the crocidolite fibres interaction with human mesothelial cells as investigated by combining...

Journal of Microscopy, Vol. 249, Pt 3 2013, pp. 173–183 doi: 10.1111/jmi.12006

Received 28 June 2012; accepted 28 November 2012

The crocidolite fibres interaction with human mesothelial cells asinvestigated by combining electron microscopy, atomic force andscanning near-field optical microscopy

L A U R A A N D O L F I ∗,¶, # , E L I S A T R E V I S A N†, # ,M A R I N A Z W E Y E R ∗, S T E F A N O P R A T O‡,B A R B A R A T R O I A N‡, F R A N C E S C A V I T A†,V I O L E T T A B O R E L L I†, M A R I A R O S A S O R A N Z O†,M A U R O M E L A T O§ & G I U L I A N O Z A B U C C H I†, ∗∗Clinical Department of Medical, Chirurgical and Healthy Science, University of Trieste, Trieste34127, Italy†Department of Life Science, University of Trieste, Trieste 34127, Italy

‡A.P.E. Research S.r.l., Area Science Park, Basovizza, Trieste 34012, Italy

§IRCCS Burlo Garofalo, Trieste 34137, Italy

¶Present address: CBM S.c.r.l. Basovizza, Area Science Park, 34149 Trieste, Italy

#They contributed equally to this work.

Key words. AFM, crocidolite, mesothelial cells, SEM, SNOM images, TEM.

Summary

In this study, we have performed a morphological analysis ofcrocidolite fibres interaction with mesothelial cells (MET5A)by combining conventional electron microscopy with atomicforce (AFM) and scanning near-field optical microscopy(SNOM). After 6-h exposure at a crocidolite dose of 5 μg cm−2,90% of MET5A cells interact with fibres that under theseconditions have a low cytotoxic effect. SEM images pointout that fibres can be either engulfed by the cells thatlose their typical morphology or they can accumulateover or partially inside the cells, which preserve theirtypical spread morphology. By using AFM we are able todirectly visualize the entry-site of nanometric-sized fibresat the plasma membrane of the spread mesothelial cells.More importantly, the crocidolite fibres that are observedto penetrate the plasma membrane in SNOM topographycan be simultaneously followed beneath the cell surfacein the SNOM optical images. The analysis of SNOM datademonstrates the entrance of crocidolite fibres in proximityof nuclear compartment, as observed also in the TEM images.Our findings indicate that the combination of conventionalelectron microscopy with novel nanoscopic techniques can beconsidered a promising approach to achieve a comprehensivemorphological description of the interaction between asbestosfibres and mesothelial cells that represents the early event infibre pathogenesis.

Correspondence to: Professor Giuliano Zabucchi, Department of Life Science,

University of Trieste, Trieste 34127, Italy. Tel: +39 040 5588660; fax: +39 040

558 4023; e-mail: [email protected]

Introduction

The interaction between asbestos fibres and mesothelial cellshas been widely studied with the aim of revealing thepathogenetic mechanisms leading to asbestos-related diseases(Roggli et al., 2008). These investigations have been focusedon several fundamental aspects such as the mechanisms bywhich the fibres interact with membrane receptors and triggertransmembrane signalling (Mossman et al., 1998; Dostertet al., 2008; Mossman et al., 2011); the pathways throughwhich the fibres can reach the cell interior (Davis et al., 1974;Malorni et al., 1990) and affect specific cell compartmentsand responses (Hesterberg et al., 1986; Jensen et al., 1999;Jiang et al., 2008); the mechanism of cytotoxicity induced bythe fibres (Courtney Broaddus et al., 1997; Aljandali et al.,2001; Jiang et al., 2008) and the influence of the fibreson cell metabolism (Riganti et al., 2002, 2003). Altogetherthese studies have shown that the fibres can trigger variousresponses from gene transcription to cell death. The mutagenicproperties of asbestos fibres and their capacity to affect thegene-profile expression (Courtney Broaddus et al., 1997;Cavallo et al., 2004; Nyrmark et al., 2007) suggest thatthey may also reach the nuclear compartment (Malorniet al., 1990). In any case, the entrance of the fibres intothe mesothelial cells is considered a first necessary stepfor asbestos-induced injury (Liu et al., 2000). The study offibres uptake is difficult to achieve in these cells. This ismainly due to their spread morphology (height less than1 μm), which makes it difficult to identify intracellularfibres and distinguish them from extracellular ones (Liuet al., 2000). To answer this question, some techniques have

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been applied, including scanning electron microscopy (SEM)combined with electron back-scattered and transmissionelectron microscopy (TEM) or simply transmission electronmicroscopy (Hesterberg et al., 1986, 1987; Malorni et al.,1990; Cavallo et al., 2004). More recent studies show thatfibres can be identified as intracellular by confocal microscopy,only in the presence of a lipid soluble fluoroprobe to visualizethe plasma membrane (Boylan et al., 1995). The applicationof scanning probe techniques as atomic force microscopy(AFM) and scanning near-field optical (SNOM) can representuseful innovative approaches for investigating the interactionof the fibres with mesothelial cells, with high resolution andwithout using labelling procedures. AFM can provide a precisemeasurement of three-dimensional map of a specimen withnanometre resolution. Besides, SNOM is the only techniqueable to combine high resolution topography image with sub-diffraction optical resolution information (below 100 nm)(Enderle et al., 1998; Nagy et al., 1999; Zenobi & Deckert,2000; De Lange et al., 2001; Rieti et al., 2004; Hoppeneret al., 2005; Ianoul et al., 2005; Zweyer et al., 2008;Dietler et al., 2010). Moreover, near-field light waves decayexponentially over distance less than 100 nm (Pohl et al.,1988) and carry highly localized information along the zaxis. This makes SNOM a surface selective technique andSNOM reflection images can provide optical informationabout a thin layer below the cell surface. For such features,together with their high sensitivity and non-invasiveness,they appear to be particularly suitable for investigatingphenomena occurring at the level of plasma membrane(Rasmussen et al., 2005; Indrieri et al., 2008; Muller, 2008;Jung et al., 2010) and for visualizing sub-membrane structuresof whole cells with a flat morphology (Trevisan et al.,2010). In this framework, we combined SEM and TEM withnanoscopic techniques as SNOM and AFM to obtain high-resolution morphological description about the interaction ofcrocidolite fibres with mesothelial cells. Our findings point outthat important details about the interaction of nanometricfibres with the cell membrane surface (entry-site, membranemodification, etc.) can be observed by AFM and SNOMtopography, while SNOM optical images allow followingcrocidolite fibres below the membrane of the whole spreadmesothelial cells. Moreover, the analysis of SNOM andTEM images provides direct evidence about the entrance ofcrocidolite fibres in proximity of the nuclear compartment.

Materials and methods

Crocidolite fibres

Crocidolite Asbestos UICC Standard fibres (SPI#02704-AB)are purchased from SPI Supplies Division, Structure Probe, Inc.(West Chester, PA 19381-0656, USA). Mineral compositionand fibres size distribution are widely characterized (Kohyamaet al., 1996; SPI Supplies data sheet). Crocidolite fibres are

suspended in sterile phosphate buffered saline (PBS). Thenumber of crocidolite fibres per weight unit are 70×103 μg−1

as determined by counting in Thoma Chamber.

Cell culture and treatments

MET5A cells are cultured on poly-lysine L coated coverslipes(18 mm diameter) (Menzler Glasser) using RPMI 1640medium supplemented with 10% heat inactivated fetal bovineserum (FBS), L-glutamine 2 mM, 100 U ml−1 penicillin and100 U ml−1 streptomycin at 37◦C in a 5% CO2 atmosphere.Semi-confluent MET5A cultures are exposed to a crocidoliteconcentration of 5 μg cm−2 for increasing incubation time(3, 6, and 12 h) in culture medium. After exposure, samplesare extensively washed, firstly with culture medium andthen with PBS. Unexposed cells are treated analogously andused as negative control. Afterwards samples are stainedwith Diff-Quik for conventional optical microscopy (OrthoplanLeitz microscope, equipped with a JVC TK-C1381EG DIGITALColour Video Camera) observations and screening of treatedsamples.

Cell viability assay (WST-1)

The assay is based on the reduction of WST-1 by viable cells(Roche Diagnostics GmbH, Germany). WST-1 is a ready-to-use substrate which measures the metabolic activity ofviable cells. The reaction produces a soluble formazan salt.It is suitable for measuring cell proliferation, cell viabilityor cytotoxicity. In order to perform viability assay, cells arecultured in a 96-well microplate (50.000 cells/wells) usingRPMI 1640 medium supplemented with 10% heat inactivatedfetal bovine serum (FBS), L-glutamine 2 mM, 100 U ml−1

penicillin and 100 U ml−1 streptomycin at 37◦C in a 5%CO2 atmosphere. After 24 h MET5A cultures are washed andresuspended in phenolred free-RPMI supplemented with 10%heat-inactivated FBS. MET5A exposed to a crocidolite (finalconcentration of 5 μg cm−2) for 3, 6 and 12 h are incubatedwith WST-1 (diluted 1:10) for 3 h at 37◦C. Quantificationof formazan salt is performed in microplate with an ELISAplate reader (Biotek Instruments INC, Luzern, Switzerland) byusing a wavelength of 450 nm. The absorbance value directlycorrelates with the viable cell number. Statistical analysis ofthese samples is performed by GraphPad Prism 3.0 (GraphPadSoftware).

SEM

Control and treated cells are fixed with 2.5% glutaraldehydein PBS pH 7.4 at room temperature for 20 min, rinsedin PBS and post-fixed in 1% osmium tetroxide in PBS for30 min. Afterwards samples are rinsed in PBS and thendehydrated in ascending ethanol concentrations (35%, 50%,70%, 90%, 100%). Samples are transferred to a critical point

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dryer (Bal-Tec; EM Technology and Application, Furstentum,Liechtenstein) in 100% ethanol and dried through CO2.Coverslips are mounted on aluminium sample stubs and goldcoated by sputtering (Edwards S150A apparatus, EdwardsHigh Vacuum, Crawley, West Sussex, UK). SEM images areobtained using a Leica Stereoscan 430i scanning electronmicroscope (Leica Cambridge Ltd., Cambridge, UK). Manymicrophotographs at different magnification are stored foreach sample. SEM imaging is performed at an acceleratingvoltages of 20 kV, working distance of 17–18 mm and beamcurrent of 0.08–0.1 nA.

Sample preparation for AFM and SNOM imaging

For AFM and SNOM imaging, cells grown on glass coverslipsare fixed with 4% formaldehyde in PBS pH 7.4 at roomtemperature for 20 min, washed three times in PBS, two timesin water, dehydrated in ascending ethanol concentrations (upto 70%) and allowed to dry.

AFM and SNOM set-up

High-resolution topography AFM images are taken by usinga A-100 AFM microscope (A.P.E. Research, Trieste, Italy)equipped with an absolute flexure scanning system witha maximum xy-scan area of 100 × 100 μm and z-scanrange of 10 μm, with strain gauge sensors to providean absolute positioning. The microscope mounts a digitalcamera that enables to select the scanning area. AFM imagesare collected both in non-contact and contact mode underambient conditions. Oxide-sharpened silicon nitride probes(Mikromasch, Tallinn, Estonia), 125 μm long with 40 N/mspring constant (for non-contact mode), and 460 μm longwith spring constants of 0.15 (for contact mode) are used; bothhaving a nominal radius of curvature about 10 nm. Duringmeasurements scanning parameters are adjusted to preventdamaging of the biological structures.

SNOM topography and near-field measurements areperformed by using a TriA-SNOM microscope (A.P.E.Research, Trieste, Italy), equipped with a flexure scanningstage with a maximum xy scan area of 100 μm ×100 μm andz-scan range of 10 μm (high voltage mode), with strain gaugesensors to provide an absolute positioning. The TriA-SNOMsetup is provided with interchangeable laser sources, coupledwith a single mode optical fibre. In this work, a laser withwavelength of 532 nm is used. Besides topography images,SNOM optical reflection and optical transmission signals aredetected with two photomultipliers (R74000, HamamatsuPhotonics KK Hamamatsu city, Japan). The SNOM probeconsists of an aluminium-coated pulled optical fibre with anominal tip aperture of 50 nm (Lovalite, Besancon, France).Two optical vision systems are integrated within the SNOMhead to control the probe position and select the scan area.An upper optical vision system is used to monitor probe

Fig. 1. Quantification of cells viability during the interaction of crocidolitefibres (5 μg cm−2) for increasing incubation times (3, 6, 12 h). Thisanalysis is performed by WST-1 assay. The values are the mean of at leastfive experiments, ±SD.

approach to the sample, while one transmission camera (withinterchangeable achromatic objectives) is utilized for a bottomview of the sample. Processing and analysis of SNOM andAFM images are carried out by using the Gwyddion Software(Gwyddion open source software, http://gwyddion .net).

TEM

The monolayers are washed twice with PBS and processedas previously described (Borelli et al., 2002). Briefly,monolayers are fixed on ice for 1 h in a solution of 2%glutaraldehyde (Serva) and 2% osmium tetroxide (1:1) in0.1 M cacodylate buffer (pH 7.3). Fixed cells are washedtwice with 0.1 M cacodylate buffer, dehydrated in ascendingethanol concentrations (35%, 50%, 70%, 90%, 100%) andthen embedded in Dow Epoxy Resin (DER332, UnioneChimica Europea, Milano, Italy) and DER 732 (Serva).The ultrathin sections (100 nm thickness), obtained bythe Ultrathome III (Pharmacia-LKB, Uppsala, Sweden), doublestained with lead citrate and uranyl acetate, are visualizedwith a transmission electron microscope (EM208; Philips,Eindhoven, The Netherlands). TEM images are taken with aMorada camera (Olympus Soft Imaging Solutions, Muenster,Germany).

Results

MET5A cells viability

In order to exclude the toxic effect in our experimentalconditions, MET5A cells viability is tested and analyzed foreach exposure time as shown in Fig. 1A. Over 80% of cells arefound to be viable after a maximum exposure time of 12 h. Inthe following morphological analysis, we focus our attentionon 6 h exposure time, being observed that at this exposuretime 90% of cells are viable.


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Fig. 2. SEM images of control (A,B) and crocidolite-treated MET5A (C-F)exposure time 6 h. Two different MET5A cell morphologies resulting fromcrocidolite exposure are reported in (C) and (E); high magnification images(D) and (F) are acquired on the cells shown in (C) and (E), respectively. Barin A = 5 μm; bar in B = 10 μm; bars in C and E = 3 μm; bars in D andF = 1 μm. SEM imaging is performed at accelerating voltages of 20 kV,working distance of 17–18 mm and beam currents of 0.08–0.1 nA.

SEM imaging

In Fig. 2 representative SEM images of untreated (A andB) and crocidolite-treated MET5A cells (C-F) are shown.In SEM images, we observe that after 3 h exposure timecrocidolite fibres are adherent on more than 60% of cells. Thispercentage rises up to 80% after 6 h, and does not increasewith longer exposure time. The analysis of the SEM imagespoints out the presence of two cell populations having differentmorphologies resulting from fibres exposure. In one case, somefibres appear to be either placed over the cell membrane, orpartially internalized into the cell, which preserves its spreadmorphology (Fig. 2C and D). In the second case, as shown inFig. 2E and F, the crocidolite fibre goes through the entirecell body, which partially retrieves the cytosolic processes

and adopts a globular shape. This morphological change isobserved in about 5% of the treated cells. Moreover, we observethat the discontinuity between fibre and membrane at themembrane entry-site is not well resolved in high magnificationSEM images (Fig. 2D and F), even if the fibre is clearly insidethe cell (see Fig. 2F).

SNOM and AFM imaging

The same cell culture observed with SEM is analyzed by AFMand SNOM. These two latter techniques do not need stainingor metal-coating procedures and MET5A are only fixed andpartially dehydrated before imaging. AFM and SNOM imagingof fixed cells is carried out under ambient conditions thatcan be considered a non-invasive high resolution approachfor topographical studies of cell membrane. Indeed, a rapidfixation preserves the membrane surface features of living cellsand provides optimal conditions for scanning, while scanningon living cells, by comparison, can provide slightly lowerresolution due to the fluid and dynamic nature of the plasmamembrane (D’Agostino et al., 2009; Walker et al., 2011). TheAFM and SNOM imaging are generally carried out on cellshaving a thickness between 1 and 1.5 μm.

Representative AFM images are shown in Fig. 3. Here wecan discern two fibres interacting with the cell membrane(Fig. 3A, B). A further zoom into this area is shown in Fig. 3C asa three-dimensional representation. The image displays that asmall fibre is placed over the membrane and has a vertical size(thickness) of about 30 nm, as evaluated by the height profileindicated by a white arrowhead (see Fig. 3C, cross section 1),while another one clearly penetrates below the membraneand it has a vertical size of about 100 nm (see Fig. 3C, crosssection 2). A further representative AFM image of the fibreentry-site at the membrane, acquired on another cell, is shownin Fig. 3D. In this case, the fibre has a vertical size of about600 nm (see cross section 3 of Fig. 3D).

In Fig. 4, representative SNOM images acquired on bothuntreated (A−C) and crocidolite-treated MET5A (D−I) cellsare shown. In the topography image of unexposed MET5Acells, we can clearly distinguish a large cytoplasmatic regionand the nucleus containing some nucleoli (Fig. 4A). In thecorresponding optical reflection (Fig. 4B) and transmission(Fig. 4C) images, these cellular components appear to displaydifferent optical contrast according to their local reflectionand absorption properties. SNOM images of crocidolite-treatedMET5A cells show the presence of fibres associated withcells (see Fig.4D−I). Similarly to what was observed forSEM images, we can recognize two cell populations resultingfrom crocidolite fibres exposure. Generally SNOM topographyimages display that some are placed over the membrane (seethe arrowhead in Fig. 4D), while others can be distinguishedas partially internalized by the cell (see the dashed arrow inFig. 4D). More rarely, we observe that the cell completelyembraces the crocidolite fibre, with a drastic cellular shape



Fig. 3. AFM image of a crocidolite-treated MET5A cell 6 h exposure time (A). On this cell a zoom-in image (B) is acquired on the area indicated by thegrey frame; a further zoom in this area is obtained and shown as a three-dimensional view in (C). A similar high resolution AFM image is acquired onanother cell and shown as a three-dimensional view in (D). Height profiles are provided to give quantitative information. They are obtained in sampleareas indicated by arrowheads (C, cross section 1) and black arrows (C cross section 2 and D cross section 3). These AFM images are obtained in contactmode under ambient conditions.

modification as shown in the topography image of Fig. 4G. Thecorresponding optical images (reflection and transmission)reveal that the crocidolite fibres have optical contrast totallydifferent from cellular components as shown in Fig. 4E, F andH, I. Notably, intracellular portion of the fibre appears to havean optical contrast different from the fibre portion outside thecell, as indicated by the white arrows in the reflection andtransmission images of Fig. 4E and F.

These optical SNOM images, combined with the topographydata, demonstrate that it is possible to follow the fibre routebeneath the plasma membrane (see the white arrow in Fig. 4Eand F). This is likely due to the changing of local opticalproperties (i.e. reflection, transmission and scattering) of thefibres when penetrates below the membrane. The observed

crocidolite fibres have generally a length of 10–50 μm witha thickness of 100–500 nm. However by SNOM imagingsmall fibres, few microns in length can also be detected,as shown in Fig. 5. They can be clearly observed both intopography (thickness of about 50 nm) and optical images(170 nm resolution), providing evidence of resolution beyondthe conventional diffraction limit that is a peculiar aspect ofSNOM. In this case, the optical contrast seems to be equivalentfor all the fibres (both in reflection Fig. 5B,C and transmissionFig. 5E,F). This, combined with topography image (Fig. 5A, D),indicates that fibres are very likely located outside the plasmamembrane.

In SNOM images, the crocidolite fibres appear to befrequently observed in proximity to the nuclear and


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Fig. 4. Representative topography and optical SNOM images of control (A−C) and crocidolite-treated MET5A cells (6 h exposure) (D−I). Topographyimages (A, D, G) show structural features of the cell surface, while reflection (B, E, H) and transmission (C, F, I) images highlight cellular structures thatexhibit different optical properties. The extracellular fibres (see arrowhead in D) can be distinguished from those partially internalized (see dashed arrowin D). In optical images (E) and (F), arrows indicate the portion of fibre internalized by the cell.

perinuclear area as shown in Fig. 6A−C. A zoomed view ofthe cell indicated by the white arrow of Fig. 6A is reportedin Fig. 6D. Here the crocidolite fibre appears to be localizedin the nuclear area. In the corresponding optical images(reflection and transmission), the fibre displays an opticalcontrast different with respect to the cell (Fig. 6E and F). In thiscase, a change in optical contrast in correspondence of nucleararea is more evident in the optical transmission images (seethe white arrow in Fig. 6F). By zooming into this area, therepetitive segmented structure of the fibre is lost at the levelof the nucleus, as indicated by the black arrow in the SNOMtopography of Fig. 6G. A good correspondence between thesite where the fibre loses its segmented structure (Fig. 6G)and the optical contrast changes in the corresponding opticaltransmission image is revealed (see the white arrow in Fig. 6I).The combination of SNOM topography and optical imagessuggests that the fibre penetrates inside the cell in proximityof the nuclear area. The crocidolite fibres that in the SNOMimages are observed to penetrate in proximity of the nuclearcompartment are found to have a length between 15 and30 μm, while the thickness is observed to ranges from 100

up to 300 nm, as evaluated from cross-section profiles in thetopography images (see Fig. 6L).

TEM analysis

The interaction of the crocidolite fibres with cell nucleusis further analyzed by performing TEM on the same cellculture observed with other techniques. High magnificationTEM images show the presence of the crocidolite fibres inside(Fig. 7A, B) or close to the nucleus of mesothelial cells (Fig. 7C).

Discussion

The entrance of asbestos fibres into the cells is believed tobe a fundamental step for asbestos-induced injury (Boylanet al., 1995; Liu et al., 2000). Some papers state that fibreshave to enter the cell to interact with chromosomes or themitotic apparatus, while others show that the fibres can exerttoxic alterations at the plasma membrane (Brody et al., 1983a,1983b; Elferink et al., 1991). However, the study of the role ofentrance has been quite limited by the difficulty of measuring



Fig. 5. Topography (A and D) and optical SNOM images (B,C and E,F) of crocidolite-treated MET5A cell show tiny fibres adherent to the plasma membrane.The arrows in (D) and (E) indicate the position of height profile (G) and (H) respectively, obtained on a small fibre. These line traces reflect the hightopographical resolution on the z axis (50 nm) (D) and the superior resolution of SNOM (170 nm) in (E) as compared to a diffraction-limited opticalmicroscope.

uptake of the fibres and by the incapacity to enhance entranceof the fibres selectively without increasing the overall dose (Liuet al., 2000). In addition, the spread morphology of mesothelialcells (1 μm thickness) does not easily enable us to visualize thefibres engulfment, and most of microscopic techniques fail todiscriminate fibre adherence to the cell from fibre entrancebeneath the cell membrane (Liu et al., 2000). This means thatit can be even more complicated to reveal the asbestos fibresinteraction with specific sub-cellular compartments.

More information about morphological features of thecrocidolite fibres interaction with plasma membrane and sub-cellular compartments of mesothelial cells can be achieved bycombining well-established techniques (electron microscopy)with more recent nanoscopic techniques (AFM and SNOM).All these techniques can resolve morphological structuresdown to nanometre level, but their mechanisms of imageformation are completely different, thus enabling us to obtaincomplementary information. The investigation of numerouslarge sample areas by SEM points out that a high percentageof cells (80%) interacts with the crocidolite fibres aftershort exposure time (6 h). The resulting cell population,frequently preserving their typical spread morphology,

exhibits numerous associated fibres, which appear eitherpartially internalized or placed over the membrane, whereassignificant cell morphology modifications are observed only ina small percentage of cells (about 5–10%). Although SEMenables us to resolve ultra-structural features of the cellsurface, in some cases it appears to be complicated to recognizethe fibre entry-site at the plasma membrane. Likely, this canbe related to the presence of metal coating required for SEMimaging. Indeed, the metal layer is observed to introducean additional surface roughness that can hide numerousnanostructural details (Paoletti et al., 2003).

In this framework, the application of AFM and SNOMopens up the possibility to gain new details about the fibresinteraction with single flat mesothelial cells. These nanoscopictechniques are well suited for the study of the morphology ofthe cells with nanometric resolution (Jung et al., 2010), sincethey require only minimal non-invasive sample preparation(Muller et al., 2011). In both techniques, a sharp probe,positioned within few nanometres from the sample surface,is scanned parallel to the surface and the x,y,z data at eachpoint are acquired. As a result, an accurate three-dimensionalmap of the specimen down to the nanometre level can be


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Fig. 6. Topography (A) and optical SNOM images (B,C) of crocidolite-treated MET5A cell that show the nuclear area of three different cells. A zoom-inimage is acquired on the nuclear area of the cell indicated by the white arrow (D); a further zoom in this area is obtained and shown in (G). The combinationof topography (A, D, G), reflection (B, E, H) and transmission (C, F, I) images provides information about the position of the fibres with respect to thecell membrane. Black dashed arrow in (G) indicates the site where crocidolite fibre seems to enter beneath the plasma membrane; white arrows (F andI) indicate optical contrast change at the membrane entry-site. Height profile in (L) is obtained on the fibre outside of the cell, as indicated by the blackarrow.

obtained. Besides that, SNOM provides simultaneously highoptical resolution in xy axes (<100 nm) and particularly zoptical resolution (order of 10 nm). This latter characteristiccan be exploited to obtain information about structures insideand immediately below the plasma membrane (Trevisan et al.,2010). This represents an important advantage of SNOM withrespect to conventional optical microscopies (i.e. confocal,phase contrast, differential interference contrast), which aretwo-dimensional techniques that provide an (xy) image ofa plane of the sample, while a 3D image can be digitallyreconstructed only subsequently.

The AFM images of MET5A cells exposed to crodidolitefibres provide the evidence of the fibre entry-site at the plasmamembrane and a wealth of details about the membrane aroundthe fibre. Although we notice that during either AFM contact

or non-contact imaging, fibres weakly bound on the surfaceof membrane can interfere with the scanning tip, while thisis not observed in SNOM imaging. This can be related tointeraction forces between tip and surface that might differ inthe SNOM and AFM scanning mode. The topography images,acquired by both AFM and SNOM, also offer the possibility todirectly measure the size (length and thickness) of the fibres.The size quantification (length and thickness) along with thepositioning of the fibres within the cell represents an importantachievement for further understanding about the role of fibressize for cytotoxic and carcinogenic action (Lippman, 1990;Lippmann, 1994; Hill et al., 1995; Fubini, 1997; Donaldsonet al., 2010).

The ability of SNOM to acquire simultaneous highresolution topography and optical images offers the possibility



Fig. 7. TEM images of cell sections of MET5A after 6 h exposure to crocidolite fibres. A nuclear membrane cleft due to the presence of the fibres is shownin (A−B). The presence of a crocidolite fibre near the nucleus can be observed in (C). N = nucleus. Bars in A, C and D = 500 nm; bar in B = 200 nm.

to discriminate the different cellular compartments ofmesothelial spread cells from crocidolite fibres, owing totheir optical properties. The optical features of the fibre areexploited to follow its route into the cytosol of the mesothelialcell, particularly in those cells that maintain their typicalspread morphology. The SNOM optical images demonstratethat fibres can penetrate in proximity of the nucleus, thussuggesting that asbestos fibres can also interact directly withthe genetic material. The direct interaction of crocidolite fibreswith the nuclear compartment is also supported by TEMdata. TEM images reveal that fibres are in close contact withthe nuclear membrane and associated with the cytoskeletalframework of the cells (Ruttner et al., 1987). However, suchelectron technique allows neither a direct z axis measure, norto completely describe the fibre partially internalized in thecell. Indeed the sectioning procedure of sample preparationdoes not enable to maintain the integrity of the fibre (i.e.length and thickness) interacting with the cell. Moreover, thethickness of the mesothelial cell is lower than 1 μm and asresult only few sections of the mesothelial cells can be obtainedby sectioning the cell monolayer (sections thickness about

100 nm). Therefore, TEM can provide only a partial view ofwhat is occurring within a cell. Hence, TEM and SNOM can beconsidered as complementary techniques in morphologicalinvestigation of MET5A cells interacting with crocidolitefibres.

In conclusion, our data indicate that the combinationof SEM, TEM, AFM and SNOM provides a comprehensivemorphological description about the interaction of crocidolitefibres with the mesothelial cells. In addition, we demonstratethat SNOM represents a non-destructive, sensitive, label-free and spatially resolved technique that can be exploited todiscriminate between intra- and extracellular asbestos fibresin spread mesothelial cells and to identify the interaction offibres with specific cellular compartments. Finally, the abilityof AFM and SNOM of measuring the size (thickness andlength) of the fibres that interact with the cells might beexploited to get further information about the relationshipbetween fibre size and toxicity. Future applications of thesenanoscopic techniques may provide with new advances inunderstanding toxic and carcinogenic effects of asbestosfibres.


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Acknowledgements

This work is supported by grants from the Regione AutonomaFriuli Venezia Giulia (D.P. Reg. no. 120/Pres. del 04.05.2007).The authors acknowledge grants from Friuli Venezia GiuliaRegion 2009 (Nanotox 0060 and Commissione AmiantoFVG).

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