line), and alveolar andinteralveolar areas (green line). The latter spectrum shows a SHG peakat 430 nm, attributed to collagen fibers, and a broad 2PEF peak at490 nm, attributed to elastic fibers in the walls of alveoli.

Fig. 1. Multimodal multiphoton imaging of normal fresh lung tis-sue. Green color corresponds to SHG, red/purple to 2PEF, yellow toSHG/2PEF colocalization, and white to THG. Acquisition time: 4.9 sper image (5853 561 pixels). 30 mWexcitation at 860 nm (a–c and f,g),and at 780 nm (d) or 100 mW at 1180 nm (h). Scale bar: 25 lm. (a,b)SHG image mapping the distribution of collagen fibers (a) in the vis-ceral pleura, and (b) in the lung parenchyma (21 lmbelow the lung sur-face); (c,d) 2PEF images recorded in the same area, revealing (c) elasticfibers in the alveolar and bronchiolar walls, epithelial cells and alveolar

macrophages (plain red circular structures) at 860 nm excitation, and(d) cellular endogenous chromophores such as NAD(P)H at 780 nm ex-citation; (e) Scheme of the experimental setup, showing epidetection ofthe multiphoton signals; (f,g) SHG/2PEF images of (f) alveoli and bron-chioles (combination of b and c), and (g) a respiratory bronchiole, 42 lmdeeper in the lung (longitudinal view); (h) THG/SHG image recorded45 lm below the surface: Round structures delineated by collagenSHG in the alveolar and bronchiolar walls correspond to residual airin the lung. Small dots correspond to lipid bodies.


164 A.-M. PENA ET AL.

Fibrosis Quantification

The relationship between the SHG signal and thequantity of collagen is not trivial because of the coherentnature of this nonlinear optical process. SHG scales asthe squared density of the harmonophores in homogene-ous media. In media that are heterogeneous at the mi-croscopic scale, SHG is strongly dependent on the spatialdistribution of the harmonophores within the focal vol-ume (Mertz and Moreaux, 2001; Stoller et al., 2003;Williams et al., 2005), and SHG signals from collagenwere shown to strongly depend on the collagen organiza-tion (Kim et al., 2000). In order to define a robust methodto process our images, we calculated the fraction of theimages occupied by a significant SHG signal, using thefollowing procedure. We restricted the analysis toregions extending from the surface down to 80 lm,where the detected SHG signals (typ. 50 counts/pixel inthe pleura and 5–10 counts/pixel in a deep fibrotic area)were above the background, and we verified that the sig-nal over background ratio did not change significantly incontrol and treated samples. We measured histograms ofbackground noise in the images and applied to theimages a threshold corresponding to the maximum back-ground value (typ. 4 counts/pixel in our conditions). Wethen calculated the fraction of the images occupied bySHG signal, and averaged the surface percentage ofSHG signal over all images in a z-stack to obtain a fibro-sis score expressed in volume percentage. We verifiedthat the exact value of the threshold did not affect ourcomparison of fibrotic versus normal tissues (see below).Finally, we systematically compared identical volumes oflung tissue observed using similar imaging conditions.To minimize uncertainties, we normalized the measuredSHG signal to the squared excitation intensity, and wetook into account possible changes in pulse duration orfocusing conditions by using a SHG reference signal pro-vided by collagen fibers in the dermis of a fixed skin sec-tion. Altogether, this phenomenological score providesan estimation of the extent of fibrosis in the tissue.

RESULTS AND DISCUSSIONMultiphoton Imaging of Unlabeled Lung Tissue

Figure 1 presents multiphoton images of normallung tissue freshly dissected from the mouse. Weimaged an intact lobe in physiological solution usingthe excitation geometry depicted in Figure 1e (860 nm,30 mW). We observed neither significant fading of themultiphoton signals nor any morphological alterationof the tissue upon continuous illumination during typi-cally 20 min.

Figures 1a and 1b show SHG images recorded in thevisceral pleura (a) and 21 lm inside the lung paren-chyma (b). The SHG signal presents a fibrillar distribu-tion that we attributed to collagen. We verified thatpoint using conventional histological techniques, asdisplayed on Figure 2a. We imaged a histological sec-tion stained with picrosirius, using SHG microscopy[Fig. 2(a1)] and transmitted-light microscopy [Fig. 2(a2)].Both images reveal the same structures, namely thevisceral pleura, which invaginates to form distinctlung lobes, and thin collagen fibers in the walls of thealveoli. It validates the attribution of the SHG signal tofibrillar collagen. SHG microscopy provides highly con-

trasted images, as seen in Figure 1: the intricate struc-ture of collagen fibers is clearly revealed in the pleura(Fig. 1a), as well as in the walls of the alveoli deeperinside the lung (Fig. 1b).

Figure 1c shows a 2PEF image of the same area as onFigure 1b, upon excitation at 860 nm. These excitationconditions are expected to reveal endogenous fluoro-phores like elastin, flavoproteins or retinol (Zipfel et al.,2003). In particular, fibrillar structures are visiblearound the alveoli, which correspond to elastic fibers.The attribution of the 2PEF signal to elastin was con-firmed by examination of stained histological sections[Fig. 2(b1) and (b2)]: we consistently observed a 2PEFsignal in the areas highlighted by orcein. Figure 2(b1)also shows endogenous 2PEF from other structures,such as bronchiolar and alveolar epithelium, or macro-phages (see next section). We note that the 2PEF signalis enhanced in stained areas, because of the fluorescenceof orcein, as shown by its spectral characterization dis-played on Figure 2(b3). For example, the branch of thepulmonary artery and the respiratory bronchiole aredelineated with thicker walls than what is observed intransmitted-light microscopy [Fig. 2(b2)].

Other structures produce 2PEF signal with 860 nmexcitation: Figure 1c reveals round structures (10–20 lm in diameter) in the vicinity of the alveoli, whichcorrespond to alveolar macrophages. We confirmedthat point using immunofluorescent labeling on thinfrozen sections with antimacrophage CD11c antibody:Figure 2(c1) shows the 2PEF image recorded at 1100nm excitation with positive staining of these struc-tures, as well as a slight nonspecific staining of othercomponents, as confirmed by in situ 2PEF spectra dis-played on Figure 2(c2). We also recorded in situ spectraof macrophages 2PEF from fresh lung tissue (Fig. 2d).The recorded spectrum peaks around 510 nm, whichmay correspond to retinoids or to a mixing of differentspecies, including flavoproteins (Zipfel et al., 2003). Itis clearly distinct from the multiphoton spectrumrecorded in the same conditions in the alveolar walls,characterized by a high collagen and elastin content(Fig. 2d). The latter spectrum exhibits a thin peak atthe harmonic frequency because of collagen, and abroad red-shifted peak around 490 nm consistent withpublished elastin 2PEF spectrum (Zipfel et al., 2003).

A unique advantage of multiphoton microscopy isthe possibility to combine different nonlinear contrastmodes, taking advantage of their specificity. Figure 1fcombines the SHG and 2PEF images from Figures 1band 1c, which were recorded simultaneously. This mul-timodal image clearly reveals the structure of the alve-olar walls that are delineated by elastic and collagenfibers. As other examples, Figure 1g shows a longitudi-nal view of a respiratory bronchiole that continues tobranch further into alveolar sacs, Figure 2(a1) showsthe peripheral lung with a pleural scissure, and Figure2(b1) shows a bronchovascular axis.

Other multiphoton signals can be exploited in lungtissue as illustrated in Figures 1d and 1h. Figure 1dpresents a 2PEF image recorded in the same area asFigures 1b and 1c with 780 nm excitation. It shows in-tracellular signals, that we attributed to NAD(P)H, ascorroborated by the 2PEF spectrum recorded in situ at720 nm excitation (Fig. 2d) and by published NAD(P)H2PEF spectra (Zipfel et al., 2003). Figure 1h presents



third harmonic generation (THG) combined with SHGat 1180 nm excitation. The THG image reveals micro-meter-sized lipid bodies (Debarre et al., 2006), as con-firmed by Nile Red staining. Larger structuresdelineated by collagen fibers are also visible and corre-spond to residual air from the alveolar spaces and thebronchiolar tree. This readily detectable THG signal isproduced by the discontinuity of the optical propertiesbetween air and tissue (Debarre et al., 2006). Figure 1halso shows that collagen SHG is efficiently obtainedover a wide range of excitation wavelengths.

These data illustrate that multiphoton microscopyprovides a rich description of lung tissue morphologyin fresh unlabeled samples. This technique opens thepossibility to visualize the 3D structure of the lung tis-sue from z-stacks of combined SHG/2PEF images. Inparticular, SHG/2PEF images in Figure 1 are extractedfrom a z-stack acquired from the pleura to 90 lmwithin the tissue. 3D reconstructions are also availablefrom these data, as exemplified in Figures 3(b1) and3(b2).

Versatility With Respect to Tissue Preparation

We recorded multiphoton images of various types oftissue preparations: ex vivo intact lobes (Figs. 1, 3aand 3b), paraformaldehyde (PFA) fixed intact lobes,PFA-fixed paraffin-embedded histological sections[Figs. 2(a1) and 2(b1), and 3c], and frozen histologicalsections (Fig. 2c). We found that multiphoton signalsexhibit similar characteristics regardless of tissue proc-essing, except for a few points related to biochemicalchanges upon fixation. First, THG signals wereobserved only in fresh unfixed tissue. This is consistentwith the fact that lipid bodies are fragile structuresthat may be disrupted upon PFA fixation, and that thespace initially occupied by air bubbles is filled withPFA or buffer in fixed samples. Second, endogenous2PEF signals are enhanced in fixed samples, and thefluorescence spectra recorded in fixed and fresh tis-sues do not match perfectly. Similar fixation-related ef-fects have been reported in conventional microscopy(Majumber et al., 2005). Third, the maximum imagingdepth is decreased by typically 25% in fixed tissue.This may be related to changes in the tissue opticalproperties (scattering, absorption, refractive indices)upon fixation. We could obtain multiphoton imagesdown to typically 100 lm in fresh lung tissue with pre-served resolution and acceptable signal over noise ra-tio. These data illustrate the superior imaging depth intissues of multiphoton microscopy when comparedwith conventional techniques. Unresolved structureswere also visible deeper inside the tissue when increas-ing the excitation power.

Multiphoton Imaging of Bleomycin-InducedLung Fibrosis

The high specificity of collagen SHG signal opens thepossibility to study the 3D distribution of collagen fibersin pathological conditions. We investigated lung fibrosisinduced by bleomycin: mice received a unique intra-tracheal dose and were sacrificed at day 3, 7, or 14. Westudied fresh and PFA fixed lobes, as we found that col-lagen SHGwas not sensitive to sample preparation.

In bleomycin-treated mice sacrificed at day 14 (D14),we observed that the SHG signal distribution clearly

differs from the one in control samples: it presents col-lagen fibers accumulation in heterogeneously distrib-uted areas in the alveolar interstitium. This heteroge-neous distribution is particularly evident in Figure 3a,which combines four images acquired in adjacent areasof fresh tissue, or in images spanning larger fields ofview. Such heterogeneity is characteristic of bleomy-cin-induced fibrosis (like IPF in humans), which makesit very difficult to assess the extent of fibrosis usingother existing techniques (Polosukhin et al., 2005).

We took advantage of the micrometer scale 3D reso-lution of the multiphoton images to assess the 3Darchitecture of the pulmonary fibrosis. Figure 3(b1)shows a 3D reconstruction from a z-stack of SHGimages in a lung fibrotic area. It provides a unique vis-ualization of the micrometer scale distribution of thecollagen fibers. Similarly, we studied the SHG signaldistribution in the pleural area (0–52 lm deep), asshown in Figure 3(b2). The density of collagen fibers ishigher than in the control tissue (Fig. 1a), and thepleura appears to be thicker compared to control lungs,in association with increased subpleural collagen depo-sition.

Bleomycin-induced lung fibrosis is accompanied byan inflammatory process, which is evidenced on multi-photon images by the presence of numerous alveolarmacrophages and an increased cellularity in the alveo-lar spaces and in the interstitium. The lung at day 14displayed on Figure 3a presents more intra-alveolarcells than similar images of control lung (see forinstance Fig. 1c). As this inflammatory process takesplace before the settling of fibrosis, we also studiedlungs at day 3 and day 7 postbleomycin instillation.Figure 3c presents multiphoton images of fixed sec-tions of control, day 3, day 7, and day 14 bleomycinlungs. We observed that the 2PEF signal densityincreases from day 3 to day 7, and decreases after-wards, in agreement with previous descriptions of thisinflammatory process (Izbicki et al., 2002). In parallel,we note that SHG microscopy can detect collagen accu-mulation as early as day 3. These data show that mul-tiphoton microscopy can reveal the inflammatory reac-tion, taking advantage of the 2PEF signal exhibited byinflammatory cells.

Altogether, multimodal multiphoton microscopy evi-dences principal features of bleomycin-induced lung fi-brosis: endogenous cellular 2PEF reveals the inflam-matory process with high sensitivity and SHG appearsas a unique tool to display the micrometer scale 3Darchitecture of the fibrosis. We will then discuss how totake advantage of the latter signal to obtain reproduci-ble scores of fibrosis.

Scoring of Fibrosis

We have shown that SHG microscopy provides ahighly specific 3D mapping of lung fibrosis, whichopens the possibility to quantitatively assess its distri-bution. For that purpose, we defined three differentscores based on the SHG signal acquired in the pleuralregion (0–52 lm deep), in the subpleural region (14–52 lm deep), and in a central region (deeper paren-chyma). The first two scores were measured on wholeintact lungs. To study deeper parenchyma, we cut thelung lobe in a sagittal plane and recorded z-stacks ofSHG images in randomized regions along the cut



(0–60 lm deep). To evaluate the reproducibility of thesescores in the bleomycin-induced fibrosis model, westudied fixed lung samples from 5 bleomycin-treatedmice and 5 control mice. We recorded multiphotonimages in four different areas for each score, whichresulted in 20 measurements in each case.

Results are displayed on Figure 4, which comparesthe statistical distributions and the quantitative valuesof the scores obtained with fibrotic and control animals.Figure 4a presents z-profiles of the SHG signal,obtained from z-stacks of SHG images recorded in dif-ferent regions of whole intact lungs. The percentage ofpixels exhibiting significant SHG signal is plotted as a

function of the depth from the pleura to within thelung, including the subpleural region, and amounts tothe surface density of collagen fibers. We observe thatfibrotic lungs present larger SHG signal densities inthe pleural and in the subpleural areas than the con-trol ones. Remarkably, all the z-profiles correspondingto the bleomycin treated mice exhibit larger SHG sig-nal densities in the subpleural area (14–52 lm deep)than the control mice profiles (see inset in Fig. 4a).Conversely, the distributions of fibrotic and controlz-profiles in the pleural region present a small regionof overlap. These observations translate in the distri-bution of the volume fraction of significant SHG signal

Fig. 3. Multiphoton imaging of bleomycin-induced lung fibrosisdistribution. SHG (green: collagen) and 2PEF (red: elastin, macro-phages) signals excited with 50 mWat 860 nm. Scale bar: 100 lm. Yel-low color: SHG/2PEF colocalization. (a) SHG/2PEF image of a day 14fibrotic lung (fresh unlabeled tissue), recorded 42 lm under thepleura, evidencing the heterogenous fibrosis distribution; (b) 3Dreconstruction of the SHG signal from the same fibrotic lung at day

14, within the tissue (b1, area depicted on Fig. 3a and in the pleuralarea (b2); (c) SHG/2PEF images of unstained histological sections em-bedded in paraffin, from (c1) control, (c2) day 3, (c3) day 7, and (c4)day 14 bleomycin lungs. Bleomycin treatment induces (i) inflamma-tion, which decreases after day 7, as evidenced by the 2PEF signal,and (ii) fibrosis, evidenced by the SHG signal detected as early asday 3.



Fig. 5. Normal and fibrotic human lung samples. (top) SHG/2PEFand (bottom) transmitted-light images of serial histological sections(scale bar: 200 lm). (top) unstained section, same code colors as inFigure 1; (bottom) HPS staining, and Masson’s trichrome staining.(a) Control lung showing a bronchovascular axis and surrounding al-veolar spaces with thin alveolar walls, as revealed by HPS staining(a2). Masson’s trichrome (a3) staining highlights fibrosis in green,which is seen mainly around the vessels and the bronchovascular

axis, and correlates to the SHG collagen distribution (a1, green andyellow). (b) IPF fibrotic lung showing marked architectural changewith fibrosis and distorted alveolar spaces. (c) Fibroblastic focuscomposed by myo-fibroblasts (c2), and surrounded by fibrosis (c3,Masson’s trichrome). SHG/2PEF image (c1) highlights the collagendistribution mainly in the periphery of the focus, with thinner fibersin its center, and the central accumulation of fibroblasts.

Fig. 4. Scoring of the lung fibrosis: fraction of pixels exhibiting sig-nificant SHG signal, measured as a surface density in z-stacks of 5403 540 lm2 SHG images recorded in 20 areas from 5 control mice(white) and 5 bleomycin-treated mice (green), and averaged over thez-stack to obtain a volume fraction. (a) Z-profiles of the SHG signaldensity from the pleura down to 50 lm within the tissue. Results in

the subpleural area (14–52 lm) are enlarged in the inset; (b–d) Dis-tribution of the SHG signal density (b) in the subpleural region (14–52 lm, see Panel a), and (d) in the central region (540 3 540 lm2

images over 60 lm depth); (c) Mean values of the three fibrosis scores.Error bars correspond to the standard error (SE) of the mean. The val-ues for the subpleural score are enlarged by a factor of 10.

averaged over the pleural or subpleural regions. Fig-ure 4b displays the histogram obtained from the sub-pleural region depicted in the insert of Figure 4a (14–52 lm deep). We observe that the volume fractions ofcollagen SHG are systematically larger in the bleomy-cin treated samples than in control samples.

We then measured volume fractions of significantSHG signal away from the pleura, in the central region(60 lm deep from the sagittal cut section). The distri-butions obtained for control and fibrotic lungs are dis-played on Figure 4d. The volume fraction of collagenSHG is larger in bleomycin-treated mice than in con-trol ones, but both histograms are broadly distributedand show a region of overlap. It is consistent with theheterogeneous nature of the bleomycin-induced fibro-sis, as visible for instance in Figure 3a. We note thatthe largest values in these distributions are due to thepresence of arteries and bronchioles in this region ofthe lung: their adventitia contains collagen whose SHGsignal artifactually increases the fibrosis score.

Figure 4c summarizes the mean values and errorbars of the three different scores for control and fibroticlungs. Most importantly, all the scores are higher infibrotic lungs when compared with controls, whichshows that SHG microscopy is a reliable method to sortout fibrotic and control mice. We attribute the perform-ance of this technique to the high specificity of the col-lagen SHG signal, and to the increase of the volumeprobed by SHG microscopy when compared with othertechniques restricted to 2D sections. However, allscores do not display the same sensitivity to fibrosis, asevidenced by the ratio of their mean values in thefibrotic lungs and in the controls: 2.7 in the pleuralregion, 14.4 in the subpleural region and 8.1 in the cen-tral region. We note that the exact value of the thresh-old used in the analysis procedure do not affect theseconclusions. We find that the score ratios are 2.8 in thepleural region, 9.5 in the subpleural region, and 6.0 inthe central region when changing the threshold from100% to 50% of the maximum background level. We at-tribute the higher sensitivity and reproducibility of thesubpleural score to a less heterogenous distribution offibrosis in the subpleural region than in centralregions. The subpleural score seems to be significanteven for small volumes (0.015 mm3), and requires inpractice only a few samples (typ. 4).

These scores provide a quantitative assessment ofthe extent of lung fibrosis in the bleomycin model, andmay be generalized to other animal models. In compar-ison, biochemical methods such as the hydroxyprolineassay or the Sircol assay are time-consuming andrequire important amounts of tissue (usually a wholelobe), which are not usable for other analysis; and his-tochemical staining methods are restricted to small 2Dareas. Consequently, fibrosis scoring using multi-photon microscopy should prove particularly useful invarious assays, including testing the effects of drugs orperforming kinetic studies.

Multiphoton Microscopy of HumanLung Fibrosis

The assessment of lung fibrosis in animal modelsraises the question of the applicability of multiphotonmicroscopy to human fibrosis. For this purpose, we an-

alyzed serial sections from human control and fibroticbiopsy specimens (formalin-fixed paraffin-embeddedlung tissue, see methods), and compared conventionaltechniques used to assess IPF with multiphoton mi-croscopy of unstained samples.

Figure 5 shows morphological staining (HPS), fibro-sis staining (Masson’s trichrome), and SHG/2PEFimages in the same areas of one control and two fibroticlungs. SHG/2PEF imaging reveals the normal lungarchitecture with a bronchovascular axis and sur-rounding alveolar spaces lined by thin alveolar walls[Fig. 5(a2)]. By comparison, in the fibrotic human lungof IPF, the lung architecture is disorganized by thickareas of fibrosis, highlighted on the Masson’s trichrome[Fig. 5(b3)], as well as distorted alveolar spaces. TheSHG/2PEF corresponding image shows identical areasof fibrosis [Fig. 5(b1)]. It also allows to characterize thestructure of the fibroblastic foci, the hallmark of usualinterstitial pneumonia, as seen on Figure 5(c1) (ATS/ERS, 2000): SHG highlights the distribution of collagenwhich is denser in the periphery of the focus comparedto its center, whereas 2PEF underlines the fibroblasticpopulation in the center. This study shows that SHG/2PEF microscopy reveals the main features of IPF.

CONCLUSION

This work demonstrates the applicability of multi-photon imaging for sorting out normal and fibroticlung tissue, in animal model as well as in human sam-ples. Endogenous SHG signal from fibrillar collagenreveals the intricate structure of collagen fibers in thepleura and in the bronchoalveolar walls, and can becombined with endogenous 2PEF signals from elasticfibers, or cells such as macrophages, in order to visual-ize the lung tissue morphology. This technique is appli-cable to most tissue preparations, including fresh andPFA-fixed tissues, cryosections, formalin-fixed paraf-fin-embedded sections, as well as histochemical or im-munofluorescent stained sections. In the bleomycin-induced lung fibrosis, 3D SHG imaging provides a pre-cise description of the fibrosis distribution and of itshighly heterogeneous nature. Moreover, we haveshown that inflammation is observable throughchanges in tissue endogenous 2PEF, which indicatesthat multimodal multiphoton microscopy can distin-guish the fibrotic and inflammatory steps of these path-ological processes.

Taking advantage of the specificity of collagen SHGsignals, we have proposed fibrosis scores based on vol-ume fractions of significant SHG signal in variousregions. These phenomenological scores showed a greatsensitivity in the sense that they required small vol-umes of tissue (typ. 0.06 mm3), in particular in the sub-pleural region, and proved to sort out control andfibrotic lung samples. They should be readily general-ized to other organs, as a sensitive method to deter-mine the extent of fibrosis and characterize its 3D dis-tribution. From both clinical and experimental per-spectives, 3D investigation and scoring of collagenproliferation and lung matrix remodeling, combinedwith other techniques including genomic tools, willhelp to develop new therapeutic approaches based on abetter understanding of pathogenic mechanisms.



ACKNOWLEDGMENTS

The authors thank J.-M. Sintes, X. Solinas, I.Lamarre-Jouenne, O. Thibaudeau and the pathologydepartment at Bichat-Claude Bernard Hospital forexpert technical assistance, and P.L. Tharaux, M.Aubier and P. Soler for fruitful discussions.

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