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International Journal of Cosmetic Science, 2008, 30, 117

Review Article

In vivo reectance-mode confocal microscopy in clinical dermatology and cosmetology


lez*, and Y. Gilaberte-Calzada S. Gonza
*Dermatology Service, Memorial Sloan Kettering Cancer Center, New York, NY, USA, Grupo Dermatologico, Madrid and Dermatology Service, Hospital San Jorge, Av. Mart nez de Velasco 34, 22005 Huesca, Spain

Received 13 May 2007, Accepted 14 July 2007

Keywords: dermatology, cosmetology, in vivo confocal microscopy, non-invasive skin pathology

Synopsis In vivo reectance confocal microscopy (RCM) is a non-invasive imaging tool that allows real-time visualization of cells and structures in living skin with near histological resolution. RCM has been used for the assessment of benign and malignant lesions, showing great potential for applications in basic skin research and clinical dermatology. RCM also reveals dynamic changes in the skin over time and in response to specic stimuli, like ultraviolet exposure, which makes it a promising tool in cosmetology, as it allows repetitive sampling without biopsy collection, causing no further damage to the areas under investigation. This review summarizes the latest advances in RCM, and its applications in the characterization of both normal and pathological skin. sume Re ectance (RCM) est La microscopie confoncale en re un outil de visualization non-invasif qui permet el des cellules et structures lobservation en temps re solution pratiquede la peau vivante avec une re te utilise e pour ment histologique. La RCM a e sions be nignes et malignes et a lobservation des le un fort potentiel pour les applications des montre
Correspondence: Salvador Gonzalez, Dermatology Service, Memorial Sloan-Kettering Cancer Center, 160 East 53rd Street, 2nd Floor, New York, NY 10022, USA. Tel.: +1 212 6100185; fax: +1 212 308 0530; e-mail: gonzals6@mskcc.org

e de base et en derdomaines de la recherche cutane galement capable matologie clinique. La RMC est e de montrer des variations dynamiques dans la peau ` des stimuli ponse a en fonction du temps et en re ciques comme une exposition aux ultra-violets. spe thode un outil prometteur en Ceci fait de cette me tologie permettant des mesures re pe titives cosme ` vements de biopsies, cest-a ` -dire sans le sans pre provoquer de dommages aux zones de la peau ` res avance tudie es. Cet article re sume les dernie es e concernant le RCM et ses applications dans la risation de la peau saine et pathologique. caracte Introduction The ability to evaluate a skin lesion microscopically in a non-invasive fashion has long been a goal for dermatologists and dermatopathologists. In the last several years, several techniques have been developed aimed to provide dynamic microscopic information about the skin to offer in vivo diagnosis and monitoring of disease evolution in real time without morbidity. These technologies include magnetic resonance [1], high frequencyultrasonography [2], optical coherent tomography [3] and, more recently, reectance confocal microscopy (RCM) [4, 5]. Currently, in vivo RCM has a wide range of applications, allowing real time non-invasive microscopic imaging with a resolution near that of conventional histology (1-lm lateral and 3-lm axial) when exploring cutaneous structures between the stratum corneum and reticular dermis

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In vivo reectance-mode confocal microscopy

lez and Y. Gilaberte-Calzada S. Gonza

[6]. RCM is based on the collection of images from light reected by living tissue [710]. In 1995, Rajadhyaksha et al. [5] rst reported the foundations of laser scanning RCM. Since then, improvements in design and optimization of optical parameters have improved image quality, reduced the size and improved ergonomics of the imaging devices. In recent years, imaging techniques have improved, increasing the number of applications in dermatology, and a signicant number of skin conditions have been objectively characterized. Foundations of RCM A confocal microscope consists of a light source, a condenser, an objective lens and a detector (Fig. 1). The light source illuminates a small threedimensional spot within a sample, such as skin. This illuminated spot is then imaged onto the detector through a small aperture (pinhole). The

small aperture allows only light that originates from the focused illumination spot to be detected, whereas the light that originates away from the spot is reected [11]. The light source, the illuminated spot and the detection aperture are in optically conjugated focal locations and this arrangement is called confocal. The illuminated spot is then scanned horizontally over a 2-dimensional grid to obtain a horizontal microscopic section. This process is known as optical sectioning [710]. Adjustments can be made in the focal length of the beam, allowing the microscope to image a series of horizontal planes stacked vertically, with and axial thickness of 25 lm (Table I). In this sense, the numerical aperture of the objective lens determines image resolution, which means that there is an inversely proportional relationship between high resolution images and small apertures (less light), and low resolution through larger apertures (more light). Confocal

Out-of-focus plane In-focus plane (object) Out-of-focus plane

Point light source

Condensder lens

Tissue sample

Objective lens

Point detector pinhole / aperuture

Figure 1 Diagram of a reectance mode confocal microscope. The diagram depicts non-invasive imaging of a thin (focused) plane of skin. Back-scattered light is detected from the skin rather than transmitted light. The small aperture (pinhole) in front of the detector collects only the light in focus, while rejecting light that is out of focus.

Wavelength of light source for 1- to 3-lm Visible 488514 nm sectioning in the epidermis 35 lm in the deeper dermis Near-infrared 8001064 nm Objective lens Magnication 30100 Numerical aperture 0.71.2 Resolution Lateral 0.51.0 lm Axial 3.05.0 lm Illumination power up to 40 mW Real time confocal images are parallel to skin surface Confocal images of normal epidermis show honeycombed appearance Melanin is the best endogenous contrast agent

Table I Summary of basic principles of reectance confocal microscopy and image interpretation

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images are obtained in greyscale. In this context, white represents total light reected and black represents no reection at all. More light is reected when the tissue contains structures of size similar to the wavelength of the light source [5, 6]. Reectance confocal microscopy systems use a laser as a light source. Best quality images are obtained with low power lasers (around 40 mW) and near-infrared wavelength (8001064 nm). With an 830 nm diode laser, skin penetration is around 400 lm [6]. Water immersion lenses are used as the refractive index of water (1.33) is close to that of epidermis (1.34). Skin movement may be a problem during in vivo imaging. To overcome this, a ring template skin contact device is used to reduce motion artefacts and to contain the immersion medium (water or ultrasound gel) for the objective lens during imaging [6]. Basic principles for image interpretation of the skin Images obtained by RCM are en face (horizontal) images when compared with vertical sections obtained by routine histology. Whereas routine histology requires tissue staining with exogenous dyes for diagnosis, confocal images are resolved in greyscale. Confocal images are based on the presence of endogenous contrast [11], which is provided by microstructures such as melanin, haemoglobin or cellular organelles [5]. Therefore, structures are visualized based on the optical properties such as the refraction index and the reectivity of the tissue under investigation [12, 13]. Epidermis Imaging normal skin in real time usually takes place from the surface and progressing deeper, thus most supercial images correspond to the stratum corneum (Fig. 2a). The stratum corneum produces the rst image of the top surface of the skin because of backscattered light at the water-to-stratum corneum interface. Corneocytes are visualized as brilliant polygonal shapes of 10- to 30-lm size, and grouped in islands separated by skin folds, which appear very dark. The next layer (section) is the stratum granulosum, regularly distributed at a depth of 1520 lm (Fig. 2b).The stratum granulosum keratinocytes are 20- to 25-lm size and their nuclei show up as dark central oval structures sur 2008 The Authors. Journal compilation

rounded by a bright grainy cytoplasm. Underneath the stratum corneum is the stratum spinosum, located 20- to 100-lm deep. It consists of a tight honeycomb pattern of keratinocytes (10- to 15-lm size) with well-demarcated cell borders (Fig. 2b). Between 50- to 100-lm depth we can nd the dermo-epidermal junction. Basal keratinocytes are small (715 lm) and bright [6], due mostly to the presence of melanin inside the cell (Fig. 2c). The melanin in basal keratinocytes is typically arranged in a supranuclear position, often referred to as melanin caps or umbrellas, implying their protective function. Melanocytes can be seen as bright, solitary nests at the dermo-epidermal junction, with round, oval, fusiform and dendritic shapes [14]. Different brightnesses correspond to endogenous variation among skin phototypes and the anatomic location. Other cells that contain melanin are melanophages which can be distinguished in the supercial dermis as large, bright cells with ill-dened cytoplasmic borders, usually located around or near vessels. Dermis Dermal papillae are observed at the dermo-epidermal junction as dark round areas surrounded by rings of bright circles of basal cells containing highly reective melanin granules. Capillary loops are located in the centre of dermal papillae as black holes, often showing bright erythrocytes rolling within them; based on their relative morphologies, sizes and numbers, these cells were identied as erythrocytes (6- to 9-lm diameter), leucocytes (630 lm) and platelets (25 lm). Below the dermo-epidermal junction, a network of collagen bres and bundles (1-lm and 5- to 25-lm diameter, respectively) can be observed within the papillary dermis and supercial reticular dermis. Eccrine ducts appear as bright central hollow structures that spiral through the epidermis and dermis. Hair shafts with pilosebaceous units can be observed [6, 15], and appear as central hollow structures with elliptical elongated cells at the circumference, with a central white structure corresponding to the hair shaft. Use of RCM in clinical dermatology RCM ndings of inammatory skin conditions Reectance confocal microscopy has been used to describe inammatory skin conditions in vivo and

te Franc tologie 2008 Society of Cosmetic Scientists and the Socie aise de Cosme International Journal of Cosmetic Science, 30, 117 3

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lez and Y. Gilaberte-Calzada S. Gonza

d (b)

B C D GK

SK (a) (c)

(d)

Figure 2 Normal skin. (a) Histological section of normal skin with lines indicating depth levels in which confocal images B, C and D have been taken. (b) Confocal image of stratum corneum level showing high refractivity. Note skin dermatoglyphs (d). (c) Slightly oblique image showing the presence of granular keratinocytes (GK) and spinous keratinocytes (SK). GK are regularly seen at depths of 1015 lm. The dark oval areas correspond to nuclei within the bright cytoplasm. SK are seen at 20100 lm below the stratum corneum. (d) Confocal image at dermo-epidermal level. Note basal keratinocytes (arrows) brighter than surrounding keratinocytes and dermal papillae openings (*). Scale bar = 50 lm.

non-invasively psoriasis [16], contact dermatitis (CD) [1720] and bacterial [21] and fungal infections [22, 23] have been evaluated to dene their characteristic confocal features (Table II). An advantage of RCM is that the majority of benign inammatory skin conditions are limited to the epidermis and upper dermis, where RCM has a resolution comparable with traditional histology.

Contact dermatitis Contact dermatitis is the most common occupational skin disease [23]. To differentiate between allergic and irritant CD (ACD and ICD, respectively) poses, a signicant challenge to the dermatologist, as the clinical presentation, immunological prole and histopathological features in ACD and ICD is remarkably similar [2428]. Correlation of RCM

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Table II Reectance confocal microscopy features of inammatory dermatosis

Skin lesion

Features

Contact dermatitis Allergic Irritant

Psoriasis vulgaris

Rosacea

Pyogenic granuloma

Spongiosis Microvesicle formation Inammatory inltrate Pronounced supercial changes in stratum corneum Intra-epidermal necrosis Prominent epidermal hyperproliferation and visible nucleoli Parakeratosis, acanthosis and papillomatosis Thinning of the granular layer Munros microabscesses Increased tortuosity of the dermal vasculature Increased diameters of the pilosebaceous ducts Tortuous capillaries Perifollicular and perivascular inammatory inltrate Increased number of dilated and tortuous blood vessels Pronounced spongiosis Exocytosis Branched hyphae Inammatory inltrate Inltrating neutrophils in the subcorneal pustule Capillary dilatation Multiple highly refractile round structures (2040 lm) Hyperkeratotic stratum corneum Pleomorphic ballooned keratinocytes Multinucleated giant cells in a loose aggregate of keratinocytes Inammatory cells and debris.

Cutaneous infections Onychomycosis Bacterial folliculitis Warts Herpes

with conventional histopathology has unveiled distinctive characteristics for ACD and ICD [17, 18, 20]. Spongiosis is noted as intercellular brightness by RCM. The presence of epidermal inammatory cellular inltrate can be visualized as bright round or oval structures 9- to 12-lm size interspersed between keratinocytes. Areas of necrotic epidermis, perivascular inammatory inltrate, and increased size and brightness of basal keratinocytes are also seen in both types of reaction. Whereas ACD predominantly exhibits vesicle formation, inammatory inltrates and spongiosis [17, 20], ICD is typically associated to pronounced supercial changes involving the stratum corneum, clear demarcation and separation of individual corneocytes, and parakeratosis; it is possible to identify parakeratotic nuclei and these appeared as bright (highly refractile) oval structures placed centrally within corneocytes (Fig. 3) [17, 20]. In these studies, both ACD and ICD have demonstrated similar degrees of spongiosis. Preliminary data on the accuracy of RCM in the diagnosis of ACD showed that spongiosis and exocytosis in the spinous layer have high sensitivity and specicity with regard to patch testing, indicating that RCM may add cellular level
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information to the clinical interpretation of patch tests, thus potentially enhancing ACD diagnostic [29]. Furthermore, kinetic evolution of ACD and ICD has been investigated by RCM, showing characteristic patterns. Firstly, the onset of disruptive changes is generally much faster for ICD when compared with ACD. The supercial changes in the stratum corneum are visible within few hours following the application of the contact irritants, whereas stratum corneum alterations are generally less prominent in acute ACD. Secondly, ACD exhibits microvesicle formation, whereas ICD also features intra-epidermal necrosis [17, 20]. Finally, epidermal hyperproliferation associated with ICD reactions can be followed using RCM, and seems to be a sensitive parameter for the activity of ICD reactions. Overall, ICD has a faster onset and resolution than the cutaneous changes in ACD. Recently, investigations have also been aimed at the evaluation of ethnic variability in skin response to experimental contact irritants such as sodium lauryl sulphate and common household detergents such as ivory dishwashing liquid. Preliminary studies performed in Caucasian and African-American volunteers have revealed

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ACD RCM

ICD RCM ACD

H&E

(a) ICD

(b) ACD

(c) ACD

(d)
Figure 3 Confocal features of allergic contact dermatitis and irritant contact dermatitis observed with reectance confocal microscopy (RCM) and correlated by routine histology. (a) Marked stratum corneum disruption with loss of the normal homogenous bright pattern and marked corneocyte demarcation and separation; Parakeratosis also is seen as bright oval structures placed centrally within corneocytes represent nuclei (indicated by arrows). (b and c) Spongiosis seen as increased intercellular brightness apparent on RCM; Inammatory cell inltrate seen as bright structures 12- to 15-lm size interspersed between keratinocytes. Arrows denote inammatory cells; (d) Intra-epidermal vesicle formation seen as dark spaces in the epidermis containing inammatory cells and necrotic keratinocytes. Arrows denote vesicles. Scale bars = 50 lm (Reproduced from Swindells et al. (2004) with permission from Mosby, Inc.).

different degrees of stratum corneum disruption and intra-epidermal changes, which suggest differences related to skin pigmentation [19, 30]. Furthermore, RCM successfully visualized subclinical degrees of cutaneous irritation, further underlining the capability of RCM to detect irritation at sub-threshold irritant concentrations [30]. Overall, RCM is a promising tool for the noninvasive evaluation of CD. Future work will be aimed at the investigation of individual allergens,

different allergen concentrations and the testing of additional contact irritants and cosmetics. Psoriasis Psoriasis is a common benign, proliferative skin disorder with histopathological features that can be visualized using RCM. In this regard, the major features that distinguish the uninvolved skin from lesional psoriatic skin are increased numbers of dermal papillae with enlarged dermal blood vessels inside of them to supply the

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proliferative lesion with circulating erythrocytes [31]. Other features are: (i) retained nuclei within the corneocytes, seen as small dark areas (parakeratosis); (ii) clusters of polymorphonuclear leucocytes forming the Munros microabscesses,

visualized as highly refractile compared with the surrounding keratinized background. It is easier to discriminate within the microabscess than in routine histology; and (iii) thinning of the granular layer (Fig. 4) [16].

(a) A B

C (b) dp

dp

(c)
Figure 4 Psoriatic lesion. Histologic section with bars indicating the depth levels in which confocal images A, B and C have been obtained. (a) Confocal image of parakeratotic stratum corneum. Note arrows pointing out nuclei. (b) Confocal image showing transverse section of Munros microabscess (arrow). (c) Confocal image at dermo-epidermal junction showing increased number density of dermal papillae (dp). Note tortuous blood vessels (arrows). Scale bar = 50 lm.

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Rosacea Acne rosacea is a benign, inammatory skin disorder of unknown aetiology with female preponderance. The disease typically presents on the face with dilated vasculature and acneiform inammatory papules and pustules. RCM histopathology reveals increased diameters of the pilosebaceous ducts, tortuous capillaries and a characteristic perifollicular and perivascular inammatory inltrate [21]. RCM ndings of cutaneous infections Fungal infections Dermatophyte infections including onychomycosis and tinea pedis, although common, can be difcult to diagnose. Diagnosis may be delayed when culture is necessary because of the time required for the hyphae to grow. RCM enables rapid real-time identication of branched hyphae, visualized as a network of long, dark, sometimes septated structures. In vivo, they appear below the surface of the nail plate, but they can also be observed in vitro in nail clippings or scrapings [22, 23]. RCM can be particularly useful for tinea unguium, in which the high percentage of false negative results in the microbiological tests makes the diagnosis difcult. Bacterial folliculitis Folliculitis imaged with RCM can be diagnosed by direct observation of a hair follicle surrounded by a signicant number of small bright granular cells (neutrophils), which can be also found within the subcorneal pustules. In addition, severe spongiotic epidermis and capillary dilatation in the dermal papillae can be observed [21]. Viral infections Warts have also been imaged with RCM. The hyperkeratotic stratum corneum and the presence of multiple highly refractile round structures measuring 20- to 40-lm size within the lesion allows a rapid and conclusive diagnosis of common wart. These typical round structures may be keratohyaline granules or putative viral particles within infected keratinocytes, based on their size (our unpubl. data). Cutaneous herpes infections may be atypical and severe, especially in immunocompromised individuals. RCM has been proved to be a useful tool in their diagnoses. Their main features are the presence of pleomorphic big round cells with dark cytoplasm, identied as ballooned keratinocytes, and

internal round bright structures corresponding to multinucleated giant cells. Both types tend to form loose aggregates interspersed with round bright cells identied as inammatory cells moving within a black, uid medium. These ndings are identical to those provided by conventional histology [32]. RCM ndings of skin neoplasms Reectance confocal microscopy characterization of neoplastic lesions is a major area for research, with the potential to aid in the non-invasive diagnosis and management of a variety of skin cancers. With the advent of newer less invasive or topical therapies, it is desirable to use non-invasive diagnostic tools that enable accurate identication of tumour subtypes and tumour margins, and response to treatment. RCM of melanocytic lesions Pigmented lesions include different cellular constituents: melanocytes, pigmented keratinocytes and melanophages. Using RCM, pigmented keratinocytes appear as polygonal cohesive cells with bright granular cytoplasms of varying intensity. Melanocytes appear as bright round, oval, fusiform or dendritic cells. They can be identied by their nested growth pattern as aggregates of bright round to oval structures at the dermo-epidermal junction or in the supercial dermis. Melanocytes are also recognizable as single cells along the dermo-epidermal junction, usually separated from each other by a variable number of keratinocytes. Finally, melanophages appear as large bright plump cells with illdened cytoplasmic borders, usually located around or near vessels of the supercial dermis. Melanocytic nevi In nevi, both spinous and granular layers show no alteration, with normal patterns in a homogenous population of well-demarcated cells (Table III). A nevus itself is made of nevomelanocytes, a homogenous population of small monomorphus round to oval bright refractive cells with centrally positioned dark round nuclei [14, 33]. Dermal papillae are uniformly distributed and circumscribed by a rim of refractive monomorphous cells (edge papillae) that correspond to small melanocytes and melanin-rich keratinocytes, without any cytological atypia. In junctional nevi, melanocytes are at the dermo-epidermal junction level [3335]. On

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Table III Reectance confocal microscopy features of melanocytic skin tumours


Skin lesion Features

Melanocytic nevi

Cutaneous melanoma

Preserved honeycomb appearance of keratinocytes in the epidermis Populations of monomorphous round to oval bright refractile cells with centrally positioned nuclei in the basal layer (pigmented keratinocytes and melanocytes) Regular and uniform dermal papillae Clusters of bright round cells in the dermis of compound nevi (nests) Disarray of keratinocytes (loss of epidermal honeycomb appearance) Coarse branching dendritic structures in the epidermis Small and irregular dermal papillae Bright grainy particles in the epidermis Dermal cell clusters (nests) of different aspect when present Enlarged round or dendritic highly refractive cells ascending in the epidermis (pagetoid spread)

the contrary, in compound and dermal nevi, they are seen within the papillary and reticular dermis, near the vessels. Sporadically, small brilliant dendrites in the epidermis can be observed. Assessment of atypical nevi by dermoscopy reveals strikingly common features with common nevi and melanoma, and occasionally the diagnosis may be extremely difcult [34]. In atypical nevi, the cell population is more heterogenous in size, shape and refractivity (different intensity in brilliance) (Fig. 5 and Table III) [3335]. However, cells tend to be round or oval as in common nevi, rather than dendritic as in melanoma. In general, atypia correlates with attenuated brightness in most melanocytes, with isolated large bright epithelioid cells with peripheral nuclei, and cell nests can be less demarcated [34, 35]. They also show focal loss of keratinocyte demarcation within the epidermis overlying the lesion and bright granules within the epidermis, probably representing melanin dust. Atypical nevi, especially on the face, may show destructuration at the dermo-epidermal

junction, and have non-homogenous dermal papillae with the absence of demarcation by refractive cells (non-edge papillae). Melanoma Reectance confocal microscopy allows the identication of features characteristic of nevi and melanoma. Pigmented melanomas and amelanotic melanomas exhibit remarkably similar features when assessed by RCM [33, 3638]. Confocal features suspicious for melanoma are shown in Table III. These include structural changes in the spinous and granular layers, keratinocyte disarrangement and loss of intercellular demarcation (disruption of the honeycomb pattern) [33]. Enlarged atypical cells with pleomorphic morphology, variable refractivity and angular nuclei may be found in several layers of the epidermis (pagetoid dissemination), and in the dermis (Fig. 6). Cells may be oval, stellate or fusiform, including coarse branching dendritic processes and

(a)

(b)

Figure 5 Displastic lentiginous nevus. (a) Histologic section. The bars in image A show the depth level in which confocal image B has been obtained. (b) shows heterogeneous brightness with irregular distribution of dermal papillae. Dermal papillae are surrounding by non-refractile rim of cells. Scale bar = 50 lm.

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* * *

(a)

(b)

(c)

Figure 6 Supercial spreading melanoma. (a) Histology section with bars indicating the depth level in which confocal images B and C have been obtained. (b) Confocal image shows the presence of atypical melanocytes and dendrites (arrows) ascending in the epidermis (pagetoid spread), within a background of marked loss of keratinocyte demarcation (*). (c) Confocal image shows the presence of non-refractile rims around dermal papillae openings (non-edge papillae, *). Also, enlarge (atypical) melanocytes are observed (arrows). Scale bar: 50 lm.

eccentrically placed large nuclei [33, 37, 39]. Indistinguishable cell borders may be evident, which contributes to the abnormal epidermal morphology. In the basal layer, cells may be grouped resembling a dysplastic nevus or isolated. In the dermis, cerebriform cell clusters appear, including thin refractive cell aggregates (poligonal or enlarged) surrounded by melanin dust [34]. Melanoma also shares some of the features of dysplastic nevi. In this regard, dermal papillae are smaller, more irregular and their borders are poorly dened when compared with common nevi. The architectural pattern is very asymmetrical in size and refractivity. Melanin dust is composed of coarse granules (13 lm) compared with atypical nevi, and is scattered along the epidermis. Linear dendrites are thicker and brighter than in healthy skin. A useful advantage of RCM is that it enables identication of abnormal intra-epidermal melanocytic proliferation, granules and dendritic structures in clinically amelanotic melanomas [39]. These features may be evident because of the presence of melanosomes in the cytoplasm, which act as endogenous source of contrast because of their size (0.61.2 lm) and refractive index (1.70), and/ or the presence of some melanin in pre-melanosomes [11, 36]. Apart from diagnosis, RCM has shown good correlation to epiluminescence microscopy during histological examination [40], and also as a selective non-invasive tool to guide biopsy collection [37], pre-surgical mapping [38, 41] and monitoring the response to treatment [38].

A limitation for the use of RCM in melanoma diagnosis is an imaging depth. Lesion depth has been shown to be a very important prognostic factor in patients diagnosed with melanoma. Available instruments can image to a depth of 200350 lm, but the presence of refractive structures in the dermis, such as inammatory cells and collagen bundles, may decrease contrast and difcult melanocyte visualization [38]. Thus, although RCM has been shown useful for the differential diagnosis of intra-epidermal processes, little or no information about dermal cells and structures can be obtained, limiting its use for the assessment of deeper lesions. RCM of non-melanocitic skin tumours Actinic keratosis The main features of actinic keratosis assessed by in vivo RCM include irregular hyperkeratosis in the stratum corneum. In addition, the stratum granulosum is almost identical to that of normal skin, with dark nuclei, contrasted against the bright refractile cytoplasm of the keratinocytes. Whereas nuclei in the stratum spinosum and stratum basale vary in shape, size and haphazard orientation, these ndings correspond to nuclear enlargement with pleomorphism in a pattern consistent with architectural disarray, which does not involve the full thickness of the epidermis usually seen in conventional histology (Fig. 7) (Table IV)

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Figure 7 Actinic keratosis. (a) Histology section showing the depth level of confocal image. (b) Confocal image shows en face confocal image with keratinocyte disarray-epidermal pleomorphism. Scale bar: 50 lm.

(a)

(b)

Table IV Reectance confocal microscopy features of non-melanocytic skin tumours


Skin lesion Features

Actinic keratosis

Squamous cell carcinoma Basal cell carcinoma

Irregular hyperkeratosis Epidermal nuclear enlargement and pleomorphism Architectural disarray limited to lower portion of epidermis Nuclear enlargement with pleomorphism Full thickness architectural disarray of epidermis Islands of tumour cells with monomorphic elongated basaloid nuclei Polarization of tumour cell nuclei along the same spatial axis throughout epidermis Prominent inammatory inltrate admixed or closely apposed with tumour cells Increased dermal vasculature with tortuous dilated blood vessels and leucocyte accumulation and rolling along endothelial lining Plemorphism and architectural disorder of overlying epidermis.

[42]. Observation of dysplastic features in the full skin thickness on RCM is suggestive of squamous cell carcinoma. Squamous cell carcinoma The shallow penetration of the RCM illuminating wavelengths prevents accurate visualization at the dermo-epidermal junction, particularly in hyperkeratotic lesions. This makes differential diagnosis between supercially invasive SCC and SCC in situ unfeasible because of lack of adequate visual assessment at the dermo-epidermal junction. Confocal features suggestive of SCC are full thickness architectural disarray and nuclear enlargement with pleomorphism observed from the basal layer to the stratum granulosum (Table IV). Other features suggestive of SCC, such as vascular patterns and keratin pearls, need further investigation. Basal cell carcinoma Reectance confocal microscopy morphological characteristics of basal cell carcinoma (BCC) have been well dened (Table IV) [43, 44], and include: (i) the presence of pleomorphism and architectural
2008 The Authors. Journal compilation

disorder of the overlying epidermis, indicative of actinic damage or the presence of the tumour; (ii) the presence of islands of refractive tumour cells with elongated monomorphic basaloid nuclei, associated with intervening areas of low refractility, which might correspond to the mucinous stroma (Fig. 8); (iii) nuclei of tumour cells that are polarized along the same axis of orientation, disrupting the normal honeycomb pattern of the epidermis and the dermal papillae architecture; (iv) increased dermal vasculature with prominent dilatation and tortuosity of blood vessels; and (v) trafcking of leucocytes is easily identied as bright, highly refractile round cells along the endothelial lining (Fig. 8C). A retrospective, multicentric study of 152 patients has shown that the presence of at least two of these criteria has a sensitivity of 100% for the diagnosis of BCC [45]. As the number of criteria present for a lesion was greater, the specicity increased, so when at least four criteria were present, the specicity was 95.7%, giving the best concordance between high sensitivity and high specicity. The most sensitive and specic criterion was the presence of polarized nuclei (91.6% and 97%, respectively), which is consistent with histological ndings as palisading is one of the most remarkable characteristics of BCC. Specic

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B C D

n
bv

n
(a) (b) (c) (d)

Figure 8 Basal cell carcinoma. (a) Histology section. The bars in image (a) show the depth level in which confocal images B, C and D have been obtained. (b) Confocal image from upper level showing a population of cells with elongated nuclei, polarized along one axis (/). (c) Confocal image shows BCC nodes (n). (d) Confocal image demonstrates dilated blood vessels (bv) with leucocyte trafcking (arrows) surrounding a BCC node (n). Scale bar: 50 lm.

additional ndings have been described in BCC subtypes: (i) pigmented BCC have been characterized by an architectural conguration including well-dened cord-like or nodular structures, which correlate with nodules and/or cords of atypical basaloid cells; peritumoural dark spaces consistent with peritumoural mucin; and intratumoural bright dendritic and granular structures which are the image of both intratumoural dendritic melanocytes and melanin dust [46]; melanophages appear as bright oval to stellate structures with indistinct borders [47, 48]; (ii) solid nodular BCCs show nesting and nodularity of epithelial cells at papillary dermis together with clefting and palisading of tumour cells (Fig. 8b,c); (iii) brotic/ inltrating BCCs have curled bundles of collagen with large cells, representing the tumour stroma [44]. Reectance confocal microscopy has the potential to become a useful tool for non-invasive monitoring of the response of skin tumours to novel non-invasive therapies. In this regard, there are ongoing RCM studies evaluating the histopathological responses of actinic keratoses treated with 5-aminolevulanic acid photodynamic therapy [49], as well as those of BCCs treated with imiquimod. In relation to imiquimod, RCM accurately predicts the presence or absence of BCC before, during and after treatment [50]. To date, RCM has proven accurate in establishing presence of BCC and its responsiveness to the treatment regimen with imiquimod [50]. Potential use of RCM in cosmetics Reectance confocal microscopy is a potentially useful tool in cosmetics, either in research or in clinical applications. In research, RCM offers the

opportunity to determine in vivo the kinetics of the skin after the application of new substances, allowing the microscopical assessment of immediate changes induced by them in the skin in real time. Reectance confocal microscopy is a sensitive tool for the characterization and quantication of histological changes of the epidermis and papillary dermis because of ageing. A signicant decrease in dermal papillary index (number of papillae per mm2) and in thickness of the basal layer, and an increase in the granular layer with age have been reported. This decrease is more pronounced between the age of 18 and 50 than in older women (65- to 80-years-old) [51]. In this group, large segments of at epidermal-dermal junctions with complete loss of papillae are observed often. In these regions, the microvasculature consisted of horizontal vessels with noticeable larger diameters than in young skin, probably venules rather than capillaries. A few curled remnants are occasionally found just beneath the atrophic epidermis. The vessel walls could not be resolved into their individual cells, including endothelium, pericytes or smooth muscle cells by confocal examination, because of low reectiveness. Changes in the epidermis and supercial dermis following treatment with anti-ageing products, lasers and pulsed light therapies can be monitored by RCM before, during and after application (Figs 9, 10). In this regard, topical vitamin C applied for 5 weeks restores the anatomical structure of the epidermal-dermal junction in young skin and increases the density of capillary containing papillae in the aged skin of post-menopausal women compared with its vehicle [51]. Therefore, RCM is a very valuable method to evaluate the cutaneous response to anti-ageing therapies.

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Figure 9 Normal dermis. Confocal images taken at upper reticular dermis level of a subject volunteer of 41 years old (a) compared with another of 84 years old (b). Scale bar: 50 lm (courtesy: C. Alessi, Lucid Inc., NY, USA).

(a)

(b)

Figure 10 Sun-exposed skin. Supercial confocal images (approximately 30-lm depth) of photodamaged skin at baseline (a) and at 1 month of treatment (b). Note thinner dermatoglyphs after treatment. Scale bar: 50 lm (courtesy: C. Alessi, Lucid Inc., NY, USA).

Prominent Dermatoglyphs

Thinner Dermatoglyphs

(a)

(b)

Comedogenecity has long been a concern in the development of cosmetic and personal care products. The hairless rhino mouse has extensively been used for comedone studies because its skin contains many horn-lled utriculi also known as pseudocomedones. These pseudocomedones appear in RCM as circular or oval structures lled with a highly refractive substance keratin and collagen and enclosed by loosely arranged dermis [52]. The skin effect of retinoic acid has been observed by RCM in these animals. Removal of follicular plugs from pseudocomedones and transformation into normal follicular structures in a dose-related manner were observed. Sebaceous duct development was registered by RCM, changes perfectly correlated with conventional histology. Additionally, small comedones, presenting as follicular plugs within the epidermis and small pustules at and below the dermo-epidermal junction, have been detected in RCM images of patients with acne (Fig. 11). Therefore, RCM is a useful tool for in vivo morphological and quantitative evaluation of skin comedones over time and can be used either for studying the comedogenecity of a cosmetic product or for monitoring the treatment of comedones.
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Reectance confocal microscopy greatly enhances assessment of dynamic pigmentary changes in human or animal skin over time and in response to specic stimuli such as ultraviolet radiation exposure [53, 54]. A study in pigmented guinea pigs has shown that after 4 days of irradiation, when only faint erythema and no clinical tanning was yet visible, RCM could already detect pigmentary changes, as follows: (i) melanocytes, visible as bright dendritic cells, have bigger cell bodies; (ii) the stratum spinosum, composed of small polygonal cells with dark nuclei and a brighter ring of cytoplasm is less bright because of epidermal hyperplasia; (iii) a dened cellular layer emerged showing keratinocytes in different degrees of melanization and cells with the size of spinous cells belong to the stratum spinosum; and (iv) many dendrites viewed in cross-section appeared as small bright dots showing increased dendricity of melanocytes. RCM images after 3 weeks after starting irradiation, when skin has clinically visible tanning, show: (i) melanocytes are increased in size and number and pigmented keratinocytes of the stratum spinosum can be seen next to them; the stratum spinosum consists out of pigmentloaded keratinocytes, but in some cells the nucleus

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(a)

(b)

Figure 11 Comedo cyst. Confocal images (a and b) at different depth levels of a comedo cyst. Scale bar: 50 lm (courtesy: C. Alessi, Lucid Inc., NY, USA).

can still be seen as a dark round center [54]. RCM has also been used to evaluate in vivo dose and time-dependent skin changes in humans following solar-simulated irradiation and to correlate them with other non-invasive techniques such as optical coherence tomography [55]. The authors demonstrated denite optical coherence tomography and RCM ndings obtained from UV exposed skin including increase epidermis thickness (hyperproliferation and acanthosis), reduction in dermal reectivity (dermal oedema), increase in brightness of the basal layer (pigmentation), and increase in vessel diameter within the dermal papillae (vasodilatation). In conclusion, RCM seems to be a very useful tool in photobiology research including sunscreen tests [53, 54]. Additionally, this tool also has been used to analyze depigmenting agents [56]. In the context of skin photoageing, solar lentigines occurring in sun-damaged skin of older adults also have been analyzed by RCM [57] (Fig. 12). RCM can be an accurate in vivo method

for assessing their response to cosmetic approaches not only for evaluating pigmentation degree, but also for monitoring their morphological changes at high resolution. Finally, RCM can be used to tattoo-related skin alterations [58]. Massive subepidermal deposits of dense, clustered pigment granules up to about 3-mm size correspond to black tattoos, with the more scarce and diffuse deposits corresponding to red, blue and green tattoos. Diffuse pigment granules tended to accumulate in the outer dermis underneath the level of the basement membrane zone. Therefore, RCM could be a useful technique for pre-evaluation of tattoos before laser removal, helping to predict their clinical outcome. Limitations of RCM The main limitation of RCM is its penetration in dermis, which nowadays reaches a maximum depth of 350400 lm. This prevents imaging of

(a)

(b)

Figure 12 Solar Lentigo. Reectance confocal images were collected from a solar lentigo located on the hand of a 60year-old volunteer. They were taken at 43 lm (spinous compartment, (a)) and 97 lm (dermo-epidermal level, (b)) below the stratum corneum. At dermo-epidermal papillae, the presence of polymorphous dermal papillae with polycyclic contours is noted. Eccrine duct (encircled) appears as a spiraling structure showing less refractivity and a donut shape at e Lauder Companies, Oevel, dermal epidermal level. Scale bar: 50 lm (courtesy: L. Declercq and C. Polleiet, Este Belgium).

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structures located in the deep dermis, especially in cases of hyperpigmented or hyperkeratotic lesions, as, in these cases, there is strong contrast attenuation because of absorption and scattering of light going through those structures. The presence of refractive structures in the dermis, such as inammatory cells and collagen bundles, may also decrease contrast and difcult melanocyte visualization.This might be improved by assaying different immersion media and illumination sources. The current contact device can be unwieldy and difcult to use on a non-at surface. A portable prototype RCM has recently been developed that will further miniaturize the microscope to produce a more manageable or hand-held device possibly using bre optics. This would make it much more user-friendly and practical. Finally, work is ongoing to facilitate the creation of vertical and 3-dimensional sections, which would vastly increase the potential of RCM. Summary and conclusions Reectance confocal microscopy has the potential to reduce the need for invasive biopsies by facilitating in vivo diagnosis of both benign and malignantly pigmented and non-pigmented lesions. Unfortunately, a major limitation to this technology is the imaging depth, especially in hyperkeratotic lesions, largely because of attenuation of the intensity, which is secondary to light absorption and scattering. Further improvements regarding increasing the RCM power and depth of optical penetration, and using optimal immersion media with good diffusion properties may help address this issue. Reectance confocal microscopy presents researchers with the opportunity to perform noninvasive evaluation of skin lesions with histological detail. It may be employed as a guide for performing biopsies, by helping to determine areas exhibiting suspicious features of malignancy and reducing sampling error [37]. Furthermore, it may be used as an adjunct to Mohs surgery and therapy [5961] by mapping out the margins or extent of involvement prior to excision or other therapies. RCM can be repeated indenitely, and therefore can be used to monitor progression or resolution of lesions through time. In this regard, RCM may be valuable to examine the histopathological response of tumours to therapy. Progressive normalization of architecture in lesions of AK trea 2008 The Authors. Journal compilation

ted with photodynamic therapy has been observed under RCM monitoring, and it has also been employed to conrm complete clearance of AK and BCC and resulting inammatory response to topical imiquimod. Finally, RCM is also a powerful tool in cosmetics not only in research, but also to monitor histological changes after cosmetic treatments in a non-invasive way. Acknowledgements This work has been partially supported by a grant from the Spanish Ministry of Health (FISS, PI060499). References
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