reflectance confocal microscopy for in vivo skin imaging

Reflectance Confocal Microscopy for In Vivo Skin Imaging†

Piergiacomo Calzavara-Pinton*1, Caterina Longo2, Marina Venturini1, Raffaella Sala1

and Giovanni Pellacani2

1Department of Dermatology, University of Brescia, Brescia, Italy2Department of Dermatology, University of Modena and Reggio Emilia, Modena, Italy

Received 18 March 2008, accepted 14 July 2008, DOI: 10.1111 ⁄ j.1751-1097.2008.00443.x

ABSTRACT

Reflectance confocal microscopy (RCM) is a novel noninvasive

technique for ‘‘in vivo’’ examination of the skin. In a confocal

microscope, near- infrared light from a diode laser is focused on

a microscopic skin target. As this light passes between cellular

structures having different refraction indexes, it is naturally

reflected, and this reflected light is then captured and recomposed

into a two-dimensional gray scale image by computer software.

Focusing the microscope (adjusting the focal point on the z-axis)

allows images to be obtained of different levels within the skin.

Commercially available microscope systems of this type can

create images with enough detail for use in histological analysis.

The first investigations using these microscopes served to identify

the appearance of the various cell populations living in the

different layers of normal skin. Today, the main interest has

become focused on the use of these microscopes as a diagnostic

tool: a means of investigating benign and malignant tumors of

melanocytes and keratinocytes, and, more importantly, the

findings of this field of study can be used to develop a diagnostic

algorithm which would be not only highly sensitive but specific as

well. The aim of the paper is to provide an updated literature

review and an in-depth critique of the state-of-the-art of RCM

for skin cancer imaging with a critical discussion of the

possibilities and limitations for clinical use.

INTRODUCTION

Even expert dermatologists can diagnose correctly only around75–80% of skin tumors, and this figure is probably even worse

for general practitioners and doctors with other specializations(1). Therefore, the final diagnosis is often based on a surgicalbiopsy. This is an invasive method, which is painful, leaves a

scar, and is time consuming because several days are neededbefore the skin sample is ready to be examined and anhistopathological diagnosis can be given by the clinician.Therefore, in vivo non-invasive diagnostic techniques, e.g.

dermoscopy and digital epiluminescence (2), high frequencyultrasonography (3), optical coherence tomography (4), mag-netic resonance imaging (5) and reflectance confocal micros-

copy (RCM), also known as confocal scanning laser

microscopy, have been developed. All of them provide

additional information that is not readily available throughmere clinical inspection, and all preserve the tissue, and canprovide not only real-time diagnostics, but they also give the

possibility of following the progression of skin lesions overtime (6).

However, among these new techniques, RCM is the only

one at this time with a power of resolution approaching thatnecessary for seeing the details necessary for histologicanalysis, and therefore the only one that could become a truealternative to a physical dissection of the skin (7–11).

THE CONFOCAL PRINCIPLE ANDRCM TECHNOLOGY

Although the general principles of RCM were described byMarvin Minsky in as early as 1957 (12), it was only in the pastdecade that advances in optical and electronic technologies

allowed a reflectance confocal microscope to be developed,suitable for clinical ‘‘in vivo’’ examination of skin lesions.

These devices use a laser as a source of monochromatic and

coherent light.Wavelength range and power of the laser have a critical

importance. Light transmittance in Caucasian skin increases

progressively with wavelength in the near-infrared region(700–1400 nm) (13), though longer wavelengths provide limi-ted lateral resolution (7). The power of the laser light-source

which can be utilized is limited by the hazard of tissue damageand skin sensitivity.

The light that these microscopes use passes through a beamsplitter, a scanning and focusing optical lens and a skin contact

device (Fig. 1).This device serves to keep the skin laterally stable, and

therefore to reduce motion blurring. It consists of a metal ring-

and-template that is placed in contact with the patient’s skinby a double-sided adhesive tape and that can be magneticallycoupled to the microscope housing. It holds the immersion

medium, usually water or water-based gels, because theirrefractive index (1.33) is close to that of epidermis (1.34),eliminating needless reflection.

In the skin, light is focused on a small tissue spot a few

microns of diameter. Reflection (back scattering) occurs at theboundary between two cellular structures having differentindexes of refraction, such as membranes, keratohyalin gran-

ules and melanosomes. Reflectance can also occur when theobject viewed has a size similar to the illuminating wavelength.

†This invited paper is part of the Series: Applications of Imaging to Biologicaland Photobiological Systems.

*Corresponding author email: [email protected](Piergiacomo Calzavara-Pinton)

� 2008TheAuthors. JournalCompilation.TheAmericanSociety ofPhotobiology 0031-8655/08

Photochemistry and Photobiology, 2008, 84: 1421–1430

1421

Light reflected from the focal point propagates back toward

the objective lens that focuses it into a small pinhole-sizedspatial filter positioned in front of the photodetector. Thepinhole acts as a physical barrier or mask, and the diameter of

its aperture is matched to that of the illuminated spot and thusdetermines the thickness of the optical slice. As a result, lightwhich is returning from out-of-focus planes, i.e. light emanat-

ing from above and below the point we are interested inviewing, is masked out by the pinhole so that the detectorreceives light only from the thin plane of the specimen that is in

focus (optical sectioning). The name ‘‘confocal’’ originates justfrom the fact that the point source of light, the illuminatedspot in the sample, and the pinhole aperture lie in opticallyconjugated focal planes.

By changing the depth at which the objective lens focuses inthe z (vertical) plane with respect to the skin, we can image anyparticular layer within the skin by scanning horizontally in two

orthogonal directions (y-and x-axis) parallel to the plane offocus, and sending these optical signals through the interfacesoftware to reconstruct a thin horizontal microscopic image.

We can create a series of these images that would stackvertically from the stratum corneum all the way to the upperpapillary dermis. The resolution of these virtual sections on the

z-axis is 2–5 lm and therefore their thickness correlates closelywith the axial thickness of the excised histologic sections,easing interpretation.

Such a sequence of confocal images can be obtained with

commercially available 8 bits ⁄ pixel frame grabber and imagingacquisition software. The grabbed images are enhanced asfollows: cropping, scaling with bilinear interpolation, linear

3 · 3 filtering (i.e. moving average of the matrix of 3 · 3 pixelsusing triangular kernels), and contrast adjustment (13). Besidesthe advantages that sequencing can provide, the confocal

image is displayed in real time on the screen and then can beprinted or recorded immediately (14–16).

The commercially available near-infrared reflectance micro-scope (Vivascope 1500, Lucid, Inc., Henrietta, NY) that we use,

is equipped with a diode laser with peak emission at 830 nmand has a maximum power of 35 mW. With this system, eachimage has an effective 500 · 500 lm field of view and the

imaging depth in normal skin is 200–300 lm, i.e. the level of

papillary dermis and upper reticular dermis and the spatialresolution in the lateral dimension is 0.5–1.0 lm (6,7,11).

CONFOCAL HISTOLOGY OF NORMAL SKIN

Keratinocytes of the various skin layers have different appear-ances, and can be identified by measuring their distance from

the stratum corneum.As we look down into the skin, the first layer we see is the

stratum corneum. It consists of large (10–30 lm), anucleatedpolygonal cells that appear very bright because the difference

in the refractive indexes at the interface between the immersionmedium (water or gel as described above) and the stratumcorneum results in a large amount of back-scattered light.

They have dark outlines and form islands separated by fissuresand wrinkles, which appear very dark. It overlies the super-ficial layers (stratum granulosum and stratum spinosum) that

appear in confocal images as a honeycombed pattern which isformed by 10–25 lm polygonal cells with dark nuclei sur-rounded by bright white thin cytoplasm.

Basal cells are even smaller in size, about 7–12 lm and arelocated at an average depth of 50–100 lm below the stratumcorneum. They are highly refractive because of the ‘‘melaninhats’’ forming bright disks on top of the nuclei (Fig. 2).

Melanocytes and pigmented keratinocytes appear as solitarybright, round or oval structures in the basal layer because of thehigh refractive index of melanin (11). In normal skin, it is

difficult to distinguish between melanocytes and pigmentedkeratinocytes, sincemelanocytes rarely show branching outlinesthat may correspond to their dendrites. In normal skin, basal

cells and melanocytes appear as round or oval rings of brightcells surrounding dark dermal papillae centered by dermalvascular loops, where individual blood cells can be identified by

their relative typical shape and size. Blood vessels are sur-rounded by collagen fibers (1–5 lm) and bundles (5–25 lm)forming a reticulated network and various cell populations (7).

Among these, melanophages are easily recognized because

they are rich in melanin. They are large, intensely reflectingcells with ill-defined cell borders and bright, coarse grainycytoplasm and are located in the vicinity of blood vessels in the

upper dermis (11). In the superficial dermis, the presence ofnonaggregated separated cells has been reported as well (17).They may be nucleated round to oval or triangular cells with

well-demarcated refractive cytoplasm and dark nucleus. Smallhyper-refractive dots and small cells with very bright cyto-plasm and small nuclei as well as plump irregularly shapedbright cells with ill-defined borders and usually no visible

nucleus are also sometimes visible in the dermal papillae (17).Superficial skin appendages can be identified with RCM as

well. Eccrine sweat ducts appear as bright, oval to round

centrally hollow structures that spiral through the epidermisand dermis. Hair shafts with pilo-sebaceous units appear asbright, circular structures whorled centrally hollow structures

with elliptical elongated cells at the circumference and a centralrefractile long hair shaft.

However, we emphasize that the confocal features of

normal skin may vary according to the different skin states.Indeed, ‘‘normal skin’’ has a great interindividual variability inthickness and other characteristics according to a person’s age,sex, race (i.e. black skin usually thicker than white skin) and

location (e.g. sun-exposed vs sun-protected skin areas).

Figure 1. Confocal scanning laser microscopy technology: after emis-sion, light passes through a beam splitter, a scanning and focusingoptical lens and a skin contact device.

1422 Piergiacomo Calzavara-Pinton et al.

In addition, previous sun exposures modify the dermoscopicfeatures within the same individual.

MELANOCYTIC LESIONS

Pattern analysis

Melanocytic lesion exploration is conducted by the examina-tion of mosaics and ⁄ or series of consecutive high resolutionimages from the surface to the dermis down to the point of loss

of resolution (approximately 200–300 lm). Mosaics areusually acquired at three levels (superficial layers, dermal-epidermal junction, upper dermis) for the evaluation of generalepidermal pattern, frequencies and distribution of pagetoid

cells, regularity of the dermal–epidermal architecture, and thepresence and distribution of nests. High resolution images areemployed for the evaluation of cyto-architectural aspects.

However, when we evaluate RCM patterns and characteristics,we must always keep in mind that RCM images of skin cancersvary greatly and these differences may be pronounced.

Superficial epidermal layers. The stratum spinosum and gran-ulosum are usually made up of large (10–30 lm) polygonalcells with dark nuclei and bright cytoplasm and cell borders

giving rise to a honeycombed appearance. In some melanocyticlesions, we see instead small polygonal cells with refractivecytoplasm separated by a less refractive border, giving rise to acobblestone appearance. Sometimes melanomas present a

disarray of the normal architecture of the superficial layers,characterized by unevenly distributed bright granular particlesand cells, irregular in shape and size (18). The presence of large

cells with bright cytoplasm and dark eccentric nuclei insuperficial layers suggests an infiltration of pagetoids. Thoughthe presence of a great amount of large pleomorphic cells

spreading upwards in pagetoid fashion is strictly correlated toa diagnosis of melanoma, the presence of a few atypical cellswithin superficial layers can be occasionally seen in benignlesions, as well (19).

The basal cell layer and the dermal–epidermal junction. Basalcells appear at a depth of approximately 50 to 100 lm belowthe stratum corneum, and are usually brighter than other

epidermal cells. Deeper down, dermal papillae appear as darkround to oval areas circumscribed by refractive cells, corre-sponding to melanocytes and melanin-rich keratinocytes.

Usually, in benign lesions dermal papillae are homogeneous,circumscribed by regular rims of refractive cells (‘‘edgedpapillae’’), lacking cytological atypia and appearing as bright

rings separated by thin structureless spaces (20). Alteration ofthe rete-ridge, corresponding to epidermal flattening andirregular papillae, shows up as small to medium-sized irreg-ularly shaped dermal papillae without a demarcated rim of

bright cells (‘‘nonedged papillae’’) and enlarged interpapillaryspaces frequently presenting some large reflecting cells (20). Insome cases, round or oval medium to large cells, with bright

cytoplasm and peripheral nuclei, corresponding to cytologicalatypia, are also observable. Atypia is graded according to theconcentration of large nucleated cells and their pleomorphism:

we grade them as either ‘‘mild to moderate’’ or ‘‘marked.’’Sometimes, cells not aggregated in clusters but closely distrib-uted in the same plane form sheet-like structures that areobservable in the transition from the epidermis to dermis (21).

Upper dermis. Immediately below the basal cell layer, thepresence of aggregates of refractive cells forming oval torounded structures has been reported in melanocytic nevi

(11,18), in Spitz nevi (22) and in lentigo maligna (23).According to their appearance, cellular clusters are classifiedinto three different types (24,25). Compact aggregates of large

polygonal cells, with hypo-reflecting nuclei and fine granularcytoplasm, forming polyhedral structures are defined as ‘‘dense

Figure 2. Normal skin. (a) Stratum corneum: large, anucleated,polygonal cells that appear very bright, surrounded by dark furrowscorresponding to skin folds (asterisks); (b) Honeycombed pattern ofsuperficial layers: polygonal cells with dark nuclei surrounded bybright white thin cytoplasm; (c) Basal cell layer: dermal papillaecorrespond to dark round to oval areas (asterisks) circumscribed byrefractive cells (arrows), corresponding to melanocytes and melanin-rich keratinocytes. Aggregate of round bright cells (dotted circle)corresponds to basal cells at suprapapillary plate (scale bar: 50 lm).

Photochemistry and Photobiology, 2008, 84 1423

clusters,’’ whereas the presence of rounded nonreflectingstructures with a well-demarcated border, containing isolatedround to oval cells with dark nucleus and reflecting cytoplasm,sometimes having a multilobate aspect, are defined as ‘‘sparse

cell clusters.’’ Whereas both nevi and melanomas presentfrequently dense nests, the latter are predominantly charac-terized by dishomogeneous and sparse cell clusters. Cerebri-

form nests are specific but not sensitive for malignancy andpredominantly observed in nodular type melanomas.

Inside dermal papillae, separated or clustered reflecting cells

are frequently observable. Single large rounded refractive cells,with bright cytoplasm and dark eccentric nucleus correspondto melanocytic cells infiltrating dermal papillae, whereas

irregularly shaped plump bright cells with ill-defined cyto-plasmic borders correspond to melanophages (11,26). Largeirregular cells with refractive cytoplasm and eccentric darknucleus infiltrating dermal papilla are observable in invasive

melanomas, whereas they are seldom present in atypical nevi.Both in benign and malignant melanocytic lesions, dilatedcapillaries with high blood flow within the papillary dermis,

sometimes associated with melanocytic nests, are sometimesobservable. Whereas no peculiarity of collagen texture hasbeen reported in the pigmented component of melanocytic

lesions, a fibrillar pattern with thin irregularly distributedrefractive bundles, sometimes intercalated by refractive cells, inassociation with the loss of typical dermal–epidermal profile, isobservable inside regression areas and photodamaged skin.

Dermoscopy-confocal-histology correlations in

melanocytic lesions

Since confocal microscopy seems to be the natural link

between dermoscopy and histopathology, thanks to its highresolution, horizontal imaging and noninvasiveness, this tech-nique can be used in vivo to carry out the analysis of the

cytological and architectural structure of characteristic dermo-scopic features, and correlate them with histopathology.Recently, Scope et al. demonstrated the correlation between

the global dermoscopic pattern and confocal mosaics at thedermal–epidermal junction, and the correspondence betweenconfocal images and specific dermoscopic features, such asatypical pigment network, pigment globules, peripheral

streaks, and blue–white veil (27). Subsequently, the sameauthors focused their analysis on seven cases presentingperipheral streaks, enabling different morphological subtypes

to be identified on the basis of their confocal appearances (28).We recently aimed at systematically exploring, with confocalimaging, substrates having specific melanocytic dermoscopic

features in over 200 lesions, obtaining a comprehensive andsystematic archive serving as a point of reference for thedescription and characterization of the underlying cytologicaland architectural aspects of various pathologies and their

histopathologic correlations (29).Typical pigment network is constituted by ‘‘edged papillae,’’

corresponding to elongated regular rete-ridges (20), whereas an

atypical pigment network is characterized by ‘‘nonedgedpapillae’’ and a disarrangement of the rete-ridge in all cases.Dermoscopic findings showing regular pigment globules at

dermoscopy is evidence of dense melanocytic clusters andtypical of monomorphous nevocytic nests upon histology. Onthe other hand, irregular pigment globules correspond to

irregularly shaped clusters, usually showing regular cytology inbenign lesions and atypical cells in melanomas. Interestingly,the histologic substrate of pigment dots is clearly identifiableby confocal microscopy, showing them to be pagetoid melan-

ocytosis in melanomas, and melanin clumps within theepidermis in common nevi. Streaks indicate different sub-strates according to their morphology. Radial streaming in

confocal microscopy images corresponds to a parallel series ofelongated lines of basal cells projected toward the peripheryand to elongated and parallel oriented epidermal cristae at the

periphery of the lesion. Peripheral globules appear as denseclusters rimming the lesion periphery and pseudopods corre-spond to those globular-like bulging structures, similar to

dense nests bridged to the lesion core.As recently demonstrated, confocal microscopy is particu-

larly useful for the interpretation of the bluish pigmentation,enabling us to distinguish between inflammatory infiltrate,

predominantly constituted by plump bright cells within dermalpapillae corresponding to melanophages, and malignantmelanocytic cells that singularly or in clusters infiltrate the

dermis in invasive melanomas (26). Within regression areasconfocal microscopy fails to identify specific aspects in themajority of cases, presenting thin reticulated collagen fibers,

frequently along with plump bright melanophages.

Confocal microscopy diagnostic algorithms

Several diagnostic confocal features of melanocytic lesionshave been investigated. Very high sensitivity and specificity

values of some confocal aspects, such as melanocyte cytology,disarray of the architecture, and poorly defined keratinocytecell borders, were identified by Gerger et al., comparing two

preselected images per lesion from a database of 27 melanomasand 90 nevi, the majority of which corresponded to clearlybenign lesions (30). Based on 102 melanocytic lesions belong-

ing to 37 melanomas and 65 equivocal nevi, six criteria wereidentified as independently correlated with a MM diagnosis,and a diagnostic algorithm was developed (21), which follows

here below:Two major criteria were scored 2 points, corresponding to

the cytologic atypia at basal cell layers (mild or marked) andnonedged papillae at dermoepidermal junction. Then four

minor criteria, represented by the presence of rounded cells insuperficial layers spreading upwards in a pagetoid fashion, orpagetoid cells widespread throughout the lesion, or cerebri-

form clusters in the papillary dermis, or nucleated cells withindermal papilla, were scored 1 point. Considering a thresholdscore equal to or >3, a 97.3% sensitivity and 72.3% specificity

were obtained, whereas when we increase the threshold,specificity increases but unfortunately not without a conse-quent decrement of sensitivity. Recently, the validity of thealgorithm was blind tested on a larger population of equivocal

melanocytic lesions, corresponding to 136 melanomas and 215nevi, showing in a reproducible clinical setting a 92%sensitivity and 70% specificity (31).

Junctional, compound and dermal nevi

Confocal features of common melanocytic nevi have beendescribed in several papers. In junctional, compound and

dermal nevi, superficial layers usually showed a regular


epidermal architecture, presenting a honeycombed or cobble-stone pattern (18,31), in conjunction with melanin pigmentpresent throughout the epidermis. Pagetoid infiltration consti-tuted by rounded cells was reported in less than only 5% of

nevi (19). At the dermal–epidermal junction, benign lesions areusually characterized by edged papillae brighter than the onesin the surrounding skin. Sometimes nonedged papillae are also

observable, especially in presence of architectural disarrange-ment, such as in dysplastic nevi (18,20). Large nucleated cellsmay also be visible, frequently in coincidence with cells defined

atypical by histology.Junctional nevi may be seen as reflecting rings surrounding

dermal papillae (Fig. 3) or may also show melanocytic nests in

connection with the basal layer, appearing as bulging struc-tures (junctional clusters) or enlargement of interpapillaryspaces (junctional thickening) (21).

Compound nevi usually augment the junctional features,

such as the presence of compact aggregates (regular densenests) within the papillary dermis (25). Since it is sometimesdifficult to distinguish between junctional and dermal clusters,

the exact diagnosis between junctional and compound nevi isnot always reliable.

Dermal nevi are characterized by the presence of large

aggregates of clustered cells, filling enlarged dermal papillaeand not connected to the basal cell layer. Some large roundednucleated cells more or less aggregated are sometimes visible in

the upper portion of the cluster, especially in congenital nevi(32). Further down clusters assume a more compact andhomogeneous aspect with no evident cell contours. Uponhistology nevus cells are orderly disposed as cords and nests of

cells, usually with the larger and more pigmented cells in theupper portion, corresponding to the ones visible throughconfocal microscopy, decreasing in size and pigmentation with

depth (29).Although unusual in common nevi, the presence of atypical

nests, such as nonhomogeneous clusters, sparse cell nests,

and ⁄ or cerebriform ones, are rarely observed in benign lesions(31). The presence of plump bright cells with ill-definedborders is seen both in junctional and compound nevi.

Spitz/Reed nevi

First described by Sophie Spitz, epithelioid and ⁄ or spindledcell nevi share some clinical and histologic features withmelanomas. Spitz nevi with a globular dermoscopic appear-

ance were the first to be studied (22), and they werecharacterized by numerous dense regular nests covering thewhole lesion area.

Confocal images of Spitz nevi show the presence of‘‘pagetoid’’ cells within the superficial layers of the epidermisin more than 20% of cases (19). In comparison to melanoma,

pagetoid melanocytosis is usually less abundant, predomi-nantly located in the center of the lesion, and constituted bysmall, elongated cells with short dendritic-like branches.Moreover, thin elongated cells with peripheral dendritic

processes at the extremities are frequently observed in thebasal and suprabasal layers, and which prove to be spindlecells upon histological exams. A rim of dense nests regularly

distributed at the lesion’s perimeter is frequently observed anddirectly correlated with the peripheral melanocytic nestssharply demarcating the lesion’s border upon histology (33).

The frequent observation of nonedged papillae at thedermal–epidermal junction, atypical cells within the epidermalbasal layer and pagetoid cells in the epidermis, have proved to

be the most frequent characteristics leading towards a misdi-agnosis of a large percentage of Spitz nevi. In fact, more than50% of Spitz nevi were classified as melanomas when using theRCM algorithm (21). Numerous plump bright cells, corre-

sponding to melanophages, may be seen in the dermis in themajority of lesions, and increased vascularity is also frequentlyobservable within the upper dermis. When dealing with these

lesions, the limited penetration of confocal microscopy ham-pered the exploration and identification of certain aspects,such as deep mitoses and cellular maturation, necessary to

finalize the diagnosis.

Melanoma

Melanoma is a malignant proliferation of melanocytes forwhich the prognosis is related to the depth of dermal invasion

(Breslow’s thickness measurement). Superficial spreading mel-anomas, characterized by a superficial radial growth phaselater followed by dermal invasion and vertical growth, account

for 70% of melanomas. Nodular melanoma is the mostaggressive type of melanoma and accounts for about 15% ofall melanomas diagnosed. Lentigo maligna, characterized by a

long-lasting radial growth phase within the epidermis, and

Figure 3. (a) Mosaic of multiple individual 500 lm · 500 lm imagesthat offer an overview of a larger field of view in a junctional nevus.Reflecting rings surrounding dermal papillae (asterisks) sharplycontrasting with the dark background (scale bar: 500 lm); (b) Edgepapillae corresponding to dermal papillae (asterisks) circumscribed bya rim of refractive cells (arrows) (scale bar: 50 lm).


lentigo maligna melanoma typically occur on sun-damagedskin in middle-aged and elderly people, and account forapproximately 10% of melanomas. Acral lentiginous mela-noma accounts for about 5% of all diagnosed melanomas.

No confocal microscopy studies have yet evaluated the acrallentiginous melanoma type because of the limited depthpenetration of the device which did not allow us to explore

into skin areas having a thick corneal layer.

In situ and superficial spreading melanomas. As a tumorproliferates its confocal architecture changes. Thin melano-

mas, histologically characterized by a malignant proliferationpredominantly located in the epidermis and at the dermal–epidermal junction, often present pagetoid cells in the super-

ficial layers and a mild to marked cytological atypia in basallayers. Architectural disarray occurs, and we can observenonedged papillae and sometimes even sheet-like structures(Fig. 4), as well as the occasional proliferation of irregular

dense nests. As the thickness of the tumor increases, epidermaldisarray, cell pleomorphism and architectural disarrangementall increase and the papillary dermis begins to be infiltrated by

single nucleated cells or atypical cell clusters in dishomoge-neous or sparse cell-nests. Therefore, the greater the thicknessof the tumor, the higher the confocal algorithm score. The five

melanomas (out of 136 cases studied) which scored 0 on the

confocal algorithm were in all cases lesions thinner than0.5 mm (31). When melanomas arise on pre-existing nevi, cellatypia may present itself only within the limited area of thetumor itself. For this reason, one must accurately explore the

entire lesion in order to identify any isolated malignant aspectsand not miss a melanoma. It is always advisable to follow upand recheck even those lesions which seem to be completely

lacking atypical features in a confocal exam.

Nodular melanomas. Nodular melanomas differ from othertypes of melanoma in that they tend to grow more rapidly in

thickness than in diameter, may not have a radial growthphase, and they do not develop from a pre-existing mole.A bad prognosis must be given when there is an elevated tumor

thickness at diagnosis, because of the rapid growth rate of thistype of tumor. In order to identify the characteristic featuresseen in the confocal imaging of nodular melanomas, 10 lesionswere recently studied and compared with nodular areas and

blue palpable areas in 20 superficial spreading melanomas (34).Nodular melanomas show a thin epidermis with a honey-combed pattern or a peculiar broadened-honeycombed pat-

tern, consisting of polygonal cells with black nuclei and thickbright borders, with a compact eosinophilic corneal layeroverlaying this thin epidermis, and there is usually a lack of

pagetoid cells. Immediately below the epidermal layersthe typical papillary architecture was no longer visible in thenodules, because of the epidermal flattening caused by themassive proliferation of malignant cells in the dermis.

Markedly pleomorphic cells with bright cytoplasm and darknuclei were also present in the basal layer, sometimes distrib-uted in sheet-like structures, but more often in the dermis,

frequently forming dishomogeneous aggregates, appearing asdishomogeneous sparse cell clusters or cerebriform nests.Though nodular melanomas are very similar to the nodules

in superficial spreading melanomas at the dermal level, signs oftheir previous radial growth phase, such as pagetoid infiltra-tion, the presence of atypical cells and residual nonedged

papillae were frequently observable, allowing us to hypothesizea different biological behavior.

Lentigo maligna and lentigo maligna melanoma. Lentigo mal-igna is a malignant proliferation of melanocytic cells charac-

terized by a prolonged intraepidermal growth phase of atypicalmelanocytes aligned along the dermal–epidermal junction andfrequently extending deep into the appendageal epithelium.

Clinically, it can resemble other pigmented lesions, such asinflamed seborrheic keratosis, pigmented actinic keratosis orsolar lentigo, therefore instrumental devices should be used to

arrive at a correct diagnosis. Since confocal microscopy allowsus to visualize the cytological and architectural aspects ofsuperficial layers and the upper dermis at a quasi-histologicresolution, it has been successfully applied here. Superficial

epidermal layers displayed a focal or global disruption of theirregular architecture and diffuse grainy textures (35). Numerouslarge pleomorphic cells with prominent dendritic processes were

found throughout all layers of the epidermis, suggesting apagetoid spread of atypical melanocytes. Atypical bright cellswere also noted surrounding hair follicles, This tendency of

atypical cells to group around the hair follicle is considered arelevant diagnostic clue (23,35). In lentigo maligna dermalpapillae were hard to see (35). In the papillary dermis

Figure 4. (a) Mosaic of multiple individual 500 lm · 500 lm imagesin a thin melanoma (Breslow thickness = 0.28 mm). Nonedgedpapillae (asterisks) corresponding to rete ridge disarrangement (scalebar: 500 lm). (b) High magnification showing junctional disarrange-ment and atypical and polymorphic large cells (arrows), with darknucleus and bright cytoplasm corresponding to malignant melanocytes(scale bar: 50 lm).


reticulated bright collagen bundles arising from solar damage,and plump bright melanophages were frequently observable.Invasive tumors show dishomogeneous clusters and atypicalnucleated cells within the papillary dermis. Confocal micro-

scopy was also employed in a few cases of lentigo maligna todelineate preoperative surgical margins and to offer a non-invasive tool for monitoring topical therapy (36). However,

more studies are needed to validate the confocal diagnosticcriteria for lentigo maligna and to further define its clinicalapplications.

BENIGN NONMELANOCYTIC LESIONS(LENTIGINES, SOLAR LENTIGO,SEBORRHOEIC KERATOSIS)

Lentigines are small, sharply circumscribed, pigmentedmacules surrounded by skin which is normal in appearance.

Hyperplasia of the epidermis and increased pigmentation ofthe basal layer are evident upon histology. Langley et al.evaluated 10 patients using confocal microscopy, including six

cases of lentigines and four lentigo maligna. The most strikingfeature of lentigines was its increased density of dermalpapillae surrounded by bright monomorphic layers of cells.Dermal papillae assumed annular, polycyclic shapes or formed

papillary projections, surrounded by single cell layers of brightmonomorphic cells (35). Melanophages are observable insidedermal papilla. Solar lentigo on the other hand is characterized

by a distinctive cerebriform appearance of the basal cell layer,presumably because of complex anastomosing of rete ridges(35). Seborrhoeic keratosis is characterized by marked acan-

thosis, where the confocal microscopic images show thickenedepidermal layers with normal honeycombed appearance (37),frequently interrupted by bright ovoidal concentric structures

corresponding to horn cysts. Pigmented and ⁄ or inflamedseborrhoeic keratoses are characterized by the presence ofplump-bright cells, which are the melanophages inside dermalpapillae, and enlarged dark canalicular structures, that is, the

blood vessels (11).

EPITHELIAL TUMORS (NONMELANOMASKIN CANCERS)

Actinic keratosis

Actinic keratosis is the most common precancerous lesion of

the skin, affecting elderly fair-skinned people who have hadexcessive exposure to ultraviolet light. The risk of progressionof a single actinic keratosis to a full-thickness squamous cell

carcinoma remains unknown, but it has been estimated atapproximately 10%. Confocal microscopic imaging of actinickeratoses was first done on seven cases. Because of the limited

depth penetration of the instrument as well as the presence ofhyperkeratosis, the basal layer was observable in only three ofthese seven cases. Irregular hyperkeratosis in the stratumcorneum, broad uniform and regularly spaced keratinocytes in

the stratum spinosum ⁄ granulosum with evident dark nuclei,sometimes larger and more irregular than in normal skin werethe characteristic signs of actinic keratoses (8). Recently, a

blinded study evaluated the presence of parakeratosis, archi-tectural disarray and keratinocyte pleomorphism in 44 cases ofactinic keratoses. The results of this study were positive in that

in almost all cases it was possible to correctly distinguishactinic keratosis from healthy skin (38).

Squamous cell carcinoma

Squamous cell carcinoma is the second most common skin

cancer, which is usually the result of long-term sun damage tothe skin. Many squamous cell carcinomas can develop fromprecancerous lesions, such as actinic keratoses. No character-

istic features in the confocal images of squamous cell carci-noma have been found so far that may be useful for theiridentification, with the exception of one case showing the

presence of a significant hyperkeratosis hampering the imagingof the epidermis to any depth (8). Greater nuclear pleomor-phism and architectural disarray in the stratum granulosum

seemed to differentiate squamous cell carcinomas from theactinic keratoses, though further studies are requested to findout if the confocal microscope can be of any use here.

Basal cell carcinoma

Among cutaneous tumors, basal cell carcinoma is the mostcommon, occurring mainly on the sun-exposed areas of elderlyindividuals. Confocal microscopy allows us to visualize char-

acteristic patterns in these tumors, leading to an accuratediagnosis. Since 2002 research articles and case reports havelinked confocal appearances of basal cell carcinoma and their

histologic counterparts. Epidermal layers show some degree ofkeratinocytic atypia with variably sized nuclei, pleomorphism,architectural disarray, and parakeratotic nuclei in the stratumcorneum. Basaloid cells appear monomorphic in shape with a

high nucleo ⁄ cytoplasmic ratio, sometimes containing brightprominent nucleoli. A characteristic cell elongation with nucleioriented along the same principal axis gives rise to a polarized

appearance. Islands of tumor cells were noted with highrefractivity, surrounded by hypo-reflecting areas correspond-ing to the mucinous stroma. Abundant and sometimes

enlarged blood vessels are seen juxtaposed to tumor cells(Fig. 5). Rolling of leukocytes along the endothelial lining issometimes observable in larger vessels (39). Also mononuclear

inflammatory cells, such as lymphocytes and granulocytes, arepresent within tumor tissue and stroma, showing up as brightparticles or small rounded cells with a bright cytoplasm. In2004, Nori et al. explored the sensitivity and specificity of

confocal microscopy for diagnosis of basal cell carcinomas in152 skin lesions, belonging to 83 cases and having otherdermatologic conditions as controls (40). The presence of a set

of five previously described confocal imaging criteria (presenceof elongated monomorphic basaloid nuclei; polarization ofthese nuclei along the same axis of orientation; prominent

inflammatory infiltrate; increased vasculature; and pleomor-phism of the overlying epidermis indicative of actinic damage)were evaluated in blind to determine their sensitivity andspecificity. The most relevant feature for basal cell carcinoma

diagnosis was the presence of polarized nuclei showing a 92%sensitivity and 97% specificity. Although sometimes present inother skin conditions, such as lichen planus, lichenoid

keratoses, molluscum contagiosum, and mycosis fungoides,elongated monomorphic nuclei turned out to be the mostsensitive criterion (100%) for basal cell carcinoma diagnosis,

but with a 71% specificity. The presence of two or more


criteria was 100% sensitive for basal cell carcinoma diagnosis,

whereas four or more RCM criteria were 95.7% specific.Focusing on pigmented type basal cell carcinoma, well-circumscribed trabeculae ⁄ cordlike structures or polypoid

islands or nodules surrounded by dark nonrefractile cleft-likedark spaces were observed at the dermo-epidermal junctionand into the papillary dermis (41). Scattered bright oval,

plump to stellate structures with indistinct borders, corre-sponding to melanophages, were seen admixed with orbetween tumor cords and islands in the papillary dermis.

Bright dendritic structures, variably appearing with a small or

plump cell body, and with branching processes, were consis-tently seen both within the epidermis overlying the tumor orwithin the tumor islands. Although confocal microscopy isunable to differentiate melanocytic dendritic cells from Langh-

erans’ cells, immunostaining showed that Langerhans’ cellspredominantly corresponded to dendritic cells located withinthe epidermis and the melanocytes were found in the tumor

islands (41,42). In pigmented basal cell carcinomas, plumpbright melanophages are frequently visible within tumorislands or in the papillary dermis.

Confocal microscopy seemed also useful to monitor efficacyof topical treatment, such as 5% Imiquimod cream (43,44), orto help surgical margin detection, especially by the use of

contrast agents, such as aluminum chloride (45,46).

CONCLUSIONS

Skin biopsy remains the golden rule for microscopic diagnos-tics up to now. However, its invasiveness creates severallimitations to the investigation of the physiology and pathol-

ogy of the skin.In vivo RCM is the only reliable alternative that is available

so far because it is the only non invasive diagnostic techniquethat allows us to investigate normal or patho-physiologic

processes with a microscopic resolution high enough to viewcellular and sub-cellular detail.

However, the stacked horizontal ‘‘en face’’ gray scale

electronic images of RCM give different information fromthat drawn from the vertical colored sections of traditionalmicroscopy. RCM images gives a better morphological char-

acterization of cells. For example, we can highlight dendriticpagetoid melanocytes, although their vertical position in theepidermis cannot be assessed exactly, and the morphological

RCM analysis can easily differentiate pigmented, melanocyticlesions from pigmented, nonmelanocytic lesions and amela-notic melanoma from other nonpigmented lesions.

However, reading gray scale horizontal images is challeng-

ing for dermatologists or pathologists that are not experts inRCM. Therefore, a prolonged and careful training programis needed before the dermatologist becomes confident with

RCM images to the same extent he is familiar with histologicalspecimens. A distant tutorial help by experts via telemedicineand the use of verified image processing algorithms can further

help beginners in the analysis of these images. The importanceof such tutorial programs via telemedicine is evident andshould parallel those excellent results which telemedicine hasoffered to radiology in these past few years.

Once the dermatologist has acquired experience with thistechnique, he can take advantage of the unique possibilitiesoffered by RCM. It enables quick real-time in vivo imaging of

large areas or volumes of tissue. This has an obvious clinicalrelevance and can also be used to guide pathology to the mostsignificant or the most suspect area of a skin lesion and to

delineate tumor margins.This approach could be still further improved by a

combination of RCM with dermoscopy that allows for a

better investigation of the morphological characteristics of thelesion (20,26–29). Combining RCM with other noninvasivetechnologies such as optical coherence tomography, ultra-sound or MRI that image deeper but with lower resolution

could be helpful in the assessment of tumor thickness before

Figure 5. Basal cell carcinoma. (a) Islands of tumor cells with highrefractivity (dotted circle), surrounded by hypo-reflecting areas (aster-isks) corresponding to the mucinous stroma. (b) Cell elongation withnuclei oriented along the same principal axis (arrows). (c) Dilatation ofblood vessel (arrowheads), surrounding islands of tumor cells (dottedcircle): blood flow and leukocyte trafficking are better visualized inreal-time (scale bar: 50 lm).


the surgical removal of a lesion that has been diagnosed withRCM. Furthermore, unlike pathology, RCM does no damageto the tissue and is therefore suitable for monitoring dynamicprocesses and follow-up of skin lesions over time. However,

RCM is still in its infancy (6) and advances in technology areneeded in order to exploit fully its tremendous potential.

A higher resolution or the development of contrast agents

should be provided in order to enhance the possibility toidentify different cell populations and their distinctive sub-cellular structures. However, higher resolution depends on

opto-electronical improvements and there is limited experi-ence with in vivo fluorescence confocal microscopy utilizingexogenous fluorophores in human skin to date (47). Another

disadvantage of RCM is its poor capacity to explore dermalchanges or infiltrates since it images epidermis well but notmedium and deep dermis. Therefore, this technology is betterused for ‘‘early-stage’’ screening of precancers or initial

cancerous lesions rather than for ‘‘late-stage’’ diagnoses. Thislimitation could be surmounted by software ⁄hardware devel-opments which could permit a higher lateral resolution while

using lasers with longer wavelengths in the infrared regionthat penetrate deeper into the skin in comparison to the redlight that is used nowadays. Software able to reconstruct

vertical and 3D images would let us exploit the criteriaof microscopic diagnosis that have been developed withhistology.

Finally, another drawback is that RCM is not yet user

friendly and practical. This may be overcome through afurther miniaturization of the microscope, the use of hand-heldfiber optics (48) and a faster speed of acquisition and

elaboration of images (48).

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reflectance confocal microscopy for in vivo skin imaging

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