Journal of Microscopy, Vol. 241, Pt 1 2011, pp. 83–93 doi: 10.1111/j.1365-2818.2010.03405.x

Received 10 November 2009; accepted 13 April 2010

Immunogold labelling in environmental scanning electronmicroscopy: applicative features for complementary cytologicalinterpretation

G . C A F I E R O , F . P A P A L E , A . G R I M A L D I , F . R O S S O ,M . B A R B A R I S I , C . T O R T O R A , G . M A R I N O & A . B A R B A R I S IIX Division of General Surgery and Applied Biotechnology, Department of Anaesthesological,Surgical and Emergency Sciences, Second University of Naples, Naples, Italy

Key words. ESEM, FNA, galectin-3, immunogold labelling (IGL), Thin Prep.

Summary

We have combined environmental scanning electronmicroscopy (ESEM) and immunogold labelling (IGL) for theanalysis of cell morphology and surface protein detectionon human fine needle aspiration, which is processed in thinuniform monolayer (a single layer of cells) on a glass slide byThin Prep technology.

Among scanning electron microscopy techniques, wechoose the environmental modality (ESEM) because it allowsa slight manipulation of biological samples and an operationaltime comparable with cytological techniques. Moreover, theThin Prep technology confirmed a reproducible cell monolayeron glass smear, minimizing problems for the determination ofappropriate amount of material per slide.

The first experimental data in ESEM–IGL on biologicalsamples with fine needle aspiration Thin Prep, in humanthyroid nodules, showed that cells retained their morphologyand provided a clear IGL. The optimization of conditions(i.e. vacuum pressure, temperature and relative humidity)confirmed the possibility to observe an immunolabelledbiological sample and morphological signal, joined withcompositional informations, due to peculiar characteristics ofgaseous secondary electron detector in ESEM.

The ESEM–IGL and fine needle aspiration Thin Prep could beused in combination for the interpretation of cell morphologyand cell surface immunolabelling. Our paper suggests this useas a powerful diagnostic tool in a pre-surgical evaluations,opening a new applicative window for electron microscopy.

Correspondence to: Alfonso Barbarisi, Department of Anaesthesological, Surgical

and Emergency Sciences, Second University of Naples, Piazza Miraglia, 1-

80138 Naples, Italy. Tel.: +39-081-5665233; fax: +39-081-5665233; e-mail:

alfonso.barbarisi@unina2.it

Introduction

The use of conventional scanning electron microscopy (CSEM)in biology research and in medicine has been delayed for muchtime. It has occurred much less than in geology, mineralogy,physics, material science, industrial production and so on,because the preparation techniques and the coercion/restraintdue to the high vacuum does not guarantee stability ofthe appearance of three-dimensional biological sample. Asimmunogold labelling (IGL) technique has been utilized bymany authors with transmission electron microscopy (TEM)to obtain the identification/localization of receptors andantigens, both in cells and tissues, the identification of cellularcomponents through IGL by CSEM has been impeded bydifficulties in dissolving the particles and by charging non-sputtered specimens under the electron beam (Hoyer et al.,1979; de Harven et al., 1984).

In addition to electron imaging, the small gold particleshave also been more promptly visualized in the CSEM aftersilver enhancing (Scopsi et al., 1986). Some workers havealso combined electron imaging of gold particles with X-ray emission using an energy dispersive analyser in theCSEM for immunolabelling studies (Eskelinen & Peura,1988). Ten years later, chitosan–GP gels were investigatedby environmental scanning electron microscope (ESEM) inconjunction with energy dispersive X-ray spectrometry, theformers permit direct observation of the ultra structure, inhydrated conditions, simulating a natural state. Throughexamination of unfixed specimens using ESEM, it was foundthat chitosan formed a network structure in both chitosan–GPgels and chitosan–GP/blood clots (Iliescu et al., 2008).

Recently, Socher & Benayahu (2008) demonstrated thatusing gold–palladium coating time of 3 min would avoid thesehindrances, leading to a good imaging of immunolabelledbiological samples in high vacuum using a field emissionapparatus. The same authors showed that SE images of

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good quality are obtained using field emission environmentalscanning electron microscopy (ESEM) and IGL of biologicalsamples. The advantages of ESEM process are to avoid thesteps of sample preparation (dehydration, metallization, etc.).

These problems can now be overcome, with the aid ofthe ESEM which allows the imaging of wet systems withno prior specimen preparation. Since the introduction ofthis instrument, many attempts have been made to performCSEM imaging of samples in a gaseous environment. Themodern ESEM was developed about 20 years ago, only afterthe introduction of the gaseous detector device (Danilatos,1988, 1990). The ESEM allows the examination of anyspecimen, wet or dry, insulating or conducting in situ andclose to its original state, while the environmental gas mediumproduces brand new possibilities of operation and imaging.The implementations of this technique go beyond the realms ofmicroscopy, such as applications to modern nanotechnologiesand micro-engineering. Through our experience as users of awide range of CSEM and of scanning transmission electronmicroscopy, we have tried to consider the advantages andhindrances that the introduction of the ESEM could giveto research in biomedicine. Especially, the benefits it wouldreceive from IGL, little applied so far in the CSEM (Muscarielloet al., 2005).

One of the main limitations quoted by biological ESEM usersis that effective resolution is reduced in ESEM compared toSEM (Kirk et al., 2009). The intrinsic resolution is actuallyuntouched as it depends on probe size, which is the same inboth instruments (given the same beam voltage and currentsettings); specifically in this investigation the same instrumentwas used for both ESEM and SEM so that direct comparisonscould be possible. The signal-to-noise ratio is degraded slightlyin the ESEM due to the beam skirt, and the nature of thesamples themselves is the key reason for the loss of finedetail (Kirk et al., 2009). Similar case to the observationof spheroids of reaggregated neuron cultures were observedthrough ESEM: live spheroids collapsed as water evaporated.The observation of fully hydrated fixed spheroids was madepossible adjusting carefully the chamber pressure. A thin filmof water would prevent a clear view of the surface detail.This would evaporate revealing the surface while preventingboth accidental shrinkage and rewetting (Uroukov & Patton,2008).

The attempt to visualize highly hydrated structures throughSEM was hindered by the necessary preparatory steps, whichcould lead to such alteration that could mask the originalstructure of the very fragile matrix. The fibrillar feature ofthe Enteromorpha spore adhesive images obtained throughstandard SEM was considered as a dehydrated alteration afterbeing visualized through ESEM as a featureless, swollen gel-like adhesive pad. In contrast to the traditional high-vacuummethod, the ESEM enables the imaging of samples in a partlypressurized gas. This eliminates most of the sample preparationsteps discussed later. However, one of the main hindrances

quoted by biology ESEM users is that effective resolution isreduced in ESEM compared to SEM (Wiegemann & Lehmann,2009).

Another question arises about the best choice of gas tobe used in ESEM conditions (gaseous electron-ion) whichshould be the most appropriate to work in low vacuum,partly because a better signal is obtained when workingwith low accelerating voltage, in order not to damage thebiological sample, especially when observing thin layer cellsand unicellular material (Morgan & Phillips, 2006; Danilatos,2009).

ESEM involves the synergy of several techniques operatingin unison. The commercial instruments have not fullyimplemented all the features for optimal use yet. As a result,a large number of users of commercial ESEM are severelyconstrained in the following conditions: maximum pressureattainable, minimum accelerating voltage possible, amountof freedom to specimen movement, detector positioning anddesign, field of view limits, column contamination and imageastigmatism, electron gun life and instrument maintenance(Danilatos, 2009). These, in turn, result in rare damage ordifficulty in handling the specimen, practical loss of resolutionand a general limitation on range of possible applications.Argon gas density along the axis of pressure that limits theaperture in ESEM was based on the Monte Carlo method,with direct simulation of the molecular gas dynamics anddirect simulation of gas flows. The electron beam transmissionwas further computed for helium, neon, hydrogen, oxygen,nitrogen and water vapour. The study constitutes the basisfor the design and construction of an ESEM with optimalelectron beam transfer, which is the primary requirement foran optimal performance instrument (Danilatos, 2009).

Our laboratory is involved in biomaterials and tissueengineering research, a field in which the use and importanceof ESEM have increased progressively during recent years:the synthesis and characterization of poly (2-hydroxyethylmethacrylate) polyelectrolyte complexes (Rosso et al., 2004),the effect of surface nanostructures on adhesion andproliferation of 3T3 Swiss albino mouse fibroblasts (Rossoet al., 2006), the cell surface protein detection with IGL inESEM (Muscariello et al., 2008), in vitro study of osteogenicdifferentiation of human adipose stem cells on β-tricalciumphosphate three-dimensional scaffold (Marino et al., 2010).The advantage of using the ESEM operating in wet modeis that it is not necessary to make non-conductive samplesconductive. Material samples do not need to be desiccatedand coated with gold–palladium, for example, and thus theiroriginal features can be preserved for further testing ormanipulation.

Relative humidity (RH) can also be controlled bymanipulation of the temperature and pressure in the ESEMchamber (Danilatos, 1981) we also need to point out thedistinction between the use of the SEM in low vacuum andESEM modality itself. The first only operates in vacuum level

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0.01–1 Torr. The second, with a Peltier Stage can vary thetemperature from−20◦C to+55◦C) and adjusting the vacuumpressure from 0.01 to 20 Torr, determining the value of RHof the sample (0–100%), giving the chance to observe thechanges occurring with cycles of dehydration and rehydrationof the sample as well.

One of the main features of ESEM is the opportunity towork on biological samples avoiding their alteration due tothe preparatory steps of the sample, in particular fixation anddehydration. Biomaterials and tissue-engineering researchis probably the field in which ESEM has found its fullestapplication, because it enables investigation of both cell andmaterial surface morphology in hydrated conditions (Stokes,2008; Iliescu et al., 2008; Wiegemann & Lehmann, 2009).

The introduction of reliable immunological methods toproduce good quality monoclonal and polyclonal antibodiesand to couple proteins with gold particles so to developimmunological probes, allows researchers to investigate awide range of antigens in cells and tissues. The most commontechniques of immunohystochemistry involve the use of twoantibodies called primary and secondary. Primary antibodyrecognizes a specific antigen and binds to it while the secondaryantibody recognizes and binds the Fc fragment of primaryimmunoglobulin. The latter is conjugated to a substancethat can be observed with a light microscope (chromophoresor fluorescent) or with an electron microscope (metallicnanoparticles). Gold particles may be conjugated to primaryantibodies for identification of antigens but are generallyemployed as secondary antibody markers. The combinationof ESEM with IGL allows an easier localization of cell surfaceproteins and elaboration of their molecular profile.

Socher & Benayahu (2008) used cellular systems formolecular imaging along with localization of a cell surfaceantigen, SVEP1 protein. SVEP1 was studied using theimmunogold technique for the identification of cell surfacefeatures and protein localization (Socher & Benayahu (2008)).They used an antibody to SVEP1 molecule, labelled withsecondary antibody conjugated with gold particles. That waythey demonstrated that silver enhanced IGL-SEM method forcell surface protein detection provides good resolution andallows micro-architecture and antigen appearance imaging.The immunogold particles mark the antigenic site whereSVEP1 is expressed with high resolution and high reliability.

Moreover, the observation through ESEM, where samplesare not coated with a conductive layer that would mask smallgold- or silver-enhanced gold particles, makes the observationclose to a ‘native state’ of the sample (Weston et al., 2009). Thistask requires a constant increase of capacity and potentialof usage of an instrument such as the ESEM, leaving to themost knowledgeable the task to find better performance of theinstrument.

With the acquisition of the ESEM, wishing to develop thedistinctive features necessary for our type of research, wefinally decided to get a more reliable and replicable results

through the IGL. Because the galactin-3 is a protein widelystudied in our laboratory, we tried to develop a protocol andmethod of use of the ESEM which led us to the results shownin Muscariello et al. (2008). There we presented the method,optimized to improve the intensity and the specificity of thelabelling signal, to obtain a semi-quantitative evaluation ofthe same. In this work we used a combination of IGL andESEM to detect the presence of a protein on the cell surface. Toachieve this target we chose as experimental system the 3T3Swiss albino mouse fibroblasts and galectin-3.

The optimization of the method and the semi-quantitativeanalysis was carried out on stock cultures of Swiss albinomouse 3T3 cells. For this positive result, operating in theproject for ‘Validation of molecular markers and technologyinnovation in ESEM, for the clinical and prognostic settingof clonal proliferative thyroid lesions’, we decided to switchthe experimental phase from ‘in vitro’ to ‘field’, using theESEM significantly as a tool but also for diagnostic support.In collaboration with clinicians it was selected through thecombination between Thin Prep technology by fine needleaspiration (FNA) on thyroid nodules and IGL in ESEM.

The possibility of using the IGL in ESEM as an appropriatediagnostic complementary test seems a valid option in thecytopathology of many tumours, particularly for some kindsof tumours of the thyroid.

FNA cytology has been the principal method of preoperativediagnosis for many years. The standardization of FNA cytologysystems can make results easier to understand for cliniciansand give clear indications for therapeutic action (Polleret al., 2008). Within the past decade, several methods basedon thin-layer preparations of cytological specimen have beenmade available to the cytopathologists with the purpose ofimproving their diagnoses. The main publications are inthe field of gynecological cytology. The major advantages ofthin-layer techniques compared to conventional preparationsare optimal viewing of cellular functions by adequate andrapid fixation and decrease in the number of red blood cells,inflammatory cells and mucus. Reported clinical evaluationsin non-gynecological cytology, especially using FNA followedby Thin Prep technique, are limited (Cochand-Priolletet al., 2003). The preparation of conventional smears frommucoid samples, despite mucolysis, can pose difficulties for thecytotechnologist, particularly in determining the appropriateamount of material per slide. When sample concentration isnot ideal, this can result in slides that are either too scanty ortoo cellular (Rana et al., 2001).

At first, the Thin Prep Processor was an automated slidepreparation unit used mainly in Pap test (Papinicolaou Test)for cervical cancer testing. Now, the Thin Prep is also usedfor non-gynecological specimens such as for the diagnosis ofcancer of the lung, bladder, and gastrointestinal tract andin the preparation of FNA of thyroid and breast. The ThinPrep Processor utilizes a computerized process and patentedmembrane technology that controls dispersion, collection

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and transfer of diagnostic cells from the sample to the slide(Cytyc Corporation, 1993). This ensures that the slide isrepresentative and can reproduce the original sample. Underthe control of the instrument’s microprocessor, a gentledispersion step separates blood, mucus and non-diagnosticdebris, and then thoroughly mixes the sample.

Sometimes good histological and histochemistry tests arenot as sharp analytical observations—conducted throughESEM—of cell markers which allow the evaluation of tumour-specific proteins particle per particle, expressed on the surfaceof each single cell.

In this work we used a combination of IGL and ESEM toevaluate cell surface galectin-3 expression, on thyroid cells byFNA cytology Thin Prep processed, trying to obtain substantialand reliable results through the method of use of the ESEM thatis also accessible to casual users of electron microscopy.

Materials and methods

Environmental scanning electron microscopy

For the choice of ESEM parameter, the imaging conditionswere optimized to keep stable level of RH of 60%, the chamberpressure and the temperature on the Peltier Stage were setto 2.5–3.6–5.5 Torr, with water vapour, respectively set to0◦C–4◦C–10◦C, varying the accelerating voltage (HV) to 7–12.5–15 keV.

After assessing the conditions to reach acceptablereproduction of the samples observed, the choice for theacquisition of micrographs in ESEM mode were: 15 keVof accelerating voltage, using Peltier Stage at 4◦C and thechamber pressure at 3.6 Torr, with water vapour and gaseoussecondary electron detector. The 50 slides of 15 FNA in ThinPrep were observed and low- and high-magnification imageswere recorded. No Critical Point Dryer or alcohol dehydrationwas carried out before ESEM observation.

High-magnification images (12 000×, working distance7.5 mm) were used as they allow distinguishing labelnanoparticles from precipitates, both for size and shape. Lowermagnification images were used to define the distribution ofthe label on single cells (3000–6000×) or all over sampleareas.

The observations were performed with an ESEM Quanta200, FEI Europe Company, the Netherlands.

Galectin-3 IGL

Galectin-3 IGL was performed as follows:1. FNAs of thyroid nodules were treated with CytolytTM

Solution (Cytyc Corporation, Boxborough, MA), a buffersolution containing methanol, mucolytic and hemolyticagents for the erythrocytes lysis supplemented with afixative solution containing methanol and then processedaccording to the Thin Prep 2000TM method (Cytyc

Co., Marlborough, MA) using Sequence 2 (FLU/FNA,evaluate).

2. Slides ejected from the Thin Prep were suitably shaped forspecimen stage of ESEM.

3. In each experimental session, as galectin-3 positivecontrol, we used Swiss albino mouse fibroblasts 3T3cells that were maintained in Dulbecco’s modified Eagle’smedium, supplemented with 10% foetal bovine serumand 1 mM sodium pyruvate in humidified 5% CO2,95% air atmosphere at 37◦C. We employed, as negativecontrol, stock cultures of D16 mouse mesoangioblast cellsderived from dorsal aorta of mouse embryo were employed(Muscariello et al., 2008). Briefly, cells seeded at 1 × 104

cell/disc were grown for 1 day on glass slides. Cells wererinsed twice with PBS (pre-heated at 37◦C) and fixed for3 min with pure methanol pre-cooled at −20◦C.

4. Thin Prep slides and 3T3-D16 glass slides were washedtwice with 20 mM Tris-HCl buffer pH 7.5 containing NaCl500 mM and between 20 0.1% (v/v) (TBST) for 5 min.

5. Blocking—with 1% normal serum (normal goat serum,sc-2043, Santa Cruz Biotechnology) and 5% non-fat driedMilk (BioRad) in TBST—was carried out for 40 min.

6. Samples were washed twice with TBST for 5 min.7. Incubation with anti-galectin-3 rabbit monoclonal

antibody (mouse monoclonal IgG, 1 mg/mL, AffinityBioReagents), diluted 1/400 in 5% non-fat dried milkTBST, was carried out for 1 h at room temperature.

8. Samples were washed three times with TBST for 5 min.9. Incubation with anti-mouse nanogold conjugate

antibody (Nanogold anti-mouse IgG, 80 µg/mL,catal#2001, Nanoprobes), diluted 1/600 in 5% non-fat dried milk TBST, was carried out for 1 h at roomtemperature.

10. Samples were washed three times with TBST for 10 min.11. Samples were washed three times with distilled H2O for

10 min.12. Silver Enhancement (Molecular Probes), 15 min at 6◦C,

reaction was carried out to obtain a cleaner signal. Thereaction was stopped with 1% acetic acid.

13. Samples were washed three times with distilled H2O for10 min, ready for observation throughESEM.

Results

ESEM technology allowed acquiring images of gold particlesand the cell surface with its morphological details in conditionsof hydration such as 3.6 Torr chamber pressure, withwater vapour, at 4◦C of temperature, corresponding to 60%RH. These conditions were chosen after the observation ofsome preliminary samples at different values of pressure,temperature, accelerating voltage, spot size, work distance, tiltangle, etc. The Peltier heating/cooling stage allows us to workin the range −25◦C/+55◦C, allows us to achieve 0%>100%RH on the sample surface, in presence of chamber pressureranging from to 0.1 to 20 Torr. Having limited the options

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Fig. 1. Test in different conditions of chamber pressure, temperature, high voltage and relative humidity. Chamber pressure, with water vapour, variedas follows: (A) 2.7 Torr, (B) 3.6 Torr and (C) 5.5 Torr. Temperature varied as follows: (A) 0◦C, (B) 4◦C and (C) 10◦C. Accelerating voltage varied asfollows: (A) 7 KeV, (D) 12.5 KeV and (G) 15 KeV. Relative humidity: constant for all: 60%. Bar = 5 µm.

to the best ones, they were considered good enough to takepictures containing good morphological information and cleardefinition of immunogold particles.

In Fig. 1, we show some preliminary test combiningpressure at 2.7–3.6–5.5 Torr with water vapour, temperatureat 0◦C–4◦C–10◦C, accelerating voltage at 7–12.5–15 keVwith RH fixed at 60%. While each micrograph was notvery different from another, our opinion was to choose theparameters of Fig. 1H: accelerating voltage 15 keV, 3.6 Torrchamber pressure, with water vapour and 4◦C of temperature,corresponding to 60% RH.

To demonstrate the specificity of the IGL, based on literaturedata, we chose 3T3 Swiss albino mouse fibroblasts asexpression of galectin-3 cell line (Moutsatsos et al., 1987;Hubert et al., 1995; Muscariello et al., 2008). D16 mousemesoangioblast cells where considered as a possible negativecheck. Different concentrations of the secondary antibodywere used to determinate the amount of IG necessary to labelall the galectin molecules without rising non-specific bindingsto the cell surface.

The accuracy of ESEM–IGL method was assessed bythe presence of evident labelling on 3T3 Swiss albinomouse fibroblast surface (positive control) (Fig. 2A),further confirmed by the absent labelling on D16 mousemesoangioblast cells (negative control) (Fig. 2B).

Specimens were first collected by FNA of thyroid nodules andtransferred in the CytolytTM Solution (Cytyc Corporation), abuffer solution for the erythrocytes lysis, containing methanolas a fixative and then processed according to the Thin Prep2000TM method (Cytyc Co.).

The FNA of the thyroid gland is a procedure for simpleimplementation, minimally invasive and virtually painless,which is done through fine needle usually by a medicalspecialist to obtain cellular material to be submitted tocytology. The Thin Prep Processor is an automated slidepreparation unit used mainly in Pap test (Papinicolaou Test)for cervical cancer testing. The Thin Prep is also used for non-gynecological specimens such as for the diagnosis of cancersof the lung, bladder, and gastrointestinal tract and in thepreparation of FNA of the thyroid and breast.

The cellular material was transferred onto a glass slide usingcomputer-controlled mechanical positioning and positive airpressure. The slide was then ejected into a cell fixative bathcontaining methanol, ready for staining and evaluation.Normally, a sufficient quantity of cells was collected on a singleThin Prep slide (Fig. 3A). The cells, separated from taintingblood, mucus and colloidal material, became visible as a thin,uniform layer of cells on a slide for microscopic observation(Fig. 3B). The slide, suitable shaped for specimen stage of ESEM(Fig. 3C), was processed for galectin-3 IGL.

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Fig. 2. (A) Immunogold labelling of galectin-3 in 3T3 Swiss albino mousefibroblast surface cell. Particles of IGL are well evident. Bar = 10 µm. (B)Are no visible particles of IGL in D16 mouse mesoangioblast cell. Bar =2 µm (B). Both micrographs were acquired with chamber pressure of 3.6Torr with water vapour, temperature of 4◦C, high voltage of 15 KeV andrelative humidity of 60%.

The labelling was evident and clearly identifiable in thecontest of the surrounding cell surface, due to the mixed SE-BSE signal detected by the gaseous secondary electron detector(Fig. 3D). As optimal conditions for silver enhancementreaction (15 min at 6◦C) were chosen those that led to theformation of round shaped, easily detectable particles with anaverage diameter of 58.4 nm, and, at the same time, producedless precipitates. Occasional particles above 100 nm wereconsidered self-nucleation precipitates.

The 50 slides by 15 FNA of thyroid nodules showed agreat variety in the structure of cells observed, but the

Fig. 3. (A) Image of a typical Thin Prep procedure with cellular materialacquired on slide with specific circular area marked. (B) Portion of ThinPrep cell layer observed to light microscope. Bar = 50 µm. (C) Similararea of Thin Prep cell layer observed to ESEM. Bar = 50 µm. (D) Singlethyrocyte cell methanol fixed in Thin Prep process, observed to ESEM.Bar = 5 µm.

majority of them (10–15 µm in diameter) were representedby typical thyrocytes (Fig. 3D). The cellular materialwas preserved and showed well-defined morphologicalfeatures, without particular damage due to FNA, ThinPrep preparation, methanol fixation and ESEM observationconditions.

IGL in ESEM has allowed the acquisition of different galactin-3 expressed on the surface of cells extracted from different FNAof thyroid nodules, almost absent on some cell types (Fig. 4A)and particularly abundant on others (Figs 4B and C).

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Fig. 4. (A) Single thyrocyte cell (10–15 µm in diameter) with lowimmunogold labelling of galectin-3. Bar = 5 µm. (B) Larger cell with moremarked. Bar = 10 µm (B). (C) Detail of other cell strongly IGL marked.Bar = 2 µm. All micrographs were acquired with chamber pressure of3.6 Torr with water vapour, temperature of 4◦C, accelerating voltage of15 KeV and relative humidity of 60%.

Discussion

The introduction of ESEM, working in gaseous atmosphere,represented a new perspective for many Biology researchers.ESEM is a very important facility in cellular biology andrepresents an ideal ‘experimental application tool’ for IGLprocedures. The absence of complex sample preparationprotocols reduces greatly the risk of antigen loss andbackground diffusion, whereas the lack of metal coating allowsidentifying and characterizing the staining particles with greataccuracy (Muscariello et al., 2008).

The use of the ESEM, although still much to improve, hasproven very useful in biology, in particular in the study ofcell cultures. It cannot replace the CSEM in full or waive theobservation of samples fixed, dehydrated and metalized. Someauthors confirmed a range of alterations generated duringfixation and dehydration of cells for SEM, including crackingand gross shrinkage, whereas ESEM shows how it is possible toproduce good images of mammals cells revealing fine detail ofdelicate features such as filopodia and membrane ruffles (Kirket al., 2009).

ESEM, invented in 1970 and marketed since 1988, still hasmany usage problems from a practical point of view. In biology,most of the studies with ESEM are based mainly on the search

for better conditions of observation rather than applicationsin biomedicine. Recent research work of environmentalscanning microscopy to determine biological surface structurediscusses and includes new evidence about the effect of ESEMimaging on the viability of mammals cells. It shows that,although specimen preparation for high-vacuum throughCSEM introduces some alterations, there are challenges in theuse of ESEM too, particularly at higher resolutions. Concludingthat all studies of the effect of ESEM imaging on biologicalsamples are necessary to develop furthermore the promisingfield of dynamic experiments currently ongoing in ESEM. (Kirket al., 2009).

Engineers and analysts have done a great job in improvingmore and more technical features of the ESEM, but muchremains to be done. However, we believe that is the taskof biologists to understand and appreciate the use of theESEM showing its practical utility. This can occur throughexperience that demonstrates easy use and ensures reliableresults. In electron microscopy, many techniques (freeze-etching, X-ray microanalysis, cryo-ultramicrotomy, etc.) werenot widely used in biomedicine for their excessive complexity;few people would be willing to spend much of their timeacquiring a technique with uncertain results: EM is an aid forsome research but it cannot be considered ‘Research’. Reliableresults should be more often obtained increasing at the sametime the easy use of the instrument.

Basically, every time you observe a new type of biologicalsample preparation is required for a better check of theprotocols, the configuration of the machine and the choiceof parameters useful to obtain the best result. We didthese operations testing some different conditions of chamberpressure, temperature, accelerating voltage, RH, as shown insequence of Fig. 1.

To define the settings, monitor and FNA-Thin Prep for theIGL, we planned to considered the protocol that includes theuse of methanol instead of more typical methods of chemicalfixation (i.e. glutaraldehyde, formaldehyde, etc.), avoidingthe accumulation of electrical charge throughout the sample(Muscariello et al., 2008). This type of fixation certainlypreserves less cell morphology but also avoids the need toneutralize the effects of high background and non-specificlabelling.

In this work we still need to recognize the morphology ofthe cells. Mainly we want to get a good immunolabelling,without background and very selective: the setting of ourESEM needs to be understood in this light. If we cared morefor the morphological aspects of the sample we should havesafeguarded it working at lower voltage and perhaps athigher RH. Instead, our setting choices were made to obtaingood immunolabelling, neatly visible for lack of backgroundand with good balance between contrast and brightness. Inview of the fact that these samples are only mildly fixedin methanol, the quality of morphological micrographs isacceptable (Figs 2A and B, 3D and 4B).

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Our choice of using ESEM technology could appearpreferential. We appreciate the various possibilities given byCSEM and LVSEM, but our strong preference for the ESEM isdue to the necessity of IGL that is reproducible, reliable andeasily obtainable in a few hours after sampling. This idea refersto the great potential of EM, ESEM in particular, in the work ofclinicians and cytopathologists. In this study, where appliedbiotechnology together with the work of clinicians in the sameproject, the chosen subject is the observation of IGL samplesfrom FNA and processed with Thin Prep.

FNA is often the first step in the management of thyroidnodules. Some authors think that accuracy of Thin on thismatter has not yet been determined (Tulecke & Wang, 2004;Dey et al., 2000). Other pathologists choose the Thin Preptechnique because it allows to reduce the number of slides andto shorten the screening time (Nasuti et al., 2001; Marandinoet al., 2006). A recent work defines Thin Prep techniquea valid method for the pre-surgery cytological diagnosisof thyroid nodules, offering the possibility of supportivetechniques, such as immunocytochemical techniques andmolecular methods that can, therefore, be complementaryto histological evaluation for further investigation of humanthyroid follicular lesions (Stamataki et al., 2008).

Thin Prep processor produces uniform thin-layer slides,virtually free from alteration factors such as blood, mucusand inflammatory cells, containing a uniform cell layerparticularly suitable for microscopic review as the majorityof cells are concentrated in a specific area of the slide (Figs 3Aand B).

The procedures of ESEM, Thin Prep, the minimumpreparation for IGL and the high capacity to collect isolatedcells (Fig. 3C), offer a perspective able to supply some help tocytological evaluation. They can be seen as a new, original andcomplementary instrument able to support cytopathologistsin their diagnostic work. They already took the opportunityto improve diagnosis when they added immunochemistry tohistological analysis. Today, the IGL–ESEM technology couldgive an effective improvement in cytological interpretation.

With the use of ESEM, we try to draw together the betterfeatures of FNA, Thin Prep and IGL. Thanks to standardprocedures, the risk of bad sample has been eliminatedremoving some systematic error due to preparations and/oroperators (Marandino et al., 2006). In fact, the shift fromFNA to Thin Prep removes variability due to the conventionalsmear preparations, one of the renowned causes of difficultiesin cytological diagnosis (Stamataki et al., 2008).

The good preservation and neat staining of the samplescould facilitate pathologists to make more accurate diagnosis,reducing the number of uncertain cases. If a slide ejectedfrom Thin Prep and treated for IGL is directly transferred toESEM for observation, we could catch the immunolabellingof cell surface of specific proteins, detectable particles withaverage diameter of 58.4 nm. Occasional particles above100 nm were considered self-nucleation precipitates. So they

were not taken into account in the semi-quantitative analysis.Longer reaction times increase the diameter of the particles,reducing the need for magnification, but raise self-nucleationinteractions between silver ions. We chose silver-enhancedmethod for cell surface protein detection because it providesgood resolution and allows imaging of micro-architecture andantigen appearance, using biz from 1.4 nm which guaranteesimproved mobility of the antibody and so a greater specificitythan in systems using larger biz. This, one might think,introduces a further stage of the silver reaction, which couldcause alterations. But in our protocol it does not happenbecause we fix the cells with cold methanol so to avoid thenon-specific deposition of electrical charge (which happenswith aldehydes), which ultimately may cause non-specificreaction in silver. Silver enhancement reagent Ultra SmallGold Conjugates R-Gent SE-EM, is currently available withthe characteristics of slow enhancement rate (Gigout et al.,2008), which makes it easier to control particle growth (silvernucleation) and is most suited for samples fixed with aldehyde,but has not been well tested yet in samples in our ESEM.

Rossi et al. (2005) considered FNA biopsy as the mostreliable diagnostic tool for thyroid nodules. In their paper,some difficult cytological diagnoses were supported by animmunocytochemical study. The efficiency of a panel madeup of RET, HBME-1 and galectin-3 molecular markers wasevaluated in smears processed by thin-layer cytology fromthyroid lesions. Moreover, galectin-3 could play a role in themalignant transformation of thyroid cells, and it is expressedin a high proportion of carcinomas, especially the papillarytype (Gasbarri et al., 1999).

The galectins are a growing family of β-galactoside-bindingproteins, widely distributed in metazoan organisms. Thegalectins are cytosolic proteins. However, there is abundantevidence for their secretion from the cytosol via non-classicalpathways or translocation to the nucleus or the other cellularcompartments (Hughes et al., 1997, 1999). They have beenshown to play roles in many biological events, such asembryogenesis, adhesion and proliferation of cells, apoptosis,mRNA splicing, bacterial colonization and modulation of theimmune response (Liu et al., 2002; Rabinovich et al., 2002).Moreover, galectins play a key role in various pathologicalstates, including autoimmune diseases, allergic reactions,inflammation, tumour spreading, atherosclerosis and diabeticcomplications (Perillo et al., 1998; Wada & Makino, 2001).Each of the 14 known galectins exhibits a specific pattern ofexpression in various cells and tissues. Galectin-3 has beendetected in activated macrophages, eosinophils, neutrophils,mast cells and the epithelium of the gastrointestinal andrespiratory tracts, the kidneys and some sensory neurons(Hughes et al., 1997, 1999).

Although galectin-3 is predominantly located in thecytoplasm, it has also been detected in the nucleus, on thecell surface or in the extracellular environment, suggesting amulti-functionality of this molecule. Its extracellular location

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on the cell surface and in the extracellular milieu indicates itsparticipation in cell–cell and cell–matrix adhesion (Krezslak &Lipinska, 2004).

The experimental observations of cellular materials showeddifferential galectin-3 expression related to the source ofobserved samples. In order to achieve a biological evaluation,we should relate the labelling signal with specific informationabout cell state and condition in thyroid nodules. InFigure 4 we can see three images of cells that are differentiatedby size, type and level of IGL. They are conditions that relateto three different FNA of thyroid nodules. Here are shownonly as an example of the potential investigation of the IGL inESEM.

Later, in a diagnostic study, a large number ofimmunolabelling will be related to all histological andcytopathological analysis made by clinicians. These imagesare presented to show how it is possible, with the ThinPrep, to observe cells that are isolated and well definedmorphologically, assessing the level of expressed protein (inthis case Galactin-3) in each cell. Gold particles may beconjugated to antibodies for identification of antigens ofany specific cell surface protein making sure not to alter orlose protein through sample preparation treatment. If theexperiment is properly performed the result is guaranteedand does not depend on the microscope used being CSEM,ESEM or LVSEM. Instead, the quality of the result dependson the quality of the microscope, on how it is set, whouses it, so that the particles are clearly visible, measurablein a cell also morphologically well preserved. Right now,with the technology available, the ESEM mode could bethe one offering the best compromise between quantity ofexperiments and quality/quantity ratio of results. So the useof electron microscopy in complementary diagnostic aspectscould be considered interesting and satisfactory. The ability todetect the expression of specific proteins on the cell surface(sometimes malignant), in an extremely fast and reliablemanner, through a molecular method by examining severalsamples in a few hours of work, gives great potential to theEM.

Work in progress about the validation of ESEM-IGL methodfor the quantitative and qualitative evaluation of a selectedmembrane molecular marker, with biological significance willbe further examined. For now, we seem to have taken a newstep towards the immediate use of the ESEM-IGL and we hopethat many other users can be involved so to increase thenumber of good and significant results.

Frequent use of optical microscopy and of confocal lasermicroscopy has reached nowadays such a widely spread levelof confidence between their users. The complexity of use andof sample preparation techniques has not yet made possible tobring the EM to a usage level, similar to the aforementionedmicroscopes. The scientific and diagnostic information thatEM can provide are, however, in many cases decisive for theformulation of a successful diagnosis.

We like our work is dissociated from the specific case ofthe Thin Prep by FNA of thyroid nodules and it is consideredas a practical means of the use of the ESEM which must beimproved in quality but can be indeed very useful for somediagnostic exam. Researchers, also using different methodsfrom FNA and Thin Prep, may open the use of ESEM and IGLfor further testing in the identification of cell markers specific ofcancers of the lung, bladder, breast, gastrointestinal tract, andso on.

Obviously, nobody can advance a cytological diagnosis withthis preliminary result of IGL-ESEM on Thin Prep by FNA.In the future a significant statistical analysis with significantnumber of FNA-Thin to create a data base for pathologists.During previous years Thin Prep was used mostly in the field ofgynecological cytology, only recently it began to be associatedto FNA, for non-gynecological specimens, in the diagnosis ofcancers of the lung, bladder, and gastrointestinal tract, and inthe preparation of FNA of the thyroid and breast. Similarly wethink that the extension of Thin Prep to IGL-ESEM could buildup a good complementary opportunity for the citopathologist.

Excellent sample preparation is absolutely critical inbiological electron microscopy of the cell to deliver meaningfuland accurate results. However, each new advance inmicroscopy and preparation technique brings a new range ofalterations, which must be carefully characterised interpretedand solved (Weston et al., 2009).

IGL in electron microscopy has always been stronglyinfluenced by sample preparation. In fact the loss of antigenis often due to fixation, embedding, metallization and other.Also the background and/or non-specific signal can derive bysample preparation methods. With the advent of ESEM thepreparatory phase of the sample was much reduced bringingimprovement of results. Our results, showed, in agreementwith a previous work (Muscariello et al., 2008), that the highresolution power of electron microscopy in ESEM modality isable to evaluate Gal-3 expression on cell surface also on humanbiological samples thin-prep treated.

In the early years of electron microscopy there wascertainly more interest for ultra structural cytomorphologyof biological samples. Later, there was an inevitable drop ininterest and fewer applications for EM in biology were found.Today, biotechnology study of nanoparticles, cell culture,biomaterials, tissue engineering research, new diagnostictools, etc. cannot do without the use of the electron microscope.All that was unthinkable before the ESEM equipment wasavailable as a facility for the observation of samples withminimum preparation.

Acknowledgements

This research has been funded through the Ministryof Instruction, University and Research (MIUR), number200734RMKE: ‘Validation of molecular markers andtechnology innovation in environmental scanning electron

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9 2 G . C A F I E R O E T A L .

microscopy (ESEM) for the clinical and prognostic setting ofclonal proliferative thyroid lesions’.

The authors thank Dr. Ida Di Vicino, Exp. Manager(London), for kind revision of English text.

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