State of the art in non‐invasive imaging of cutaneous melanoma

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/49856215 State of the art in non-invasive imaging of cutaneous melanoma Article in Skin Research and Technology · February 2011 DOI: 10.1111/j.1600-0846.2011.00503.x · Source: PubMed CITATIONS READS 38 162 2 authors: Louise Elizabeth Smith Sheila Macneil 41 PUBLICATIONS 348 CITATIONS 455 PUBLICATIONS 7,923 CITATIONS University of South Australia SEE PROFILE The University of Sheffield SEE PROFILE Some of the authors of this publication are also working on these related projects: UK-India Centre for Advanced Technology for Minimizing Indiscriminate Use of Antibiotics View project All content following this page was uploaded by Louise Elizabeth Smith on 08 December 2014. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately. Skin Research and Technology 2011; 17: 257–269 Printed in Singapore
All rights reserved doi: 10.1111/j.1600-0846.2011.00503.x r 2011 John Wiley & Sons A/S Skin Research and Technology Review Article State of the art in non-invasive imaging of cutaneous melanoma Louise Smith and Sheila MacNeil Department of Engineering Materials, Kroto Research Institute, University of Sheffield, Sheffield, UK Background: This review focuses on looking at recent developments in the non-invasive imaging of skin, in particular at how such imaging may be used at present or in the future to detect cutaneous melanoma. Methods: A MEDLINE search was performed for papers using imaging techniques to evaluate cutaneous melanoma, including melanoma metastasis. Results: Nine different techniques were found: dermoscopy, confocal laser scanning microscopy (including multiphoton microscopy), optical coherence tomography, high frequency ultrasound, positron emission tomography, magnetic resonance imaging, and Fourier, Raman, and photoacoustic spectroscopies. This review Skin Imaging F imaging of skin, we want to be able to correctly identify the epidermis and dermis and to identify relatively large features such as rete ridges and blood vessels. Ideally, we would also like to be able to distinguish where the morphology of individual cells or groups of cells differs from their immediate neighbours as is the case in cutaneous invasive carcinomas, of which melanoma is the most difficult to diagnose and stage. The current gold standard for morphological investigation of the skin is achieved by taking a small biopsy for standard histology (1). This allows the visualisation of structures through a vertical section of the skin, i.e. from the epidermis through to the reticular dermis or even subcutaneous tissue. However, this is invasive and relatively time consuming and the accuracy of the detection of invasive melanoma relies on biopsying the right area at the right time. There is a real need for non-invasive techniques for in vivo imaging of skin to assist dermaOR NON-INVASIVE contrasts the effectiveness of these techniques when seeking to image melanomas in skin. Conclusions: Despite the variety of techniques available for detecting melanoma, there remains a critical need for a high-resolution technique to answer the question of whether tumours have invaded through the basement membrane. Key words: melanoma – non-invasive imaging – confocal microscopy – dermoscopy – PET/CT – spectroscopy & 2011 John Wiley & Sons A/S Accepted for publication 6 November 2010 tologists and pathologists in the diagnosis of skin conditions such as melanoma and basal cell carcinoma. Current techniques for the diagnosis of these conditions depend on the skill of the clinician visually diagnosing the progression of the disease, following the ABCD rules for melanoma detection (2) and/or by the removal of a biopsy, which can cause pain, expense, and scarring (3). Crucially, the biopsy involves delayduring this time, the melanoma may be progressing through the basement membrane into the dermis with its rich blood supply – a critical stage in melanoma progression. At the time of writing, not all melanoma are being correctly diagnosed. Amelanotic malignant melanoma presents a unique diagnostic challenge to physicians. While these amelanotic melanoma share certain features with other malignant neoplasms, namely large lesional size, asymmetrical architecture, and poor circumscription (4), the lack of pigment can lead to the diagnosis of a less serious cutaneous disease. According to McCalmont (5), 257 Smith and MacNeil conventional microscopy has remained the gold standard for melanoma diagnosis for several decades, and a diagnosis of melanoma is optimally based on a summation of microscopic features that are evaluated as objectively as possible by an experienced histopathologist. Photography, including whole-body photography, is also commonly used in the detection and diagnosis of melanoma and other skin cancers. However, this technique does not allow imaging below the surface of the stratum corneum and therefore will not be included in this review. Dermoscopy Epiluminescence microscopy or dermoscopy is a relatively new multispectral imaging technique used to image skin. Over the past 10 years, this non-invasive technique has been proven to be very valuable in the in vivo evaluation of pigmented skin lesions (6). The first dermoscopes used nonpolarised light and used a hand-held magnification device. This was placed on the skin after the application of a liquid such as oil or alcohol gel to reduce the diffraction of the light from the stratum corneum. However, newer instruments do not need the liquid layer (2, 7). All multispectral imaging techniques have limited depth penetration and the depth of chromophores is determined algorithmically and not optically [as it is done in optical coherence tomography (OCT) and confocal microscopy]. They are relatively inexpensive in terms of components. They consist of a good CCD camera, a multispectral source (white lamp), and some simple intermediary optics. There have also been efforts to combine confocal microscopy to provide multispectral information (8). Dermoscopy allows the visualisation of the horizontal plane of a given skin lesion but only to the level of the papillary dermis (9). There are several commercial dermascope instruments available to aid clinicians in their diagnosis such as Melafind (Mela Sciences, Inc, Irvington, NY, USA) and SIAscope (Biocompatibles International plc, Farnham, UK). s The MelaFind system consists of a hand-held imaging device, which is comprised of several components: an illuminator that filters 10 different specific wavelengths of light ranging from 430 to 950 nm (10); a lens system composed of nine elements that creates images of the light reflected from the lesions; a photon (light) sensor; and an image processor using proprietary algorithms to extract many discrete characteristics or features 258 from the images. The images provided are horizontal images through the skin and images can be obtained from as deep as the reticular dermis or subcutaneous tissue (11). In feasibility studies, MelaFind attained 95% sensitivity and close to 70% specificity, at 95% confidence levels (12). Non-contact SIAscopy or spectrophotometric intracutaneous analysis uses a digital camera to capture cross-polarised images of a scene. A flash gun is used as a light source, providing light over the entire visible spectrum. The camera provides raw information of the imaging chips’ response to the light, which results in three pictures being produced from the camera. Red, green, and blue images are produced, each covering a different region of the visible spectrum. These images are then analysed, producing non-contact SIAscans that represent the concentration of haemoglobin and melanin within the skin. This technique has been used in the imaging of pigmented skin lesions and melanoma (13, 14), diagnosis of basal cell carcinoma (15), and analysis of skin colour (16). Confocal Scanning Laser Microscopy (CSLM) Confocal microscopy has been used in dermatology in two modes: fluorescence and reflectance (9, 11, 17, 18). While dermatological research tends to use fluorescence, clinical practice uses reflectance microscopy as this does not require fluorescent labelling of cells and tissues and therefore is a viable technique for in vivo imaging. Confocal microscopes consist of a light source, a condenser, an objective lens, and a detector. The basic premise of confocal microscopy is the selective collection of light from in focus planes in the tissue. With the use of a low power near-infrared (IR) laser, a beam of light is focused tightly on a specific point in the skin. Light scattered or reflected from this illuminated point is collected through a pinhole-sized aperture by a detector. The light source, illuminated point, and detector aperture are in optically conjugate focal planes (i.e., confocal planes). This allows for the collection of light from the single in-focus plane and the rejection of light from all out-of-focus planes (19). The imaging depth of a CSLM is limited by the wavelength of the laser chosen with a 488 nm laser imaging 50–100 mm into skin as reported by Gareau et al. (20). However, others have been able to image at depths of up to 300 mm (7, 9, 11, 19, 21), providing images of the basement membrane State of the art in non-invasive imaging of melanoma area and down into the papillary dermis. This again provides two-dimensional horizontal images through the skin. If a series of images are taken, these can be stacked and a three-dimensional rendering can be compiled. CSLM can be used either in the reflectance or in the fluorescence mode. The reflectance mode relies on inherent differences in the reflectivity of the structures in the skin. Free cytoplasmic melanin pigment and cytoplasmic pigmented and non-pigmented melanosomes provide strong contrast for near-IR laser light sources (11). Generally, CSLM are not considered to be portable. However, fibre-optic confocal imaging uses a single optical fibre to both illuminate and detect the laser light in place of the detector aperture (pinhole) in CSLM. This has allowed miniaturisation of the system, thus producing a hand-held device more suitable for in vivo clinical use (11). CSLM has shown promise in the detection of both pigmented and amelanotic melanoma (7, 11, 19–21) as well as basal cell carcinoma and pathological inflammatory conditions (19, 21). CSLM can also potentially be used in the evaluation of topical treatments and in pharmacological applications, especially when combined with Raman spectroscopy. However, these instruments are generally expensive and therefore often beyond the budget of the practicing general dermatologist (2). Commercially, there have been several reports s on the use of VivaScope (Lucid Inc., Rochester, MN, USA) as a reflectance mode confocal microscope and Stratum (Optiscan Pty Ltd, Melbourne, Australia) in the fluorescence mode. Reflectance mode CSLM is a non-invasive imaging tool that allows real-time visualisation of cells and structures with near histological resolution (0.5–1 mm lateral and 3–5 mm axial when imaging from the stratum corneum to the reticular dermis) (9, 21). Initial work on reflectance mode confocal microscopy has been reported in (22). They could achieve cellular resolution into the dermis with a video rate laser scanning confocal microscope. This research group followed up on this with many studies (23–25). Contrast agents can be used to provide clearer images in both reflectance and fluorescence modes (26). s VivaScope is a clinical confocal microscope that allows imaging of individual cells in skin and other living tissues. The non-invasive nature s of VivaScope allows repeated imaging of a single tissue site, which enables monitoring natural biological processes in vivo over time, and provides real-time visualisation of processes such as blood flow in capillary loops. However, the s s VivaScope is not portable. VivaScope has been used to investigate the tissue surrounding facial pores (27) and to monitor the effectiveness of anticancer drugs in patients with actinic keratosis and basal cell carcinoma (28). Figures 1–3 below illustrate the effectiveness of CSLM in obtaining cellular resolution throughout the epidermis and in imaging melanoma. Multiphoton laser scanning microscopy (MPLSM) is similar to CSLM; however, in MPLSM, excitation of fluorophores is obtained by a non-linear multiphoton process, as opposed to the onephoton excitation used in conventional microscopy. Two-photon excitation occurs when two photos of approximately half the one photon energy are absorbed practically simultaneously by the fluorescent molecule. This means that near-infra-red radiation can be used to excite fluorophores, either endogenous autofluorescent substrates such as the reduced coenzyme NAD(P)H, pigmented basal cells, and collagen (29) or fluorescent markers added to the substrate. For this to occur, high-intensity radiation from a femtosecond pulsed laser, i.e. Ti-Sapphire, is required (30). Figure 4 illustrates the ability of multiphoton confocal microscopy to image with cellular and subcellular resolution just using the natural autofluorescence of the sample. In the case illustrated in Fig. 4, the sample is human skin. While multiphoton confocal microscopy allows the visualisation of structures with greater resolution deeper into the skin than conventional CSLM, it can still image only relatively shallow structures. For imaging deeper into skin tissue, OCT is perhaps better suited. OCT can be applied for the investigation of vertical cross sections, instead of the horizontal sections provided by microscopy images, up to a depth of about 2 mm. These images are of a much lower resolution than those given by confocal scanning laser and multiphoton confocal microscopy (31). OCT OCT is analogous to ultrasound B imaging, except that it uses light rather than sound waves (11). With respect to its resolution, it is intermediate between CSLM and ultrasound, theoretically allowing the visualisation of structures with a resolution of approximately 1 mm. OCT is, as the name suggests, a coherence technique based on 259 Smith and MacNeil Fig. 1. Confocal images of normal human skin: a stratum corneum in forearm skin: (a) the corneocytes appear as bright, polygonal cells forming islands separated by dark wrinkles and creases; (b) stratum corneum in acral (skin from the palms or soles of the feet) skin: the corneal outlines are more clearly demarcated; (c) stratum granulosum: the nucleus of the granular cells appears as a dark oval structure surrounded by a bright, grainy rim of cytoplasm. The granular cells are arranged in a cohesive pattern; (d) stratum spinosum: the cells look similar to the granular cells but are smaller in size and have a less refractive cytoplasm. The cells are arranged in a honeycombed pattern; (e) stratum basale: the basal cells form bright rings around the dark dermal papillae; (f) hair follicle: these appear as round dark whorled structures in the depth of the dermis; (g) sweat gland: sweat glands appear as oval to round, centrally dark structures spiralling to the depth. Scale bar, 50 mm. Image from Branzan et al. (65). the principle of Michelson interferometry. Light from a low-coherence length light source is split evenly between the sample and a reference mirror. By measuring the interference between light backscattered from the tissue and from the reference mirror, the distance and magnitude of optical scattering within the tissue can be measured with micrometre-scale precision. The axial resolution is determined by the coherence length 260 of the light used; typically, this is 10–20 mm but this could be improved to 2–4 mm by the use of ultra short pulse lasers (11, 32). The images obtained are two-dimensional vertical slices through the tissue as shown in Fig. 5 below. OCT is well established as a tool in ophthalmology and is being used increasingly in dermatology (1, 32–34), evaluation of wound healing (35), and particularly in the diagnosis of mela- State of the art in non-invasive imaging of melanoma Fig. 2. Dermoscopic and confocal images of pigmented skin lesions and melanocytic nevus: (a) nests of bright monomorphous structures corresponding to nevus cells and pigmented keratinocytes forming rings around the dark dermal papillae at the dermo–epidermal junction of a compound nevus; (b) lentigo maligna: prominent dendritic refractive cells and loss of keratinocyte demarcation; (c) superficial spreading melanoma: a nest of irregular bright structures corresponding to melanoma cells within the stratum spinosum (pagetoid spreading), with a blurred honeycombed pattern. The dermoscopic images were taken with the Dermogenius ultrat (Rodenstock Präzisionsoptik Linus, Munich, Germany). Scale bar, 50 mm. From Branzan et al. (65). noma (11). Unfortunately, while the layers of the skin, i.e. epidermis and dermis as well as adnexal structures and blood vessels can be seen, the basement membrane zone and cellular features cannot be visualised and so OCT is of limited use in the early detection of melanoma (9). OCT setups with sophisticated lasers have not yet achieved cellular resolution images (36, 37), although their imaging characteristics indicate that this should be possible. For example OCT has been used successfully to detect advanced basal cell carcinoma, but it was not able to distinguish between basal cell carcinoma subtypes (3). resolution (axial approaching 10 mm and lateral o30 mm) at a depth of 1.1 mm (9, 11). Figure 6 shows a typical HFUS scan through a malignant melanoma and the corresponding histology image. While the major features of the sample can be seen, HFUS does not provide cellular resolution. HFUS can be used in the follow-up of chronic and inflammatory skin diseases, pre- and postoperative assessments of skin tumours, especially malignant melanoma and basal cell carcinoma (Fig. 7), and can also be used as a guide during surgical procedures (38). Ultrasound Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET) High-frequency ultrasound (HFUS) uses ultrasound frequencies of between 3 and 100 MHz to evaluate skin morphology (9, 11). Images, as in OCT, are vertical slices through the skin, with the depth of penetration and resolution dependent on the frequency of the sound used. Low-frequency (3–10 MHz) scanners are used for subcutaneous or lymph node imaging. Twenty to 50 MHz scanners are used for sharp focusing at superficial regions, with 20 MHz generally considered the best compromise between resolution (axial o80 mm lateral 200 mm) and penetration (  3.8 mm). Higher frequency scanners (50–100 MHz) have been used to scan melanocytic lesions and have an improved MRI and PET have also been used in the in vivo diagnosis of skin cancer and malignant melanoma. The application of MRI to dermatology has become practical with the use of specialised surface coils that allow higher resolution imaging than standard MRI coils (11, 39). Bittoun et al. (40) have obtained images of normal skin with highly anisotropic voxels of 70  390  3000 mm3, with the smallest dimension being perpendicular to the skin surface, allowing the different skin layers to be visualised; see Fig. 8. Micro-MRI has also been used to investigate the courses of cellulite in humans (41) and the effects of photoageing in a murine model (42). The use of magnetisation 261 Smith and MacNeil Fig. 3. The centre panel (d) shows the refined border of a lentigo maligna melanoma (LMM) on the scalp of a patient as determined by confocal scanning laser microscopy (CSLM). The CSLM-examined foci are numbered 1–16 and are colour-coded to indicate areas that were negative (green) and positive (purple) for LMM on CSLM images. Five pairs (marked in yellow) of these foci on either side of the border were biopsied for histological confirmation. (a–c) show the confocal, histology, and Melan-A-immunostained sections of one representative area of normal skin (long arrow in d). (a) Shows the epidermal layer and demonstrates the honeycomb pattern of keratinocytes and well-defined cell-to-cell demarcations, which represent the characteristic architecture of normal skin. (b,c) The haematoxylin and eosin (H&E)stained and Melan-A-stained histological sections of normal skin, respectively. (e–g) Confocal, histology, and Melan-A immunostained sections of one representative area of skin with LMM (long arrow in d). (e) shows the spinous layer and demonstrates pagetoid spread of atypical, dendritic melanocytes (short arrow), loss of the normal architecture, and a grainy background – all features consistent with LMM. (f,g) H&E-stained and Melan-A-stained histological sections of LMM, respectively. The Melan-A staining (g) shows the dendrites of the melanoma cell and correlates with the dendritic malignant melanocyte (arrow) seen in (e). Original magnification: (b)  100; (c)  100; (f)  200; (g)  400. From Chen et al. (66). transfer contrast provides data enabling the evaluation of how the tissue in skin layers interacts with the interstitial fluids. Details obtained from high-resolution high-quality in vivo skin images with different contrasts allowed for the differentiation of skin layers, sub-layers and excellent correlation of MR data with known histological features and water constituent of skin layers (43). MRI has also been used to image melanoma metastasis in a murine model (44) as well as the imaging of individual skin cancers and the surrounding structures (45, 46); see Fig. 8. PET, a whole-body imaging technique, is widely used in the diagnosis of metastatic cancer. A tracer is used to label the cancer cells. 18Ffluoro-deoxy-glucose (18F-FDG) is one of the most widely applied PET tracers used to survey cell metabolism. The metabolic turnover of tumour cells usually exceeds physiological metabolic activity. Excessive 18F-FDG uptake has consequently been demonstrated in most cancers in vivo, rendering whole-body 18F-FDG-PET an excellent tool for the diagnosis of malignant skin cancers. Non-specific uptake of 18F-FDG, how- 262 ever, has also been reported in various inflammatory conditions (47). While PET is considered a non-invasive imaging technique, the patient does have to take in the tracer, usually by ingestion. The sensitivity of PET depends on the location, and the size, of the tumour; however, a resolution of 4–6 mm is usual. This means that PET may not be sensitive enough to detect small nodel melanoma metastases, which are usually 1–2 mm in size (48). PET by itself does not provide images containing a great deal of detail and is therefore usually combined with computerised tomography (CT) scanning (49, 50). See Fig. 9 for examples of PET, PET/CT images. Spectroscopic Imaging Techniques [Fourier Transform Infrared (FTIR), Raman, and Photoacoustic Spectroscopy] Spectroscopic imaging techniques are not routinely used in the clinical diagnosis of melanoma or for imaging the skin. FTIR and Raman are State of the art in non-invasive imaging of melanoma Fig. 4. In vivo high-resolution multiphoton imaging of human skin. The figure shows the structure of skin with complementary images of twophoton-induced autofluorescence of the different layers down to a depth of 200 mm (elastic fibres in the dermis), l 740 nm. Additional images show the bright luminescence of pigmented cells due to melanin at the stratum basale of a nevus and the infiltration of inflammatory cells (monocytes and granulocytes) into skin tissue at a depth of 65 mm. Image from Schenke-Layland et al. (29). Fig. 5. Optical coherence tomographic (OCT) image over 6 mm of human finger tip skin, palm side. Image obtained using a Michelson diagnostics EX1301 OCT system. Note the lack of cellular resolution compared with confocal and multiphoton microscopy but the increased detail of the image in terms of the dermo–epidermal junction and the presence of rete ridges when compared with the image in Fig. 3. Image courtesy of Dr M. Bonesi. probably the best known of these techniques due to their regular use in chemistry and materials science. FTIR is based on the absorption of IR energy. The IR radiation is split as in OCT so that half goes to a reference arm and half to the sample. The excitation of molecules by IR light causes the covalent bonds in these molecules to move. Different bonds absorb energy at different characteristic wavelengths. When the light is recombined in a Michelson interferometer, an interferogram is produced. This is processed using a Fourier transform to produce an absorbance spectrum that is unique to the compound being analysed. This technique is rarely used in vivo. However, FTIR has the potential to provide information from within a sample, i.e. depth profiling, if modes such as attenuated total reflectance or photoacoustic are used. 263 Smith and MacNeil Fig. 6. Twenty megahertz B-scan (DUB 20, Taberna pro Medicum, Lueneburg, Germany) of malignant melanoma (a) with the corresponding histology (b) (  5 objective lens). E, entry echo; T, tumour; K, corneum/dermis; S, subcutis; B, blood vessel; F, fascia. Image from Marghoob et al. (11). Fig. 7. Twenty megahertz ultrasound of a basal cell carcinoma on the nasal tip in cross sections: Discontinous entrance echo, hypoechoic tumour with mixed echogenicity infiltrating half of the dermal depth. Lateral demarcation of tumour borders remains uncertain (yellow arrows). The cartilage (C) is visualised as an echo-poor structure. In the transversal section (left), a small dermal vessel (V) is marked with a black arrow. The echo-rich band below the cartilage corresponds to the endonasal skin in the vestibulum. Image from Schmid-Wendtner and Dill-Müller (38). Fig. 8. Sagittal and axial T2-weighted images show invasion of a giant basal cell carcinoma on the lower leg through the anterolateral compartments of the leg. Note the anterior invasion of the tibia (arrowheads) and the osteolytic lesion of the fibular bone (*). Image from Arnaiz et al. (46). Raman spectroscopy is based on the principle of Raman scattering, the inelastic scattering of electromagnetic radiation. In Raman spectro- 264 scopy, the sample is illuminated by a monochromatic visible or near IR light from a laser source and its vibrations during the electrical polarisability changes are determined (51). Raman spectroscopy combined with confocal microscopy is rapidly becoming a useful tool. Not only are images obtained potentially at a depth of up to approximately 200 mm within the skin but chemical information is also obtained. It has been shown that the chemical signature of cancer cells is different from that of normal, non-tumour, cells, e.g. basal cell carcinoma (52). The vast majority of the dermatological studies published using this technique focus on the evaluation of topical agents for either cosmetic or pharmacological applications (51, 53–58), and the majority of these are either ex vivo or on embedded histology sections. However, Caspers et al. (53) obtained in vivo measurements while State of the art in non-invasive imaging of melanoma Fig. 9. (a,b) PET-negative/CT-positive metastases of malignant melanoma in a 44-year-old woman with a history of malignant melanoma. (a) The CT component of PET/CT showed nodular opacities in both lungs (arrows in upper panel), which showed metastases of malignant melanoma on biopsy. (b) A transaxial 18F-FDG PET scan (lower panel) showed normal 18F-FDG uptake in both lungs, probably because the lesions were smaller than the resolution limit. (c,d) PET-positive/CT-negative metastases of malignant melanoma in a 53-year-old woman with a history of malignant melanoma. (c) A transaxial 18F-FDG PET image showed increased 18F-FDG uptake in the left cerebellum (arrow in the upper panel), which was consistent with a metastasis. (d) The CT component of PET/CT did not show any abnormality in the same area (arrow in the lower panel). (e) PET scan demonstrates multiple sites of metastatic melanoma. (e) Whole-body PET scan showing hepatic and bilateral pulmonary metastases (arrows). Images (a–d) from Akcali et al. (49) image E from Essner et al. (50). Fig. 10. Left: In vivo confocal image and Raman spectroscopy of a sweat duct on the palm of the hand, 30 mm below the skin surface. The bright area shows a sweat duct. The arrows mark the spots from which the Raman spectra were obtained. Right: The asterisk marks the prominent Raman band of lactate at 856 cm 1. (a) Raman spectrum measured in the sweat duct. (b) Raman spectrum measured outside the sweat duct. (c) Difference spectrum (a b). (d) Fit result of spectrum (a) with spectrum (b) and spectra of natural moisturising factor and sweat constituents. (e) In vitro Raman spectrum of lactate. (f) In vitro Raman spectrum of urea. Image from Caspers et al. (53). looking at the concentration of water and other moisturising agents in the stratum corneum; see Fig. 10. Several molecular species were measured in vivo including keratins, b-carotene, and water, as well as exogenous materials applied to the skin (59). Photoacoustic spectroscopy uses the photoacoustic effect to investigate samples. As the name suggests, this is the generation of acoustic waves from the absorption of electromagnetic radiation. Photoacoustic microscopy and spectroscopy have mainly been used to investigate/image 265 Smith and MacNeil Fig. 11. In vivo non-invasive photoacoustic images of melanoma and vascular distribution in nude mouse skin. (a,b) Enface photoacoustic images for the NIR light source (a) 5 764 nm and visible light source (b) 5 584 nm, respectively: 1, melanoma; 2, vessels perpendicular to the image plane; 3, vessels horizontal to image plane; 4, skin. (c,d) Photoacoustic B-scan images from the NIR and visible light sources, respectively, for the dotted lines in (a) and (b). (e) A cross-sectional histology image (H&E staining): E, epidermis; D, dermis; M, muscle. (f,g) Depthwise enface photoacoustic images from the NIR and visible light sources, respectively; A, 0.15–0.30 mm; B, 0.30–0.45 mm; C, 0.45–0.60 mm; D, 0.60–0.75 mm from the skin surface. Image from Oh et al. (60). blood vessels within skin and in small animals; see Fig. 11 (60, 61) as well as for the detection of melanoma cells in the circulation (62–64). Conclusion Dermoscopy and Confocal Laser Scanning Microscopy are currently in use in the clinic, aiding in 266 the diagnosis of skin cancers, both melanoma and non-melanoma. However, while these techniques are useful in the evaluation of superficial skin cancers, they are not able to image at depth. Currently, the non-invasive techniques, OCT, HFUS, MRI, PET, and the spectroscopic techniques do not have the resolution to detect earlystage skin cancers. These techniques can, however, be used to detect and therefore aid in the State of the art in non-invasive imaging of melanoma diagnosis of late-stage metastatic cancer, i.e. when the secondary tumours become large enough to be detected. In this respect, PET and MRI are currently used to detect advanced metastasis of skin cancers. While both HFUS and OCT have the potential to be used to detect local superficial metastasis, they currently lack the resolution to detect early-stage metastasis of melanoma. In References 1. Gambichler T, Matip R, Moussa G, Altmeyer P, Hoffmann K. In vivo data of epidermal thickness evaluated by optical coherence tomography: effects of age, gender, skin type, and anatomic site. J Dermatol Sci 2006; 44: 145–152. 2. Menzies SW. Cutaneous melanoma: making a clinical diagnosis, present and future. Dermatol Ther 2006; 19: 32–39. 3. Gambichler T, Orlikov A, Vasa R, Moussa G, Hoffmann K, Stücker M, Altmeyer P, Bechara FG. 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