FACT AND ARTEFACT IN CONFOCAL MICROSCOPYT.F. WATSON

Department of Conservative DentistryUnited Medical and Dental Schoolsof Guy's and St Thomas's Hospitals

Guy's Dental School, London BridgeLondon SEI 9RT, United Kingdom

Adv Dent Res 11(4):433-44J, November, 1997

Abstract—High-resolution confocal microscopic imagesmay be made of either the surface of a sample or beneath thesurface. These images can be likened to optical tomograms,giving thin (> 0.35 urn) slices up to 200 um below thesurface of a transparent tissue: With microscopes runningunder normal conditions, the optical section thickness will be>1 um and the effective penetration into enamel and dentin amaximum of 100 um. For maximum resolution, high-quality,high-numerical-aperture objectives should be used. Refrac-tive index matching of the lens immersion media and thesubstrate will avoid distortions of images in the optical axis.Such errors could occur when imaging a considerabledistance (> 40 um) into a cell containing water, with an oilimmersion objective above the cover slip. Care should betaken in the interpretation of computerized z axis recon-structions made from serial optical sections: Their validityshould be checked with equivalent views made with thesample oriented in the same direction as the reconstruction.The use of fluorescent dyes in microscopy is a very powerfulinvestigative technique. It is important that the dyes used notbe labile and that they be well-fixed to the materials beingexamined, or the images may indicate the dye distributionrather than the material to which it is "attached". Multiplelabeling experiments must have crossover controlexperiments to verify the distribution of the individual dyes.Valuable information can often be gained by combininginformation from both reflection and fluorescence images.Two-photon laser excitation of dyes gives the potential forgreater depth penetration and improved resolution.

Key words: Confocal, scanning, optical, fluorescence,microscopy.

Presented at "Advances in the Characterization of Surfaceand Subsurface Areas of Dental Hard Tissues", a workshopsponsored by the Council of Europe and the DeutscheForschungsgemeinschaft (German Research Agency),November 13-17, 1996, at the University of Mainz, Germany

Confocal microscopy has been quite slow to beapplied in dental research, even though theprinciple behind this technique was discovered in1955 (Minsky, 1961, 1988) and the first functional

confocal microscope in a dental school was delivered in late1983 (Boyde et ai, 1985; Watson and Boyde, 1985).

Review articles have been written about the potentialapplications of the technique in dentistry (Watson andBoyde, 1991; Watson, 1994), but little has been written aboutpotential problem areas and how they may be avoided orovercome. The aim of this paper is not to describe the manyapplications of which confocal microscopes are capable, or toenter into lengthy discussions of the science behind theiroperating principles, but to highlight some of the practicalaspects of their use in dental research. Examples will bedrawn mainly from the work that has been undertaken overthe last 13 years, looking at adhesive restoration/toothinterfaces. The reader who specifically wants to find outmore about the technology and its associated techniques isrecommended to consult the original references cited inPawley (1995).

OUTLINE OF THE TECHNIQUE

The expression "confocal" derives from the use of anaperture in the conjugate focal plane of an objective lens inboth the illumination and imaging pathways of a microscope:The area surrounding the aperture rejects stray light returningfrom areas which are not in the focal plane of the lens (Fig.1). For a useful image to be made, some form of scanningdevice is required. Expressed simply, this type of scanningoptical microscopy enables high-resolution images to bemade of samples, often below the surfaces of translucentmaterials, with minimum requirements for specimenpreparation.

High-resolution confocal microscopic images may bemade of either the surface of a sample or beneath thesurface. For subsurface images, an immersion medium isused to couple the objective lens optically with the sample,to produce reflection or fluorescence images of structureswithin semi-transparent materials such as cells and dentalhard tissues. These images can be likened to optical tomo-grams, giving thin (> 0.35 um) slices up to 200 um belowthe surface of a transparent tissue. Such figures are the bestthat can be obtained, with optical systems speciallyconfigured. With microscopes running under normalconditions, the optical section thickness will be > 1 um, andthe effective penetration into enamel and dentin a maximumof 100 um. The technique generates significant improve-ments in resolution, lying somewhere between that ofconventional light microscopy and TEM/SEM (Watson andBoyde, 1991).

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434 WATSON ADV DENT RES NOVEMBER 1997

in focus Lens/Microscope

Aperture

Light source

out of focus out of focus

Fig. 1—Simplified diagram of the confocal microscope principle. The microscope isrepresented as a single lens. Light from the light source is focused within the sampleby the objective as a small patch of light corresponding to the aperture. Only lightreturning from the focused-on plane can return via the lens and aperture. Any lightfrom planes above or below the plane of focus is blanked off by the opaque portionof the aperture.

Fig. 2—Composite (C)/dentin (T) interface recorded as a combined reflectionfluorescence image with oil immersion lens (20X/0.8 NA) but with oil below thecoverslip on the LHS and water below on the RHS. The dentin tubules can be seenmore easily with the oil immersion, whereas the smeared layer produced when thesample was sectioned with a diamond saw prevents good contrast from the waterimmersion. If the sample had been polished, the water immersion image would havebeen significantly better. The yellow structure at the interface is a rhodamine-labeledbonding agent. Green, 546-nm illumination, Noran TSM. Fieldwidth, 130 pm.

RESOLUTIONAND CONTRAST

Factors affecting the resolution ofoptical microscopes (the ability todiscriminate between two pointsources of light) have been dis-cussed at great length in text-booksof microscopy (Inoue, 1986). Reso-lution is a feature inextricablylinked with image contrast. By theconfocal principle, both resolutionand image contrast can be improvedwhen compared with conventionallight microscopy. However, otherbasic microscopy principles canhave a profound effect on thequality of results achieved. Forinstance, it is imperative that theoptical components be well-alignedand clean. Vibration is one of thebiggest problems confronting reso-lution. Purchasing an expensiveconfocal microscope is a waste ofmoney without putting it on an anti-vibration table. It has been said that'you don't know you have aproblem with vibration until you getrid of it!'

The theoretical resolution of aconfocal system is primarily afunction of the numerical aperture(NA, light-gathering ability) of theoptical system and the wavelength(\) of the light. Reducing X. toincrease resolution is not alwayseasy because of the problems withhandling ultraviolet light and thespecial optical components this mayrequire in the microscope. Theimportance of high-NA, high-quality objectives should not beunderestimated for successful con-focal imaging. The best results areobtained with a lens with as high anNA as possible, bearing in mind therefractive index of the mountingmedia used for our samples (oil,1.5; glycerine, 1.4; water, 1.3). Forspecimens of enamel and dentin,immersion oil can work very effec-tively as a mounting medium. How-ever, if left in place for a long time,it will tend to 'clear' the sample asstructures such as dentin tubulesdisappear from view once the oilsoaks into the sample. Glycerine is a

VOL. 11(4) PRACTICALITIES OF CONFOCAL MICROSCOPY 435

very useful material for examininghydrated dental samples, becausefollowing microscopic examination,it can be washed off easily so thatexperiments can continue. It shouldbe noted that this hygroscopic fluidcan have a profound dehydratingeffect on water-sensitive materialssuch as glass-ionomer cements, butthis effect can be used to advantagefor dehydration stress studies ofthese materials (Watson et al.,1996). Water immersion objectivesdo not always produce good imagesof dentin structure, when comparedwith oil immersion (Fig. 2).

Problems arise if structureswhich contain a lot of water areimaged deeply below the surface bymeans of oil immersion objectives.Refractive index matching of thelens immersion media and thesubstrate is therefore of greatimportance, to avoid distortions ofimages in the optical axis, as thelens is focused progressively deeperinto the sample. An example wheresuch errors would be inevitablewould be in the imaging of aconsiderable distance (> 40 urn) intoa cell containing water, with an oilimmersion objective above the cover

Fig. 3—Image of rhodamine-labeled beads, 2 pm in diameter, suspended in a solutionof carboxyfluorescein dissolved in a glycerol-based mountant. Dual-fluorescenceimage shown as an x-z reconstruction. Notice the slight elongation of spheres in theoptical axis of the microscope, due to reduced z axis resolution and the 'shadowing'effect from the spheres, as shown by the reduced intensity of light emitted by thefluorescein solution. They were excited with the 488-nm line of an Argon-ion laser andvisualized through a 540 ± 30 nm bandpass filter and a 630-nm longpass filter with anMRC-500 confocal visualization system (Bio-Rad) mounted over an Axipoplanmicroscope fitted with an x63 NA 1.4 oil immersion objective (OI). (photo courtesy ofDr. Alan Entwhistle, Ludwig Cancer Institute, London)

glass(Vissere/a/., 1992).It should also be remembered that there will always be

less resolution in the optical axis of the microscope, and sogreat care should therefore be taken in the interpretation ofcomputerized z axis reconstructions made from serial opticalsections (Fig. 3). Wherever possible, equivalent views shouldbe made with the sample oriented in the same direction as thereconstruction, to confirm the validity of the latter. Forexample, potential problems would be indicated by sphericalobjects appearing elongated in the optical axis of themicroscope.

Reflection images or images derived from light back-scattered from the sample are a very powerful means ofdetermining the integrity of a sample. The best images areobtained with microscopes illuminated incoherently (bywhite light), such as the tandem scanning microscopes(TSMs) rather than the confocal scanning laser microscopes(CSLMs). Laser light produces 'speckle', and this tends tointerfere with image quality and can make image inter-pretation difficult. Notwithstanding, such a surface view isuseful when used in conjunction with fluorescence images(Fig. 4).

One of the most difficult problems in confocal microscopyis the imaging of small reflective objects which are associatedwith a substrate of low inherent contrast. Toothpaste slurries

on dentin illustrate the problem well: The slurry is highlyreflective on the dentin surface. Focusing deeper into thesample (even when a water immersion objective is used) willgive very misleading information about the depth penetrationof the particles, because of massive diffraction effects(Fig. 5). Scattering of light from planes which are out offocus would be exacerbated by the use of an oil immersionobjective in this situation.

SURFACE RECONSTRUCTIONAND MEASUREMENT

A major application of confocal microscopes is the imagingand measuring of surfaces, being widely used in theelectronics industry where integrated circuits are evaluated.Surface profiles can be generated from extended-focuscomputer images derived from multiple image planes,without the need for the specimen to be touched. These havethe advantage of generating many useful measurements, suchas the roughness parameter (Ra), over the complete area inview (Radford et al., 1997). The extent of further 3-Dcomputerized reconstructions that can be made from thesemulti-dimensional data is limited only by the power of thecomputer (White, 1995). Virtually all confocal microscopes

436 WATSON ADV DENT RES NOVEMBER 1997

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F/g. 4—Montage showing four images of a composite (C), primer (P), adhesive (A), and dentin tubules (T) interface. Top left:combined three-color image: P = yellow/green, A - red, T & C = blue (scarcely visible, but appearing pale blue in the air-inhibited zone at the adhesive/composite junction). While this image is attractive, it loses valuable information when comparedwith the individual images that produce it. Top right: reflection image showing tubules (T) and reflective surface of composite(C). Laser speckle is evident in this image. Bottom left: fluorescence image of primer (P), labeled with lucifer yellow. Finestructures, highlighted with open arrows, are less apparent in the combined view. 488-nm line Ar ion laser, 520-nm bandpassfilter. Bottom right: fluorescence image of adhesive (A) labeled with rhodamine B. The presence of part of this layer is maskedin the combined image. 514-nm line Ar ion laser, 600-nm longpass filter. (Imaged with Noran Odyssey, x60 OI/1.4 NA,fieldwidth 80 urn.)

are sold with some form of image processing equipment, anatural corollary of the digitized images that are produced.Storage of these images is getting easier and cheaper ascomputer technology advances. Video rate microscopy willrequire very large data transfer and image storage capacities(Watson, 1994).

As for all imaging with confocal microscopy, the higherthe NA of the objective lens, the better the image quality

(Fig. 6). For all samples, a dry lens (maximum NA, 0.95) willgenerally be effective. However, oil immersion objectivesshould offer advantages because of their improved light-gathering ability (NA < 1.4). The oil contamination of asample may be unacceptable, and if the substrate underexamination is translucent (e.g., resin composite, dentin,enamel), subsurface structures will confuse the image(Watson and Boyde, 1991). Some form of reflective surface

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Fig. 5—Reflection image of a calcium carbonate toothpasteslurry on dentin (maximum particle size, 10 /urn). Thecircular image results from the insertion of an aperture inthe microscope to improve contrast. Top: surface view ofslurry. Bottom: focused nominally 8 jum below the surface.The reflective particles have a profound scattering effect, asshown by the light scattered laterally around this aperturedimage; a diffraction ring can also be seen (arrow). (Imagedwith Noran TSM, x40 WI/0.75 NA; fieldwidth, 100urn[photos courtesy of Dr. Mark Ide].)

(e.g., gold sputter-coating, as for SEM) will then be necessaryto improve light reflection and prevent its absorption by thesample (Fig. 7).

Monochromatic light should be used to reduce the effectsof lens chromatic aberration (Fig. 8), although with full-spectrum light sources, this can be used to give a color-codedimage of the sample (Browne et al., 1992). Color changes canalso be useful for helping to position 'flat' specimens at 90° tothe optical axis of the microscope (Watson and Cook, 1995).

FLUORESCENCE MICROSCOPY

The use of fluorescent dyes in microscopy is a very powerfulinvestigative technique. This has become particularly usefulin confocal microscopy, especially with laser scanningmicroscopes which have tended to rely heavily on fluo-rescence techniques. Laser illumination has the advantage of

Fig. 6—Diagram showing how light reflected by near-vertical walls of a sample may not be collected by a lens oflow numerical aperture (after Boyde and Jones, 1995). Thiswill produce dark areas in the confocal image at each planeof focus and can cause difficulties in interpretation of thedata.

high potential light intensities with the laser beam, but alsothe concurrent disadvantage of rapid photo-bleaching of dyes.The techniques involved are discussed at length in Pawley(1995).

The improved resolution and image contrast can indicatethe distribution of sample components. An example of thiscould be the interface of a fluorescent-labeled adhesivewithin tooth tissue, where extended focus images candramatically illustrate the penetration of these materials (Fig.9). It is important that dyes used should not be labile and bewell-fixed to the materials being examined. For instance,fluorescein is highly soluble in water; therefore, images ofthis dye may indicate its distribution rather than the materialto which it is 'attached'. A control experiment for deter-mining the distribution of resin-based materials is to removethe mineralized tissue at the interface by etching. A reflectionimage is then made of the free-floating tags of material bymeans of a water immersion objective (Watson and Boyde,1987). The tags should be the same length as the tags in thefluorescence image: If they are shorter, there is a strongpossibility of dye partition from the labeled material.

Dyes which have a great chemical affinity for the materialunder examination will be advantageous and may give otherinformation if they are also sensitive to local environmentalfactors such as pH (Fig. 10) (Bhawalkar et al., 1996).Overloading of materials with dye can lead to 'quenching'(an anomalous reduction in fluorescence intensity), whichcan lead to misleading results (Fig. 11). In such conditions,the dye molecules may be too close to one another. Photonsemitted from a dye molecule, following interaction with theexcitation illumination, are absorbed by closely neighboringmolecules, rather than emitted as fluorescent light. Materialproperties should also be checked to ensure that the dye is notchanging the characteristics of the substance underinvestigation.

438 WATSON ADV DENT RES NOVEMBER 1997

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F/g. 7—Profile view (LHS) and extended focus reconstruction from which it is derived(RHS). The white line shows the position of the profile trace on the image. The verticaldistance between the arrowheads is 18 /urn. Hydrofluoric-acid-etched porcelain, sputter-coated with gold, imaged with xlOO O1/1.4 NA. 546-nm illumination, CCD camera,Noran TSMDP software: MISIS Image. Fieldwidth, 50 pm.

Lens Foci

Aperture

White light

B G R

Fig. 8—Diagram (after Boyde and Jones, 1995) showing color coding for depth in aconfocal microscope illuminated with white light such as a TSM. R = red, G = green,B = blue. Such chromatic aberration will be a feature of nearly all lens/microscopesystems and will affect the detection of light returning from the sample to themicroscope. If fluorescent dyes of widely separate wavelengths are used in multiplelabeling specimens, then the images obtained may be derived from different focalplanes in the same sample, unless this problem is corrected for.

Multiple labeling experimentscan produce attractive images, butit is important that control experi-ments be conducted to verify thedistribution of the individual dyes.Such controls would be the use ofdye R in one component and dye Fin another in one sample, and thenreversing the order of the dyeswithin the control sample.

The spectral excitation andemission characteristics of dyemolecules may not be optimallyseparated by the dichroic mirrors,filters, and laser lines available inthe confocal microscope in use.'Cross talk' or mixing of theimages of fluorescent dyes isalways a phenomenon to be awareof in multiple labeling experi-ments. It can be recognized bymaking equivalent samples butonly with single-labeled com-ponents. Similarly, 'reflectionbreakthrough' may occur withfluorescent samples which havestrongly reflective objects in thesame field of view (Fig. 12). Thefilters may be unable to stop all ofthe reflected light in these situa-tions and can lead to misinter-pretation of results. The strength ofthe reflection signal should beindependently assessed from thefluorescence signal if there is anypossibility of strong back-scattering of light by the specimen.

Shadowing effects occur inconfocal imaging, especially where'dense' materials are examined.While light may penetrate atransparent material relativelyeasily, it will be attenuated, or evenstopped, by superficial structures(Fig. 3). This will be apparent onthrough-focusing, as shown by areduction in light intensity for thedeeper layers. However, priorknowledge of the structure wouldindicate that an image should beproduced from these planes offocus. Vertical (x-z) computerreconstructions derived from astack of image planes should betreated with circumspection whenthese images are made of materialsthat are highly light-absorbing and

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-scattering (i.e., most dentalbiomaterials).

Greater depth penetration andresolution are obviously going to beof benefit, and new imaging tech-niques such as 2-photon laser exci-tation of dyes have this potential(Denk et al., 1990). The use of long-wavelength excitation removes theproblems of photo bleaching of dyemolecules. However, the lawsgoverning the transmission of lightstill remain. With the longerexcitation (near-infrared) wave-lengths used in 2-photon imaging,there will be greater sample pene-tration by the exciting light, but it isstill a requirement that the emittedfluorescent light be able to passback through the sample. Therefore,opaque or light-scattering structureswill still produce shadowing effectsin the image.

FOURTH DIMENSION:TIME

Microscopic images will record theappearance of a structure which mayor may not be changing. Classicmicroscopic techniques have tendedto look at 'fixed' tissues, but morerecently, the imaging of dynamicchanges within objects has gained inpopularity. Applications such as themapping of intra-cellular ionic flux(e.g., calcium ratio imaging) havebecome practical with the advent ofsuitable fluorescent dyes (seerelevant sections in Pawley, 1995).Capturing such fast-moving eventspresupposes a detection systemwhich is sufficiently fast andsensitive to record the events as theyhappen. Even when working atvideo-rate (25 frames/s), manydynamic events will still be unde-tectable: 'Absence of proof is notproof of absence'. Two-photonimaging techniques offer significantadvantages in high-speed imaging(Denk etai, 1990).

Many conventional confocalmicroscopes are incapable ofproducing full-frame images atvideo-rate and so will rely on 'linescans' to pick up rapid changes

Fig. 9—Single optical section (top) and extended focus (bottom) images of adhesivepenetration into dentin. 20 slices, reconstruction 20 pm thick. Notice the lateral tubulesof the dentin penetrated with adhesive but the voids in some of the main tubules. Anacid erosion experiment would confirm or deny the presence of resin in the tubules,rather than dissociated dye. Adhesive resin labeled with APSS dye (4-[N-(2-hydroxyethyl-amino)styryl]-N-methylpyridinium tetraphenylborate) (courtesy of Dr.P.C. Cheng and Dr. P.N. Prasad, SUNY, Buffalo, NY, USA). Imaged with OlympusFluoview, 488-nm line Ar ion laser, 520 bandpass filter. x60 OI/1.4 NA. Fieldwidth,130 pm.

440 WATSON ADV DENT RES NOVEMBER 1997

Fig. 10—A resin-modified glass-ionomer adhesive, labeled with APSS which has beendosed with the liquid component of the cement. This dye has a great affinity for HEMA.Paniculate distribution can be seen close to the dentin interface, with the glassparticles displaced from the cavity surface by a swelling of the matrix component ofthe system: the 'absorption layer'. The dye is showing fluorescence around the glassparticles: This may be related to its fluorescence being strongest in an alkaline-localized environment. The dye was originally developed for 2-photon imaging, andwhen these samples are imaged with this technique, high-resolution images can beproduced up to 80 fjm below the surfaces of these dense materials. When imaged witha conventional confocal microscope, as here, the maximum useful penetration will beonly about 10 to 20 pm. Imaged with Olympus Fluoview, 488-nm line Ar ion laser,combined reflection/fluorescence image. x60 OI/1.4 NA. Fieldwidth, 75 p.m.

(Watson, 1994).While video-rate imaging offers

many exciting possibilities, timelapse studies, of which all confocalmicroscopes are capable, will alsogenerate interesting informationregarding changes within materialsover time. The major advantage ofconfocal imaging will be that thesamples can be kept in near-normalconditions. However, as mentionedearlier, the microscope operatingconditions must be stable, with nodrift in the microscope stage orillumination intensity (Watson et ai,1996).

CONCLUSIONS

As with all new techniques, theinexperienced investigator should beaware of some of the artefacts inhe-rent within the system. However, thewidespread availability of confocalmicroscopes should give ampleopportunity for dental researchers tocapitalize on this technology.

ACKNOWLEDGMENTS

I would like to thank Dr. AlanEntwhistle for the image in Fig. 3and Peter Pilecki for continuingskilled technical support.

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Fig. 11—Composite (C), adhesive (A), dentin tubules (T) interface. The adhesive hasbeen overdosed with rhodamine and is showing 'quenching'. The fluorescence signal isproduced only where dye concentration is reduced, as in the air-inhibited layer andhybrid zone (arrowed). Fluorescence image LHS, combined reflection/fluorescenceRHS. Imaged Noran TSM, x20 OI/0.8 NA, Fieldwidth 300 pm. 546/600 nm.

Fig. 12—Amalgam (AM) and adhesive interface with dentin. Red/orange adhesivelabeled with rhodamine. 'Reflection breakthrough' shows as a green component of theimage. Imaged Noran TSM, x20 OI/0.8 NA, Fieldwidth 300/urn. 546/600 nm.

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