J. Biophotonics 9, No. 4, 376–387 (2016) / DOI 10.1002/jbio.201500124
FULL ARTICLE
Identification of ex-vivo confocal scanning microscopic features and their histological correlates in human skin Daniela Hartmann*, Cristel Ruini, Leonie Mathemeier, Andreas Dietrich, Thomas Ruzicka, and Tanja von Braunmühl Department of Dermatology and Allergology, Ludwig-Maximilian University, Munich, Germany Received 13 March 2015, revised 29 April 2015, accepted 30 April 2015 Published online 22 May 2015
Key words: reflectance confocal microscopy, ex-vivo, fluorescence, histopathologic correlates, diagnostic technique
Ex-vivo confocal laser scanning microscopy (CLSM) is an emerging diagnostic tool allowing fast and easy microscopic tissue examination. The first generation of ex-vivo devices have already shown promising results in the exvivo evaluation of basal cell carcinoma compared to Mohs surgery. Nevertheless, for the diagnostics of pathological skin lesions the knowledge of normal skin features is essential. Therefore we examined 50 samples of healthy skin from various donor sites including head and neck (n = 25), trunk (n = 10), upper (n = 10) and lower extremities (n = 5) using a new generation ex-vivo CLSM device offering three different laser wavelengths and compared the findings to the corresponding histological sections. In correlation with the histopathology we identified different layers of the epidermis, differentiated keratinocytes from melanocytes and described in detail skin appendages including hair follicle, sebaceous and sweat glands. Furthermore, structures of the dermis and subcutis were illustrated. Additionally, artefacts and pitfalls occurring with the use of ex-vivo CLSM have been documented. The
1. Introduction In the last decade the confocal laser scanning microscopy (CLSM) or reflectance confocal microscopy (RCM) has developed into a widespread und useful diagnostic method in dermatology and other fields of medicine, as well as in various research areas. It has been successfully used mainly in the field of der-
study offers an overview of the main ex-vivo CLSM skin characteristics in comparison to the standard histological examination and helps to recognize and avoid common artefacts.
Anatomy of a hair follicle in the reflectance mode (RM) CLSM, fluorescence mode (FM) CLSM and in a routine hematoxylin-eosin stained histological section (H).
mato-oncology but also in inflammatory dermatoses and therapeutic monitoring [1–10]. The in-vivo CLSM allows a non-invasive optical biopsy, generating fast, reproducible and painless images of the skin up to the papillary dermis. The first description of in-vivo CLSM of human skin was provided by Rajadhyaksha and colleagues in 1995 [11, 12]. Since then, the number of publications on
* Corresponding author: e-mail: [email protected], Phone: +49 (0)89 4400-56602, Fax: +49 (0)89 4400-56624 © 2015 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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this topic has constantly increased, showing a growing interest of the scientific community towards this technique [13–18]. The CLSM is based on a diode laser emitting monochromatic light that is directed through an interconnected lens system onto the area of interest of the skin and then reflected into a small pinhole (bean-splitter) detector forming an image. Only the light coming from the focal area of interest can be detected and form an image (confocal). The commonly used in-vivo CLSM device, Vivascope 1500® (MAVIG GmbH, Munich, Germany) uses an 830 nm near-infrared laser with a maximum power of 35 mW, generating images at cellular resolution, comparable with histological slides. Morphological details can be defined up to a resolution of 0.5–1.0 μm in the lateral section and 4–5 μm in the axial section [19, 20]. The laser power causes no damage to human tissues but limits the penetration depth to around 250 μm [11]. The imaging technique is based on the different refraction indices of different tissue structures; the highest refraction index is provided by melanin and melanosomes that appear extremely bright, while skin folds and cytoplasm are dark. For the image acquisition in the in-vivo CLSM a metal ring is attached to a polymer window and is fixed to the skin. Through stepwise movement of the object table, standard 500 × 500 μm basic images can be acquired to form composite mosaics of horizontal (VivaBlock®) and vertical (VivaStack®) images of the epidermis and dermis up to the papillary layer. Compared to its in-vivo counterpart, ex-vivo CLSM gives the opportunity to examine all skin layers without limitation of the penetration depth in the traditional vertical view that can be easily compared to histology. This is achieved by fixing vertical slices of excised tissue in an observation chamber and scanning through the whole sample horizontally. Another advantage of ex-vivo CLSM compared to the in-vivo use is the possibility of tissue staining. Various staining methods for ex-vivo CLSM have already been developed [21–23]. Among them, the acridine orange stain has shown promising results in the ex-vivo guided Mohs surgery of basal cell carcinoma (BCC) [24–29]. As a selective staining for nucleic acids, it highlights the nuclei of BCC cells and provides strong contrast compared to the surrounding dermis, allowing an easy detection of tumor islands without altering the tissue for further histopathological processing. Possible pitfalls that can hinder the correct identification of tissue structures in the reflectance mode (RM) are for example images of sebaceous glands that can mimic islands of basal cell carcinoma due to their roundish appearance, while an intense stromal reaction may simulate basaloid cords [24, 25, 30]. In such cases the use of fluorescent dye may be very helpful. Acridine orange but also fluorescein, toluidine blue, methylene
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blue, acetic acid and citric acid were used as staining in other preliminary studies and gave variable results [21–23]. Ex-vivo CLSM has a unique possibility compared to other invasive procedures – the specimen examined with ex-vivo CLSM can be re-examined using traditional histology including immunohistochemistry. The aim of this study was to describe the anatomy of healthy skin and its appendages as seen in exvivo CLSM in correlation to the corresponding histopathology as basic knowledge for further microscopic examinations of pathological skin lesions. Furthermore pitfalls and artefacts experienced during ex-vivo CLSM imaging have been collected and recommendations for correct handling and prevention of problems are given.
2. Material and methods From September 2014 to January 2015 we examined 50 samples of healthy skin from various donor sites using ex-vivo CLSM (Vivascope 2500®; Lucid Inc; Rochester NY). The skin probes were taken from the Dermatosurgery Section of the Department of Dermatology and Allergology, Ludwig-Maximilian University, Munich, Germany after obtaining written informed consent from each patient. The study was conducted following the principles of Helsinki. The skin probes were immediately processed after excision for the CLSM analysis. The preparation of the skin samples, the staining and scanning with the ex-vivo CLSM was performed by one specially trained dermatologist (D.H.). Two trained CLSM specialists with histological skills performed independent confocal and histological examination of the specimens and evaluated it. All samples were sectioned and scanned in the vertical mode to enable the exact correlation to traditional histology. Standard staining with hematoxylin-eosin (H&E) was used for the histological sections. The ex-vivo CLSM used here (Vivascope 2500®; Lucid Inc; Rochester NY) combines three different lasers with wavelengths of 488 nm, 658 nm and 830 nm and allows to examine the tissue in the reflectance (RM, 830 nm) and in the fluorescent mode (FM, 488 nm und 658 nm). The maximum examination depth is limited to 250 μm and the vertical resolution to 5 μm. The water-immersion objective lens has a numeric aperture of 0.9 and allows magnification ranging between 30× and 400×. The sample size of 0.63 mm × 0.63 mm may be examined at once. The microscope reconstructs a mosaic of 1089 single images (33 times 33) and allows continuous scanning of an area of 20.8 mm × 20.8 mm. In this study the software VivaScan® (Version 7, Mavig GmbH, Mu-
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nich, Germany) was used and the tools of VivaBlock® and VivaStack®, as well as conventional photography and snap shots served for obtaining and saving the images of the examined tissue. Fresh tissue, transported in physiological saline solution, was immediately sectioned, stained, examined in the CLSM and then fixed in formalin for the
histological examination. The fluorescein staining methods included acridine orange (0.6 mM; SigmaAldrich®) and fluorescein (0.4%; ALCON®) examined with the wavelength of 488 nm and 95% Nile blue A (Sigma-Aldrich®; 2 mg/ml; wavelength of 658 nm). During the staining process the 10% citric acid (Sigma-Aldrich®) for aceto-whitening and Dul-
Table 1 List of ex-vivo CLSM features of healthy skin and their histological correlates in hematoxylin-eosin (H&E) staining. Arrow ↓ – depth under surface. Anatomical/histological structure
Ex-vivo CLSM features in RM and FM
Histological correlates (H&E staining)
Stratum corneum ↓ 0–15 μm
Multiple layers of flat, non-nucleated polygonal dark grey corneocytes (dimension: 10–30 μm) surrounded by thin, bright walls.
Multiple layers of pink, flat, dead cornified, non-nucleated keratinocytes.
Stratum granulosum ↓ 15–20 μm
Polygonal greyish cells (25–35 μm) with well demarcated outlines, grainy cytoplasm and round to oval nuclei.
Nucleated keratinocytes showing pink (eosinophil) cytoplasm with dark granules and dark blue or purple nuclei.
Stratum spinosum ↓ 20–100 μm
Large (15–25 μm), greyish polygonal cells with bright and centrally located nuclei.
Few layers of polygonal prickle cells with purple nuclei in the center of the pink cytoplasm.
Stratum basale ↓ 50–100 μm
Clusters of bright small (7–12 μm) cuboidal keratinocytes, prominent nucleus and little cytoplasm.
Cuboidal-shaped cells of the germinal layer appearing darker than stratum spinosum.
Dermo-epidermal junction ↓ 100-120 μm
Wavy to straight line or band of grey to black color lying between epidermis and dermis.
Interdigitating light pink, continuous band.
Papillary dermis ↓ 100–200 μm
Reticulated network of fine grey fibers or thicker bundles (collagen); bright particles (inflammatory cells); dark round spots or wavy to coiled tubes (vessels).
Fine textured light to dark pink collagen fibers; purple dots and spots representing inflammatory cells; dark pink blood and lymph vessels; light pink nerves; sweat and sebaceous glands.
Reticular dermis ↓ 200–400 μm
Loose but thicker bright collagen fibers; vessels and skin appendages.
Coarsely textured pink collagen fibers; vessels and skin appendages.
Subcutis
Regular net-like layers of large dark cells with very thin bright walls and bright nuclei pushed to the outer edge by lipid vacuoles that appear as empty space.
Regular net-like layers of unilocular large cells with whitish homogeneous lipid-rich vacuoles and a purple colored nucleus pushed to the outer edge.
Hair follicle
Dark hair shaft surrounded by bright root sheaths with cells arranged in columns. The matrix around the papilla is the brightest structure. Arrector pili presents as set of fine grey fibers with tiny bright dots or short lines (nuclei).
Hair shafts as columns of pink to brownish keratinized cells, gradually extruded from the follicle. Arrector pili as fine sets of eosinophilic fibers with scattered elongated purple nuclei.
Sebaceous gland
Large, round, sharply demarcated structures filled with multiple round cells with bright nuclei and grey granular cytoplasm filled with vacuoles.
Sebocytes are large light cells packed with lipids and centrally located dark nuclei.
Sweat gland
Convoluted tubes with a duct formed by two layers of small nucleated cells, lining the lumen.
The secretory portion is comprised of larger cuboidal cells, lies deep in the dermis, where the tubule is twisted into a compact coiled tangle. The duct consists of a two-layered stratified cuboidal epithelium.
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becco’s Phosphate buffered saline (Sigma-Aldrich®) were used. The proper choice of the laser wavelength is based upon the staining method of the tissue. Wavelength of 488 nm is suitable for tissue stained with acridine orange or fluorescein, wavelength of 658 nm for examining samples stained for example with Nile blue. Acridine orange and fluorescein have been tried several times and the results have been compared with each other. Since acridine orange stains exclusively DNA it provides a strong contrast between nuclei-rich and nuclei-poor structures. Fluorescein stains besides DNA also extracellular matrix and shows dermis in a bit brighter way. Acridine orange revealed the clearest results and was therefore preferentially used for further staining in our study for the comparison with the histology.
3. Results We examined 50 samples of healthy skin (male : female ratio = 28 : 22; skin type I to V; age between 20 to 92 years, median 57.5 years.) from various donor sites using ex-vivo CLSM, performed 100 full sample scans, as well as hundreds of partial scans and closeups, and compared them to histology. The skin samples originated from: head and neck (n = 25), trunk (n = 10), upper extremities (n = 10) and lower extremities (n = 5). We concentrated on the identification of the epidermal layers and on the differentiation of keratinocytes and melanocytes. Further we described the CLSM images of various skin appendages (hair follicle, sebaceous and sweat glands). The structures of the dermis and subcutis, including the fatty tissue, collagen and elastic fibers, vessels, nerves and muscles were examined. The main ex-vivo CLSM skin characteristics were compared to the standard histological examination and re-evaluated. As basis for the ex-vivo CLSM image evaluation the histopathological morphology of healthy skin will be described shortly in the following section and correlated to the standard histopathological description of skin structures [31]. The main ex-vivo CLSM microscopic characteristics with their histological correlates are presented in Table 1.
3.1 Skin overview The morphology of healthy skin is presented in Figure 1a–d. First, an overview image of the whole specimen was obtained using the Vivablock® tool (Figure 1a) in both RM and FM for the orientation within the sample and to exclude possible skin patholo-
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gies. In the RM the full width of the skin is well displayed, including the subcutis. In the FM, certain structures of the skin are highlighted depending on the staining. With acridine orange, the cell- and nuclei-rich structures are most prominent. Close-ups under magnification give the possibility to study all skin layers in detail (Figure 1b–d), including the skin appendages (Figure 2a–c). Structures of the subcutis are also visible (Figure 3a–d). The fatty tissue (Figure 3a) presents with almost black cytoplasm and fine bright cell membranes with small, but prominent and bright cell nuclei. In sebaceous glands (Figure 2b) with high numbers of overlapping cells, the bright nuclei do not allow the recognition of further morphological characteristics. The collagen fibers (Fig. 1b) are very well displayed in the FM and this allows the evaluation of the unique tangled complex of collagen fibers in the dermis together with the nuclei of the fibroblasts. In the reflectance mode, the collagen fibers are also visible, but much less bright, less clear and less sharp than in the FM.
3.2 Epidermis After evaluating the overview image all skin layers were studied in detail, starting with the epidermis (Figure 1b). The keratinocytes (Figure 1c) present in the ex-vivo CLSM RM as cuboidal and polygonal to flat (depending on the layer) cells with bright nucleus and dark grey cytoplasm. The largest oval formed nuclei are seen in stratum basale. In the upper parts of the epidermis the nuclei become smaller and thinner until they get totally lost in the basket-weave-like stratum corneum. This corresponds very well to histological correlates (Figure 1e). In contrast to the reflectance mode, where the cytoplasm of the keratinocytes appears dark grey, in the FM it presents light grey and more homogeneous. In the FM the nuclei are very bright and the most prominent structures especially in the acridine orange staining. Also, cell membranes are more clearly visualized in the FM than in the reflectance mode. Stratum corneum (Figure 1a), consisting of flat non-nucleated cells, appears in the ex-vivo CLSM highly refractive with dark spots representing the cytoplasm. Stratum granulosum shows greyish keratinocytes with grainy cytoplasm (Figure 1b). Sometimes a regular honeycomb pattern may be displayed similar to the horizontal images in the in-vivo CLSM. Stratum spinosum (Figure 1b) shows few layers of prominent polygonal cells with bright central nuclei. Stratum basale (Figure 1b), if sectioned at an oblique angle, may show a cobblestone pattern, and is formed by clusters of small bright, cuboidal cells with little cytoplasm, similar to in-vivo CLSM. Melanin has an even higher refraction index © 2015 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 1 Morphology of healthy human skin in ex-vivo CLSM (Vivablock® by VivaScan®, acridine orange, RM (830 nm) and FM (658 nm) and histological correlates (H); (a) Skin biopsy from male scalp shows multiple hair follicles and normally structured epidermis, dermis and subcutis in RM and FM. Upper layers of stratum corneum are much darker in FM in contrast to RM due to nuclei-free corneocytes. Morphology of skin appendages can be better compared to histology in RM than in FM; (b) Close-up of the epidermis, dermo-epidermal junction (DEJ) and upper dermis in RM and FM. Upper layers of stratum corneum are much darker in FM in contrast to RM due to nuclei-free corneocytes. Morphology of skin appendages can be better compared to histology in RM than in FM. Collagen fibers, fibroblasts and sparse inflammatory infiltrate in the dermis highlighted in the FM; (c) Display of keratinocytes in RM in different epidermal layers presenting bright large nuclei in stratum basale and loss of nuclei in the upper parts of the epidermis; (d) Display of melanocytes (arrows) scattered among keratinocytes in FM in the basal parts of the epidermis with melanocytes showing brighter and larger nuclei.
(1.72) than keratinocytes (1.51) and therefore melanocytes (Figure 1d), scattered among the basal keratinocytes (Figure 1c) appear much brighter than keratinocytes.
3.3 Dermo-epidermal junction and dermis The dermo-epidermal junction (Figure 1b) consists of the epidermal basal membrane and dermal papillae. In the reflectance ex-vivo CLSM it might appear as dark greyish to black line or linear space between epidermis and dermis. The dark line may be interdigitating, wavy or almost straight. In the papillary dermis (Figure 1a and b) occasional bright dots representing nuclei of sparse inflammatory cells and fibroblasts, as well as dark roundish spots corresponding to the vessels may be observed. The papillary
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dermis shows a network of reticulated light grey fibers and capillary vessels. Sometimes the ring like pattern of papillary dermis known from in-vivo CLSM is also seen in ex-vivo CLSM images in case of oblique cutting angle. The ex-vivo CLSM images of reticular dermis are dominated by collagen and elastic fibers. Collagen has a relatively high refraction index of 1.43 and therefore collagen fibers can be easily distinguished from the stroma. In the FM the dermo-epidermal junction is usually dark grey to black and can be misinterpreted as empty space between epidermis and dermis (Figure 1b). In that case, examining the tissue above and underneath this ‚black line’, either manually or via VivaStack® tool, may help to visualize the anchoring fibrils, collagen fibers and the matrix. In the FM the dermis displays by the image of bright intertwined collagen fibers mixed with brightly shining nuclei of fibroblasts.
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Figure 2 Morphology of skin appendages in ex-vivo CLSM (Vivablock® by VivaScan®, acridine orange, RM (830 nm) and FM (658 nm) and histological correlates (H); (a) Anatomy of a hair follicle in RM and FM. RM showing dark hair shafts surrounded by bright root sheaths with cells arranged in columns. In FM the matrix around the papilla is the brightest structure; (b) Anatomy of a sebaceous gland in RM and FM presents as large, round, sharply demarcated structures filled with multiple round cells showing bright nuclei and grey granular cytoplasm filled with vacuoles. Clusters of brightly shining nuclei dominate the FM image; (c) Anatomy of a sweat gland in RM and FM presenting a clump of convoluted tubes with a duct formed by two layers of small nucleated cells, lining the lumen.
3.4 Skin appendages and subcutis The anatomy of skin appendages (Figure 2a–c) can be easily viewed and studied by ex-vivo CLSM. In the RM the hair shafts appear dark, the cells of the outer and inner root sheath are bright and the hair matrix surrounding the hair papilla presents with the highest brightness. In the FM the lining and orientation of the nuclei, especially in the outer root sheath, is well displayed (Figure 2a). The sebaceous glands (Figure 2b) in the RM are large, round, sharply demarcated structures filled with multiple large round cells with small bright nuclei and grey granular cytoplasm filled with sebum vacuoles. The bright nuclei in the FM are dominating the image of the whole structure of a sebaceous gland and the cytoplasm is presented as light grey homogeneous mass. The sweat glands (Figure 2c) can be well visualized in both modes as tortuous, convoluted tubes representing the secretory portion and a duct connecting the secretory portion with the epidermis. The duct is formed by two layers of small cells with bright nuclei. The lumina of the ducts are round dark circles lined by cells of the duct walls. The subcutis and its fatty tissue (Figure 3a) present in the
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FM as a network of regular, large, bright, thin loops on a dark, almost black background. The loops represent cell membranes, whereas nuclei are shown as small bright dots pushed to the side, as if lying within the membranes. These seemingly vacant dark spaces in the loops represent lipid vacuoles. In the RM the image of the adipocytes is similar, with the difference that the cytoplasm seems to be filled with a light grey to bright granular mass and the cell nuclei are not easy to distinguish. Other structures of the subcutis including vessels (Figure 3b), nerves (Figure 3c) and muscles (Figure 3d) are clearly visible in both modes. All samples of healthy skin have been correlated to the histology using H&E staining. The main confocal features and characteristics of healthy human skin and their histological correlates are listed in Table 1.
3.5 Donor sites and skin types Comparison of samples originating from different donor sites showed the same differences as those known from the histologic examination. The identification of the donor site is based on the thickness of © 2015 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 3 Morphology of subcutis and its structures in ex-vivo CLSM (Vivablock® by VivaScan®, acridine orange, RM (830 nm) and FM (658 nm) and histological correlates); (a) Image of the fatty tissue in RM and FM. Regular net-like layers of large dark cells with very thin bright walls and bright nuclei pushed to the outer edge by lipid vacuoles which appear as empty spaces; (b) Anatomy of a vessel (arteria temporalis superficialis) in RM and FM presents as large, oval, sharply demarcated structure with thick walls and large lumen; (c) Cross-section of a nerve presenting as a round spot filled with nerve fascicles colored in RM from dark grey to almost black and in FM grey with only few small bright nuclei; (d) RM and FM images of a smooth muscle (arrector pili) show a set of fine, light grey fibers in a cross-section with tiny bright dots or short lines (nuclei) localized on the borders of the muscle fascicle.
the stratum corneum and other parts of the epidermis, on the presence and form of the rete ridges, on the thickness and composition ratio of the dermis and subcutis, as well as on the representation of skin appendages, vessels and muscles. One of the clearest examples represents the image of scalp skin (Figure 1a). The average thickness of the skin (including epidermis and dermis) originating from the trunk was 2.3 mm and from the face 1.1 mm. In the facial skin of elderly patients solar elastosis in the dermis could be identified, seen especially well in the FM. The degenerated elastotic fibers presented as accumulation of a disorganized and irregularly thickened mass with only remainders of partly recognizable band-like form. It was challenging to find the subtle differences among the skin samples from skin types I to V. Therefore we simplified the classification into light (I–III) and dark skin types (IV–V), which enabled the recognition of the more refractive and more fluorescent stratum basale in ex-vivo CLSM due to © 2015 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the larger number and size of melanosomes in keratinocytes of darker skin types (Figure 4) compared to lighter skin types.
3.6 Artefacts During the study several artefacts have occurred. These can easily mislead in the process of identifying relevant microscopic features and even of making the correct diagnosis in samples of diseased skin. In order to check the nature of the causing agent, we generated all of the described artefacts at the end of the study on spare specimen and correlated the images with those artefacts observed during the study. Table 2 lists the most common artefacts, as well as the frequency of their occurrence during our trial. In addition the causes and the prevention of artefacts are proposed. The most frequently observed artefacts in our study were caused by inappropriate thickness of the specimen and by contami-
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Figure 4 Dark skin type (IV–V) displayed in ex-vivo CLSM (Vivablock® by VivaScan®, acridine orange, RM (830 nm) and FM (658 nm) and a histological correlate); (a) RM image of epidermis and upper dermis showing highly refractive stratum basale; (b) FM image of epidermis and upper dermis showing normal and not elevated numbers of melanocytes in stratum basale; (c) Histological correlate (H) of a darker skin sample with typically pigmented basal layer of the epidermis due to increased and enlarged melanosomes in the keratinocytes.
nation of the specimen with cotton gauze fibers. During our trial we observed no changes in the microscopic features of frozen versus fresh tissue sections. This enabled postponing the scanning of the tissue until the examiner was available. Nevertheless, here we present only data from examination of freshly excised, unfrozen tissue.
4. Discussion Our results show that for the general overview of the basic confocal features of healthy skin the RM gives better morphological view of all skin structures including skin appendages and it is easier to correlate these images to traditional histology than images obtained in the FM. In case of finding a particular cell or structure, it can be helpful to switch from the RM to the FM in order to separate the structure of
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interest from the background. This particularly applies for fibroblasts in the dermis and for melanocytes in the epidermis. Further the collagen and elastic fibers are much better displayed in the FM. The dermo-epidermal junction can be easier correlated to histological images if displayed in the reflectance mode. We could demonstrate that with some degree of practice it is possible to switch from the pink and blue histological depiction to the black and white confocal world. The ex-vivo CLSM enables very similar examination of the skin as dermatohistopathology. With the use of the VivaStack® tool the ex-vivo imaging allows to portray the examined structures in a three-dimensional view. The fluorescent dyes give the opportunity to selectively bind to the structures of interest and therefore might be used in ex-vivo CLSM in the future in a similar way as immunochemistry is being used today in traditional histology. More studies on this subject are warranted. Currently the ex-vivo CLSM device that combines the reflectance and FM is being tested and results are compared to traditional histology in order to elaborate a faster, easier and more precise method of examination of freshly excised tissue. In contrast to in-vivo CLSM it is an invasive diagnostic tool which, owing to that, allows examining the tissue sample in a vertical view. Therefore the results enable more accurate correlation to histological images. In this study we used mostly acridine orange, a metachromatic stain selective for nucleic acids. It binds to both dsDNA, emitting a green fluorescence (525 nm) very similar to fluorescein, and ssDNA or RNA, emitting a red fluorescence (>630 nm). Thanks to these features it can be used in the study of cell cycle to distinguish between cells in proliferation and in quiescence. As it is able to penetrate lysosomes, it is also useful in identifying apoptotic cells. It is mainly used in research activities [30]. Previous studies with an older version of the exvivo CLSM device which offered an examination only with two instead of three different laser sources have been published. These include studies exploring digital staining, confocal-histological correlations and methods to assimilate images of ex-vivo CLSM and histology [32, 33]. Furthermore studies from medical fields other than dermatology have been concentrating on pathologies of different organ tissues and their evaluation with ex-vivo confocal microscopy [32, 34–39]. To date there have been only few published dermatological articles which evaluated ex-vivo CLSM images of basal cell carcinoma, its diagnosis and monitoring of therapy [24–28, 40– 42], as well as other types of non-melanoma skin cancer, including squamous cell carcinoma [43–45]. The limitations of our study were mainly of technical nature. The preparation of tissue can be quite time consuming when examining large tissue samples, especially if there is the need for preserving the
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Table 2 List of the most common artefacts in ex-vivo CLSM, their causes, frequency and how to prevent them. Artefact
Cause
Prevention/solution
Number (n) and frequency (%) of the artefacts among all 50 samples
Frequency (%) of the artefacts among all 22 artefacts
Dark sharply demarcated lagoons of homogeneous mass with white loops in the center (Figure 5a).
Silicone on the specimen.
Careful handling of the silicone tube during the fixation process.
n = 2 (4%)
9%
Bright, dust-like, smudgy, homogeneous masses sprinkled with snow-like flakes together with blurred microscopic features (Figure 5b).
Too much silicone on the sides of the slide.
Using only small drops of silicone on each side of the slide in order to be able to squeeze the sample between the slides evenly, gently but firmly.
n = 3 (6%)
13%
Bright, long, sharply demarcated, uniform, rod-like structures arranged next to each other (Figure 5c).
Contaminating the sample during the cutting process with the fibers of a cotton gauze piece used as a cutting mat.
Cutting and slicing the specimen on a solid and stable mat and not on a piece of gauze.
n = 5 (10%)
23%
Blurred microscopic features, unable to focus properly (Figure 5d).
Wrong unstained side of the specimen being scanned.
Careful handling of the specimen and the slides, marking the stained side, if noted flipping the slide upside down.
n = 1 (2%)
5%
Dark and blurred microscopic features.
No or too little ultrasound gel used as medium for the water-immersion objective.
Adding more ultrasound gel before cleaning the slide in case of noticing dried rests of the gel on the slide.
n = 2 (4%)
9%
Unclear and extensively blurred microscopic features visualizing only the ultrasound gel and not the specimen, unable to fixate the slide into the eligible stage opening.
Thickness of the specimen is too large.
Slicing the specimen evenly and as thin as possible, not more than 0.3 to 0.5 cm.
n = 6 (12%)
27%
Unable to visualize the specimen in the FM, even though the specimen could be viewed in the reflectance mode.
The specimen has been stored in a formalin solution/ the fluorescent stain has been depreciated by light if stored inappropriately.
Keep the specimen stored in a saline solution and do not permit contact to formalin/store the stains in a dark place.
n = 1 (2%)
5%
Low quality of scanned image and black rims around the specimen.
The specimen is dried-up.
Processing the preparations and scanning as fast as possible and switching off the laser source in case of a scanning pause.
n = 2 (4%)
9%
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Figure 5 Images of artefacts produced during ex-vivo CLSM tissue examinations (Vivablock® by VivaScan®, acridine orange, RM (830 nm) and FM (658 nm); (a) Black, sharply demarcated lagoons of homogeneous mass with white loops in the center represent an artefact caused by silicone drops misplaced on top of the tissue sample (RM); (b) Bright, dust-like, smudgy and homogeneous masses that are sprinkled with snow-like flakes present an artefact caused by large amount of silicone placed next to the sample (RM); (c) Display of bright, long, sharply demarcated, uniform, rod-like structures arranged next to each other, caused by pieces of gauze on the sample (FM); (d) Blurred microscopic features and inability to focus properly are artefacts produced by inverting the slide upside down and scanning the unstained side of the specimen (RM).
orientation of the sample edges, as in the case in Mohs surgery. The tissue to be sliced and cut is not fixed in formalin and therefore needs handling with special caution. The tissue slices should be even and thin, ideally not exceeding a thickness of 3 mm. Specimens thicker than 3 mm usually do not allow proper mounting of the tissue between the slides. The size of the scanning area limits tissue samples to be examined to 20.8 mm × 20.8 mm. The scanning of such an area lasts 7 minutes, which needs to be multiplied by the number of the sample sections. The scanning speed, the size of the scanned area, mounting of the specimen on the slide, fixing of the slides on the microscope stage and a wider choice of suita-
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ble stains are crucial points in further development of this method. To our knowledge this is the first study which concentrated on the ex-vivo confocal images of healthy skin and its appendages. It is also the first study which was performed with the latest version of an ex-vivo CLSM device and allowed examination with three different laser wavelengths. This allowed using more types of staining and allowed the visualization of skin structures in different ways. New staining methods and protocols are expected. The aim of this work was to determine and describe the main ex-vivo confocal features and characteristics of human skin and to correlate them with traditional his© 2015 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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tology. We also gave an overview on possible artefacts and how to prevent them. The results of this study should serve as a guide to distinguish normal from pathological findings. Good knowledge of exvivo CLSM healthy skin images may lead to a faster and more precise understanding of pathological processes.
5. Conclusion Ex-vivo CLSM is a new emerging diagnostic tool which allows fast and easy microscopic tissue examination. Correlations to classical histological images have been systematically studied and advice for correct handling could be provided. Ex-vivo CLSM is not yet able to replace the traditional histology and immunohistochemistry but might play an important role in future tissue sample diagnostics implementing and casting a new light on bedside surgery. In the clinical practice, it might be used by properly trained dermato-surgeons to optimize the surgical procedures and the definitions of surgical margins. Exvivo CLSM’s extremely quick acquisition and evaluation time (minutes) may allow surgeons to perform a confocal-guided bedside Mohs surgery. Avoiding the long tissue sample processing would strongly shorten inter-operative waiting times, number of surgical procedures and hospitalization days. In conclusion, ex-vivo CLSM has revealed itself as a promising diagnostic tool, with potential major benefits for time sparing and cost effective surgery. Conflict of interest The Vivascope® device was provided by Mavig GmbH for the time of the study from September 2014 until January 2015. Author biographies online.
Please see Supporting Information
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FULL ARTICLE
Identification of ex-vivo confocal scanning microscopic features and their histological correlates in human skin Daniela Hartmann*, Cristel Ruini, Leonie Mathemeier, Andreas Dietrich, Thomas Ruzicka, and Tanja von Braunmühl Department of Dermatology and Allergology, Ludwig-Maximilian University, Munich, Germany Received 13 March 2015, revised 29 April 2015, accepted 30 April 2015 Published online 22 May 2015
Key words: reflectance confocal microscopy, ex-vivo, fluorescence, histopathologic correlates, diagnostic technique
Ex-vivo confocal laser scanning microscopy (CLSM) is an emerging diagnostic tool allowing fast and easy microscopic tissue examination. The first generation of ex-vivo devices have already shown promising results in the exvivo evaluation of basal cell carcinoma compared to Mohs surgery. Nevertheless, for the diagnostics of pathological skin lesions the knowledge of normal skin features is essential. Therefore we examined 50 samples of healthy skin from various donor sites including head and neck (n = 25), trunk (n = 10), upper (n = 10) and lower extremities (n = 5) using a new generation ex-vivo CLSM device offering three different laser wavelengths and compared the findings to the corresponding histological sections. In correlation with the histopathology we identified different layers of the epidermis, differentiated keratinocytes from melanocytes and described in detail skin appendages including hair follicle, sebaceous and sweat glands. Furthermore, structures of the dermis and subcutis were illustrated. Additionally, artefacts and pitfalls occurring with the use of ex-vivo CLSM have been documented. The
1. Introduction In the last decade the confocal laser scanning microscopy (CLSM) or reflectance confocal microscopy (RCM) has developed into a widespread und useful diagnostic method in dermatology and other fields of medicine, as well as in various research areas. It has been successfully used mainly in the field of der-
study offers an overview of the main ex-vivo CLSM skin characteristics in comparison to the standard histological examination and helps to recognize and avoid common artefacts.
Anatomy of a hair follicle in the reflectance mode (RM) CLSM, fluorescence mode (FM) CLSM and in a routine hematoxylin-eosin stained histological section (H).
mato-oncology but also in inflammatory dermatoses and therapeutic monitoring [1–10]. The in-vivo CLSM allows a non-invasive optical biopsy, generating fast, reproducible and painless images of the skin up to the papillary dermis. The first description of in-vivo CLSM of human skin was provided by Rajadhyaksha and colleagues in 1995 [11, 12]. Since then, the number of publications on
* Corresponding author: e-mail: [email protected], Phone: +49 (0)89 4400-56602, Fax: +49 (0)89 4400-56624 © 2015 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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this topic has constantly increased, showing a growing interest of the scientific community towards this technique [13–18]. The CLSM is based on a diode laser emitting monochromatic light that is directed through an interconnected lens system onto the area of interest of the skin and then reflected into a small pinhole (bean-splitter) detector forming an image. Only the light coming from the focal area of interest can be detected and form an image (confocal). The commonly used in-vivo CLSM device, Vivascope 1500® (MAVIG GmbH, Munich, Germany) uses an 830 nm near-infrared laser with a maximum power of 35 mW, generating images at cellular resolution, comparable with histological slides. Morphological details can be defined up to a resolution of 0.5–1.0 μm in the lateral section and 4–5 μm in the axial section [19, 20]. The laser power causes no damage to human tissues but limits the penetration depth to around 250 μm [11]. The imaging technique is based on the different refraction indices of different tissue structures; the highest refraction index is provided by melanin and melanosomes that appear extremely bright, while skin folds and cytoplasm are dark. For the image acquisition in the in-vivo CLSM a metal ring is attached to a polymer window and is fixed to the skin. Through stepwise movement of the object table, standard 500 × 500 μm basic images can be acquired to form composite mosaics of horizontal (VivaBlock®) and vertical (VivaStack®) images of the epidermis and dermis up to the papillary layer. Compared to its in-vivo counterpart, ex-vivo CLSM gives the opportunity to examine all skin layers without limitation of the penetration depth in the traditional vertical view that can be easily compared to histology. This is achieved by fixing vertical slices of excised tissue in an observation chamber and scanning through the whole sample horizontally. Another advantage of ex-vivo CLSM compared to the in-vivo use is the possibility of tissue staining. Various staining methods for ex-vivo CLSM have already been developed [21–23]. Among them, the acridine orange stain has shown promising results in the ex-vivo guided Mohs surgery of basal cell carcinoma (BCC) [24–29]. As a selective staining for nucleic acids, it highlights the nuclei of BCC cells and provides strong contrast compared to the surrounding dermis, allowing an easy detection of tumor islands without altering the tissue for further histopathological processing. Possible pitfalls that can hinder the correct identification of tissue structures in the reflectance mode (RM) are for example images of sebaceous glands that can mimic islands of basal cell carcinoma due to their roundish appearance, while an intense stromal reaction may simulate basaloid cords [24, 25, 30]. In such cases the use of fluorescent dye may be very helpful. Acridine orange but also fluorescein, toluidine blue, methylene
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blue, acetic acid and citric acid were used as staining in other preliminary studies and gave variable results [21–23]. Ex-vivo CLSM has a unique possibility compared to other invasive procedures – the specimen examined with ex-vivo CLSM can be re-examined using traditional histology including immunohistochemistry. The aim of this study was to describe the anatomy of healthy skin and its appendages as seen in exvivo CLSM in correlation to the corresponding histopathology as basic knowledge for further microscopic examinations of pathological skin lesions. Furthermore pitfalls and artefacts experienced during ex-vivo CLSM imaging have been collected and recommendations for correct handling and prevention of problems are given.
2. Material and methods From September 2014 to January 2015 we examined 50 samples of healthy skin from various donor sites using ex-vivo CLSM (Vivascope 2500®; Lucid Inc; Rochester NY). The skin probes were taken from the Dermatosurgery Section of the Department of Dermatology and Allergology, Ludwig-Maximilian University, Munich, Germany after obtaining written informed consent from each patient. The study was conducted following the principles of Helsinki. The skin probes were immediately processed after excision for the CLSM analysis. The preparation of the skin samples, the staining and scanning with the ex-vivo CLSM was performed by one specially trained dermatologist (D.H.). Two trained CLSM specialists with histological skills performed independent confocal and histological examination of the specimens and evaluated it. All samples were sectioned and scanned in the vertical mode to enable the exact correlation to traditional histology. Standard staining with hematoxylin-eosin (H&E) was used for the histological sections. The ex-vivo CLSM used here (Vivascope 2500®; Lucid Inc; Rochester NY) combines three different lasers with wavelengths of 488 nm, 658 nm and 830 nm and allows to examine the tissue in the reflectance (RM, 830 nm) and in the fluorescent mode (FM, 488 nm und 658 nm). The maximum examination depth is limited to 250 μm and the vertical resolution to 5 μm. The water-immersion objective lens has a numeric aperture of 0.9 and allows magnification ranging between 30× and 400×. The sample size of 0.63 mm × 0.63 mm may be examined at once. The microscope reconstructs a mosaic of 1089 single images (33 times 33) and allows continuous scanning of an area of 20.8 mm × 20.8 mm. In this study the software VivaScan® (Version 7, Mavig GmbH, Mu-
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nich, Germany) was used and the tools of VivaBlock® and VivaStack®, as well as conventional photography and snap shots served for obtaining and saving the images of the examined tissue. Fresh tissue, transported in physiological saline solution, was immediately sectioned, stained, examined in the CLSM and then fixed in formalin for the
histological examination. The fluorescein staining methods included acridine orange (0.6 mM; SigmaAldrich®) and fluorescein (0.4%; ALCON®) examined with the wavelength of 488 nm and 95% Nile blue A (Sigma-Aldrich®; 2 mg/ml; wavelength of 658 nm). During the staining process the 10% citric acid (Sigma-Aldrich®) for aceto-whitening and Dul-
Table 1 List of ex-vivo CLSM features of healthy skin and their histological correlates in hematoxylin-eosin (H&E) staining. Arrow ↓ – depth under surface. Anatomical/histological structure
Ex-vivo CLSM features in RM and FM
Histological correlates (H&E staining)
Stratum corneum ↓ 0–15 μm
Multiple layers of flat, non-nucleated polygonal dark grey corneocytes (dimension: 10–30 μm) surrounded by thin, bright walls.
Multiple layers of pink, flat, dead cornified, non-nucleated keratinocytes.
Stratum granulosum ↓ 15–20 μm
Polygonal greyish cells (25–35 μm) with well demarcated outlines, grainy cytoplasm and round to oval nuclei.
Nucleated keratinocytes showing pink (eosinophil) cytoplasm with dark granules and dark blue or purple nuclei.
Stratum spinosum ↓ 20–100 μm
Large (15–25 μm), greyish polygonal cells with bright and centrally located nuclei.
Few layers of polygonal prickle cells with purple nuclei in the center of the pink cytoplasm.
Stratum basale ↓ 50–100 μm
Clusters of bright small (7–12 μm) cuboidal keratinocytes, prominent nucleus and little cytoplasm.
Cuboidal-shaped cells of the germinal layer appearing darker than stratum spinosum.
Dermo-epidermal junction ↓ 100-120 μm
Wavy to straight line or band of grey to black color lying between epidermis and dermis.
Interdigitating light pink, continuous band.
Papillary dermis ↓ 100–200 μm
Reticulated network of fine grey fibers or thicker bundles (collagen); bright particles (inflammatory cells); dark round spots or wavy to coiled tubes (vessels).
Fine textured light to dark pink collagen fibers; purple dots and spots representing inflammatory cells; dark pink blood and lymph vessels; light pink nerves; sweat and sebaceous glands.
Reticular dermis ↓ 200–400 μm
Loose but thicker bright collagen fibers; vessels and skin appendages.
Coarsely textured pink collagen fibers; vessels and skin appendages.
Subcutis
Regular net-like layers of large dark cells with very thin bright walls and bright nuclei pushed to the outer edge by lipid vacuoles that appear as empty space.
Regular net-like layers of unilocular large cells with whitish homogeneous lipid-rich vacuoles and a purple colored nucleus pushed to the outer edge.
Hair follicle
Dark hair shaft surrounded by bright root sheaths with cells arranged in columns. The matrix around the papilla is the brightest structure. Arrector pili presents as set of fine grey fibers with tiny bright dots or short lines (nuclei).
Hair shafts as columns of pink to brownish keratinized cells, gradually extruded from the follicle. Arrector pili as fine sets of eosinophilic fibers with scattered elongated purple nuclei.
Sebaceous gland
Large, round, sharply demarcated structures filled with multiple round cells with bright nuclei and grey granular cytoplasm filled with vacuoles.
Sebocytes are large light cells packed with lipids and centrally located dark nuclei.
Sweat gland
Convoluted tubes with a duct formed by two layers of small nucleated cells, lining the lumen.
The secretory portion is comprised of larger cuboidal cells, lies deep in the dermis, where the tubule is twisted into a compact coiled tangle. The duct consists of a two-layered stratified cuboidal epithelium.
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becco’s Phosphate buffered saline (Sigma-Aldrich®) were used. The proper choice of the laser wavelength is based upon the staining method of the tissue. Wavelength of 488 nm is suitable for tissue stained with acridine orange or fluorescein, wavelength of 658 nm for examining samples stained for example with Nile blue. Acridine orange and fluorescein have been tried several times and the results have been compared with each other. Since acridine orange stains exclusively DNA it provides a strong contrast between nuclei-rich and nuclei-poor structures. Fluorescein stains besides DNA also extracellular matrix and shows dermis in a bit brighter way. Acridine orange revealed the clearest results and was therefore preferentially used for further staining in our study for the comparison with the histology.
3. Results We examined 50 samples of healthy skin (male : female ratio = 28 : 22; skin type I to V; age between 20 to 92 years, median 57.5 years.) from various donor sites using ex-vivo CLSM, performed 100 full sample scans, as well as hundreds of partial scans and closeups, and compared them to histology. The skin samples originated from: head and neck (n = 25), trunk (n = 10), upper extremities (n = 10) and lower extremities (n = 5). We concentrated on the identification of the epidermal layers and on the differentiation of keratinocytes and melanocytes. Further we described the CLSM images of various skin appendages (hair follicle, sebaceous and sweat glands). The structures of the dermis and subcutis, including the fatty tissue, collagen and elastic fibers, vessels, nerves and muscles were examined. The main ex-vivo CLSM skin characteristics were compared to the standard histological examination and re-evaluated. As basis for the ex-vivo CLSM image evaluation the histopathological morphology of healthy skin will be described shortly in the following section and correlated to the standard histopathological description of skin structures [31]. The main ex-vivo CLSM microscopic characteristics with their histological correlates are presented in Table 1.
3.1 Skin overview The morphology of healthy skin is presented in Figure 1a–d. First, an overview image of the whole specimen was obtained using the Vivablock® tool (Figure 1a) in both RM and FM for the orientation within the sample and to exclude possible skin patholo-
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gies. In the RM the full width of the skin is well displayed, including the subcutis. In the FM, certain structures of the skin are highlighted depending on the staining. With acridine orange, the cell- and nuclei-rich structures are most prominent. Close-ups under magnification give the possibility to study all skin layers in detail (Figure 1b–d), including the skin appendages (Figure 2a–c). Structures of the subcutis are also visible (Figure 3a–d). The fatty tissue (Figure 3a) presents with almost black cytoplasm and fine bright cell membranes with small, but prominent and bright cell nuclei. In sebaceous glands (Figure 2b) with high numbers of overlapping cells, the bright nuclei do not allow the recognition of further morphological characteristics. The collagen fibers (Fig. 1b) are very well displayed in the FM and this allows the evaluation of the unique tangled complex of collagen fibers in the dermis together with the nuclei of the fibroblasts. In the reflectance mode, the collagen fibers are also visible, but much less bright, less clear and less sharp than in the FM.
3.2 Epidermis After evaluating the overview image all skin layers were studied in detail, starting with the epidermis (Figure 1b). The keratinocytes (Figure 1c) present in the ex-vivo CLSM RM as cuboidal and polygonal to flat (depending on the layer) cells with bright nucleus and dark grey cytoplasm. The largest oval formed nuclei are seen in stratum basale. In the upper parts of the epidermis the nuclei become smaller and thinner until they get totally lost in the basket-weave-like stratum corneum. This corresponds very well to histological correlates (Figure 1e). In contrast to the reflectance mode, where the cytoplasm of the keratinocytes appears dark grey, in the FM it presents light grey and more homogeneous. In the FM the nuclei are very bright and the most prominent structures especially in the acridine orange staining. Also, cell membranes are more clearly visualized in the FM than in the reflectance mode. Stratum corneum (Figure 1a), consisting of flat non-nucleated cells, appears in the ex-vivo CLSM highly refractive with dark spots representing the cytoplasm. Stratum granulosum shows greyish keratinocytes with grainy cytoplasm (Figure 1b). Sometimes a regular honeycomb pattern may be displayed similar to the horizontal images in the in-vivo CLSM. Stratum spinosum (Figure 1b) shows few layers of prominent polygonal cells with bright central nuclei. Stratum basale (Figure 1b), if sectioned at an oblique angle, may show a cobblestone pattern, and is formed by clusters of small bright, cuboidal cells with little cytoplasm, similar to in-vivo CLSM. Melanin has an even higher refraction index © 2015 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 1 Morphology of healthy human skin in ex-vivo CLSM (Vivablock® by VivaScan®, acridine orange, RM (830 nm) and FM (658 nm) and histological correlates (H); (a) Skin biopsy from male scalp shows multiple hair follicles and normally structured epidermis, dermis and subcutis in RM and FM. Upper layers of stratum corneum are much darker in FM in contrast to RM due to nuclei-free corneocytes. Morphology of skin appendages can be better compared to histology in RM than in FM; (b) Close-up of the epidermis, dermo-epidermal junction (DEJ) and upper dermis in RM and FM. Upper layers of stratum corneum are much darker in FM in contrast to RM due to nuclei-free corneocytes. Morphology of skin appendages can be better compared to histology in RM than in FM. Collagen fibers, fibroblasts and sparse inflammatory infiltrate in the dermis highlighted in the FM; (c) Display of keratinocytes in RM in different epidermal layers presenting bright large nuclei in stratum basale and loss of nuclei in the upper parts of the epidermis; (d) Display of melanocytes (arrows) scattered among keratinocytes in FM in the basal parts of the epidermis with melanocytes showing brighter and larger nuclei.
(1.72) than keratinocytes (1.51) and therefore melanocytes (Figure 1d), scattered among the basal keratinocytes (Figure 1c) appear much brighter than keratinocytes.
3.3 Dermo-epidermal junction and dermis The dermo-epidermal junction (Figure 1b) consists of the epidermal basal membrane and dermal papillae. In the reflectance ex-vivo CLSM it might appear as dark greyish to black line or linear space between epidermis and dermis. The dark line may be interdigitating, wavy or almost straight. In the papillary dermis (Figure 1a and b) occasional bright dots representing nuclei of sparse inflammatory cells and fibroblasts, as well as dark roundish spots corresponding to the vessels may be observed. The papillary
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dermis shows a network of reticulated light grey fibers and capillary vessels. Sometimes the ring like pattern of papillary dermis known from in-vivo CLSM is also seen in ex-vivo CLSM images in case of oblique cutting angle. The ex-vivo CLSM images of reticular dermis are dominated by collagen and elastic fibers. Collagen has a relatively high refraction index of 1.43 and therefore collagen fibers can be easily distinguished from the stroma. In the FM the dermo-epidermal junction is usually dark grey to black and can be misinterpreted as empty space between epidermis and dermis (Figure 1b). In that case, examining the tissue above and underneath this ‚black line’, either manually or via VivaStack® tool, may help to visualize the anchoring fibrils, collagen fibers and the matrix. In the FM the dermis displays by the image of bright intertwined collagen fibers mixed with brightly shining nuclei of fibroblasts.
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Figure 2 Morphology of skin appendages in ex-vivo CLSM (Vivablock® by VivaScan®, acridine orange, RM (830 nm) and FM (658 nm) and histological correlates (H); (a) Anatomy of a hair follicle in RM and FM. RM showing dark hair shafts surrounded by bright root sheaths with cells arranged in columns. In FM the matrix around the papilla is the brightest structure; (b) Anatomy of a sebaceous gland in RM and FM presents as large, round, sharply demarcated structures filled with multiple round cells showing bright nuclei and grey granular cytoplasm filled with vacuoles. Clusters of brightly shining nuclei dominate the FM image; (c) Anatomy of a sweat gland in RM and FM presenting a clump of convoluted tubes with a duct formed by two layers of small nucleated cells, lining the lumen.
3.4 Skin appendages and subcutis The anatomy of skin appendages (Figure 2a–c) can be easily viewed and studied by ex-vivo CLSM. In the RM the hair shafts appear dark, the cells of the outer and inner root sheath are bright and the hair matrix surrounding the hair papilla presents with the highest brightness. In the FM the lining and orientation of the nuclei, especially in the outer root sheath, is well displayed (Figure 2a). The sebaceous glands (Figure 2b) in the RM are large, round, sharply demarcated structures filled with multiple large round cells with small bright nuclei and grey granular cytoplasm filled with sebum vacuoles. The bright nuclei in the FM are dominating the image of the whole structure of a sebaceous gland and the cytoplasm is presented as light grey homogeneous mass. The sweat glands (Figure 2c) can be well visualized in both modes as tortuous, convoluted tubes representing the secretory portion and a duct connecting the secretory portion with the epidermis. The duct is formed by two layers of small cells with bright nuclei. The lumina of the ducts are round dark circles lined by cells of the duct walls. The subcutis and its fatty tissue (Figure 3a) present in the
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FM as a network of regular, large, bright, thin loops on a dark, almost black background. The loops represent cell membranes, whereas nuclei are shown as small bright dots pushed to the side, as if lying within the membranes. These seemingly vacant dark spaces in the loops represent lipid vacuoles. In the RM the image of the adipocytes is similar, with the difference that the cytoplasm seems to be filled with a light grey to bright granular mass and the cell nuclei are not easy to distinguish. Other structures of the subcutis including vessels (Figure 3b), nerves (Figure 3c) and muscles (Figure 3d) are clearly visible in both modes. All samples of healthy skin have been correlated to the histology using H&E staining. The main confocal features and characteristics of healthy human skin and their histological correlates are listed in Table 1.
3.5 Donor sites and skin types Comparison of samples originating from different donor sites showed the same differences as those known from the histologic examination. The identification of the donor site is based on the thickness of © 2015 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 3 Morphology of subcutis and its structures in ex-vivo CLSM (Vivablock® by VivaScan®, acridine orange, RM (830 nm) and FM (658 nm) and histological correlates); (a) Image of the fatty tissue in RM and FM. Regular net-like layers of large dark cells with very thin bright walls and bright nuclei pushed to the outer edge by lipid vacuoles which appear as empty spaces; (b) Anatomy of a vessel (arteria temporalis superficialis) in RM and FM presents as large, oval, sharply demarcated structure with thick walls and large lumen; (c) Cross-section of a nerve presenting as a round spot filled with nerve fascicles colored in RM from dark grey to almost black and in FM grey with only few small bright nuclei; (d) RM and FM images of a smooth muscle (arrector pili) show a set of fine, light grey fibers in a cross-section with tiny bright dots or short lines (nuclei) localized on the borders of the muscle fascicle.
the stratum corneum and other parts of the epidermis, on the presence and form of the rete ridges, on the thickness and composition ratio of the dermis and subcutis, as well as on the representation of skin appendages, vessels and muscles. One of the clearest examples represents the image of scalp skin (Figure 1a). The average thickness of the skin (including epidermis and dermis) originating from the trunk was 2.3 mm and from the face 1.1 mm. In the facial skin of elderly patients solar elastosis in the dermis could be identified, seen especially well in the FM. The degenerated elastotic fibers presented as accumulation of a disorganized and irregularly thickened mass with only remainders of partly recognizable band-like form. It was challenging to find the subtle differences among the skin samples from skin types I to V. Therefore we simplified the classification into light (I–III) and dark skin types (IV–V), which enabled the recognition of the more refractive and more fluorescent stratum basale in ex-vivo CLSM due to © 2015 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the larger number and size of melanosomes in keratinocytes of darker skin types (Figure 4) compared to lighter skin types.
3.6 Artefacts During the study several artefacts have occurred. These can easily mislead in the process of identifying relevant microscopic features and even of making the correct diagnosis in samples of diseased skin. In order to check the nature of the causing agent, we generated all of the described artefacts at the end of the study on spare specimen and correlated the images with those artefacts observed during the study. Table 2 lists the most common artefacts, as well as the frequency of their occurrence during our trial. In addition the causes and the prevention of artefacts are proposed. The most frequently observed artefacts in our study were caused by inappropriate thickness of the specimen and by contami-
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Figure 4 Dark skin type (IV–V) displayed in ex-vivo CLSM (Vivablock® by VivaScan®, acridine orange, RM (830 nm) and FM (658 nm) and a histological correlate); (a) RM image of epidermis and upper dermis showing highly refractive stratum basale; (b) FM image of epidermis and upper dermis showing normal and not elevated numbers of melanocytes in stratum basale; (c) Histological correlate (H) of a darker skin sample with typically pigmented basal layer of the epidermis due to increased and enlarged melanosomes in the keratinocytes.
nation of the specimen with cotton gauze fibers. During our trial we observed no changes in the microscopic features of frozen versus fresh tissue sections. This enabled postponing the scanning of the tissue until the examiner was available. Nevertheless, here we present only data from examination of freshly excised, unfrozen tissue.
4. Discussion Our results show that for the general overview of the basic confocal features of healthy skin the RM gives better morphological view of all skin structures including skin appendages and it is easier to correlate these images to traditional histology than images obtained in the FM. In case of finding a particular cell or structure, it can be helpful to switch from the RM to the FM in order to separate the structure of
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interest from the background. This particularly applies for fibroblasts in the dermis and for melanocytes in the epidermis. Further the collagen and elastic fibers are much better displayed in the FM. The dermo-epidermal junction can be easier correlated to histological images if displayed in the reflectance mode. We could demonstrate that with some degree of practice it is possible to switch from the pink and blue histological depiction to the black and white confocal world. The ex-vivo CLSM enables very similar examination of the skin as dermatohistopathology. With the use of the VivaStack® tool the ex-vivo imaging allows to portray the examined structures in a three-dimensional view. The fluorescent dyes give the opportunity to selectively bind to the structures of interest and therefore might be used in ex-vivo CLSM in the future in a similar way as immunochemistry is being used today in traditional histology. More studies on this subject are warranted. Currently the ex-vivo CLSM device that combines the reflectance and FM is being tested and results are compared to traditional histology in order to elaborate a faster, easier and more precise method of examination of freshly excised tissue. In contrast to in-vivo CLSM it is an invasive diagnostic tool which, owing to that, allows examining the tissue sample in a vertical view. Therefore the results enable more accurate correlation to histological images. In this study we used mostly acridine orange, a metachromatic stain selective for nucleic acids. It binds to both dsDNA, emitting a green fluorescence (525 nm) very similar to fluorescein, and ssDNA or RNA, emitting a red fluorescence (>630 nm). Thanks to these features it can be used in the study of cell cycle to distinguish between cells in proliferation and in quiescence. As it is able to penetrate lysosomes, it is also useful in identifying apoptotic cells. It is mainly used in research activities [30]. Previous studies with an older version of the exvivo CLSM device which offered an examination only with two instead of three different laser sources have been published. These include studies exploring digital staining, confocal-histological correlations and methods to assimilate images of ex-vivo CLSM and histology [32, 33]. Furthermore studies from medical fields other than dermatology have been concentrating on pathologies of different organ tissues and their evaluation with ex-vivo confocal microscopy [32, 34–39]. To date there have been only few published dermatological articles which evaluated ex-vivo CLSM images of basal cell carcinoma, its diagnosis and monitoring of therapy [24–28, 40– 42], as well as other types of non-melanoma skin cancer, including squamous cell carcinoma [43–45]. The limitations of our study were mainly of technical nature. The preparation of tissue can be quite time consuming when examining large tissue samples, especially if there is the need for preserving the
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Table 2 List of the most common artefacts in ex-vivo CLSM, their causes, frequency and how to prevent them. Artefact
Cause
Prevention/solution
Number (n) and frequency (%) of the artefacts among all 50 samples
Frequency (%) of the artefacts among all 22 artefacts
Dark sharply demarcated lagoons of homogeneous mass with white loops in the center (Figure 5a).
Silicone on the specimen.
Careful handling of the silicone tube during the fixation process.
n = 2 (4%)
9%
Bright, dust-like, smudgy, homogeneous masses sprinkled with snow-like flakes together with blurred microscopic features (Figure 5b).
Too much silicone on the sides of the slide.
Using only small drops of silicone on each side of the slide in order to be able to squeeze the sample between the slides evenly, gently but firmly.
n = 3 (6%)
13%
Bright, long, sharply demarcated, uniform, rod-like structures arranged next to each other (Figure 5c).
Contaminating the sample during the cutting process with the fibers of a cotton gauze piece used as a cutting mat.
Cutting and slicing the specimen on a solid and stable mat and not on a piece of gauze.
n = 5 (10%)
23%
Blurred microscopic features, unable to focus properly (Figure 5d).
Wrong unstained side of the specimen being scanned.
Careful handling of the specimen and the slides, marking the stained side, if noted flipping the slide upside down.
n = 1 (2%)
5%
Dark and blurred microscopic features.
No or too little ultrasound gel used as medium for the water-immersion objective.
Adding more ultrasound gel before cleaning the slide in case of noticing dried rests of the gel on the slide.
n = 2 (4%)
9%
Unclear and extensively blurred microscopic features visualizing only the ultrasound gel and not the specimen, unable to fixate the slide into the eligible stage opening.
Thickness of the specimen is too large.
Slicing the specimen evenly and as thin as possible, not more than 0.3 to 0.5 cm.
n = 6 (12%)
27%
Unable to visualize the specimen in the FM, even though the specimen could be viewed in the reflectance mode.
The specimen has been stored in a formalin solution/ the fluorescent stain has been depreciated by light if stored inappropriately.
Keep the specimen stored in a saline solution and do not permit contact to formalin/store the stains in a dark place.
n = 1 (2%)
5%
Low quality of scanned image and black rims around the specimen.
The specimen is dried-up.
Processing the preparations and scanning as fast as possible and switching off the laser source in case of a scanning pause.
n = 2 (4%)
9%
© 2015 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 5 Images of artefacts produced during ex-vivo CLSM tissue examinations (Vivablock® by VivaScan®, acridine orange, RM (830 nm) and FM (658 nm); (a) Black, sharply demarcated lagoons of homogeneous mass with white loops in the center represent an artefact caused by silicone drops misplaced on top of the tissue sample (RM); (b) Bright, dust-like, smudgy and homogeneous masses that are sprinkled with snow-like flakes present an artefact caused by large amount of silicone placed next to the sample (RM); (c) Display of bright, long, sharply demarcated, uniform, rod-like structures arranged next to each other, caused by pieces of gauze on the sample (FM); (d) Blurred microscopic features and inability to focus properly are artefacts produced by inverting the slide upside down and scanning the unstained side of the specimen (RM).
orientation of the sample edges, as in the case in Mohs surgery. The tissue to be sliced and cut is not fixed in formalin and therefore needs handling with special caution. The tissue slices should be even and thin, ideally not exceeding a thickness of 3 mm. Specimens thicker than 3 mm usually do not allow proper mounting of the tissue between the slides. The size of the scanning area limits tissue samples to be examined to 20.8 mm × 20.8 mm. The scanning of such an area lasts 7 minutes, which needs to be multiplied by the number of the sample sections. The scanning speed, the size of the scanned area, mounting of the specimen on the slide, fixing of the slides on the microscope stage and a wider choice of suita-
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ble stains are crucial points in further development of this method. To our knowledge this is the first study which concentrated on the ex-vivo confocal images of healthy skin and its appendages. It is also the first study which was performed with the latest version of an ex-vivo CLSM device and allowed examination with three different laser wavelengths. This allowed using more types of staining and allowed the visualization of skin structures in different ways. New staining methods and protocols are expected. The aim of this work was to determine and describe the main ex-vivo confocal features and characteristics of human skin and to correlate them with traditional his© 2015 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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tology. We also gave an overview on possible artefacts and how to prevent them. The results of this study should serve as a guide to distinguish normal from pathological findings. Good knowledge of exvivo CLSM healthy skin images may lead to a faster and more precise understanding of pathological processes.
5. Conclusion Ex-vivo CLSM is a new emerging diagnostic tool which allows fast and easy microscopic tissue examination. Correlations to classical histological images have been systematically studied and advice for correct handling could be provided. Ex-vivo CLSM is not yet able to replace the traditional histology and immunohistochemistry but might play an important role in future tissue sample diagnostics implementing and casting a new light on bedside surgery. In the clinical practice, it might be used by properly trained dermato-surgeons to optimize the surgical procedures and the definitions of surgical margins. Exvivo CLSM’s extremely quick acquisition and evaluation time (minutes) may allow surgeons to perform a confocal-guided bedside Mohs surgery. Avoiding the long tissue sample processing would strongly shorten inter-operative waiting times, number of surgical procedures and hospitalization days. In conclusion, ex-vivo CLSM has revealed itself as a promising diagnostic tool, with potential major benefits for time sparing and cost effective surgery. Conflict of interest The Vivascope® device was provided by Mavig GmbH for the time of the study from September 2014 until January 2015. Author biographies online.
Please see Supporting Information
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