Line‐field confocal optical coherence tomography—Practical applications in dermatology and comparison with established imaging methods

Non‐invasive diagnostic techniques in dermatology gained increasing popularity in the last decade. Reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) are meanwhile established in research and clinical routine. While OCT is mainly indicated for detecting non‐melanoma skin cancer, RCM has proven its usefulness additionally in distinguishing melanocytic lesions. Line‐field confocal optical coherence tomography (LC‐OCT) is an emerging tool combining the principles of both above‐mentioned methods.


| INTRODUC TI ON
Optical coherence tomography (OCT) is well established in non-invasive diagnostics, especially for epithelial skin tumours. It enables the visualization of architectural changes down to the middle dermis, but without cellular resolution. Primarily, depth section images are displayed, whereby three-dimensional overview images are possible. This allows basal cell carcinomas (BCCs) to be diagnosed and distinguished from other epithelial tumours and precancerous lesions. It is also possible to determine the tumour thickness and the subtype of basal cell carcinoma. In the case of non-surgical therapies such as photodynamic therapy or topical imiquimod, OCT is also suitable for monitoring the course of the disease. [1][2][3][4][5] Reflectance confocal microscopy (RCM) has also been established in routine diagnostics for many years. Its domain is the differential diagnosis of pigmented lesions, as it offers a high resolution with visibility of cytological details. However, its penetration depth is limited to the stratum papillare of the dermis, missing deeper parts of tumours. RCM provides horizontal images of small sections that can be combined to form a larger mosaic. 6,7 Line-field confocal optical coherence tomography (LC-OCT) allows the simultaneous display of horizontal and vertical images with a higher resolution than conventional OCT, but also with a higher detection depth compared to RCM.
A LC-OCT prototype (DAMAE, Paris) was used in this study to image healthy skin at different locations, to display a basal cell carcinoma, an actinic keratosis and a malignant melanoma exemplarily and to compare the findings with OCT and RCM. The intention was to evaluate the potential of LC-OCT in comparison to OCT and RCM regarding diagnosis of skin cancer. 8,9 2 | MATERIAL S AND ME THODS 2.1 | Imaging devices

| LC-OCT
The LC-OCT system is based on a two-beam interference microscope with line illumination and line detection using a central wavelength of spatially coherent light source and a line-scan camera. The light source is classified as a class 1 supercontinuum laser with a central wavelength of 800 nm. The real-time image acquisition is non-invasive, painless for the patient and provides no tissue damage, so that it is also allowed to be applied in pregnant women and children.
LC-OCT measures the time of flight and amplitude of light, which is backscattered from the tissue microstructures. It substantially combines the principle of OCT interferometry with the spatial filtering of RCM. The device collects multiple A-scans in parallel from the skin surface to a depth of ∼500 μm, constantly adjusting the focus. The LC-OCT device (DAMAE Medical, Paris) has three imaging modalities: vertical or en-coupe (similar to OCT and histology), en-face (comparable with RCM) and 3D stack for a three-dimensional reconstruction. Both short movies and stacks from surface to depth can be recorded. It has an axial resolution of 1.1 μm, a lateral resolution of 1.3 μm and a field of view of 1.2 mm × 0.5 mm (vertical) and 1.2 mm × 0.5 mm (en-face), respectively. Images are displayed in a grey scale. It provides a simultaneous live acquisition and imaging evaluation, thanks to its acquisition speed of 10 images/second (2D) resp. 15 seconds for a 3D stack. The device consists of a central unit connected to a handheld probe and a monitor, with Bluetooth keyboard and mouse. Immersion oil is applied between the glass window of the probe and the skin surface for index matching. It is provided with a wheeled cart, so that it can be easily moved towards the patient. Complete technical details are described elsewhere. 10-12
The penetration depth reaches the superficial dermis at 200-250 μm. Skin images are displayed in a grey scale, where melanin and keratin serve as endogenous chromophores, provide the most contrast and appear bright, in contrast to water (cytoplasm), which appears black. There are essentially two available types of the device (VivaScope ® 1500, fixed to a flexible arm, and VivaScope ® 3000, a handheld probe) and a combination of both of them. The 1500 device has a field of view of 500 × 500 μm and can build a mosaic grid of single greyscale contiguous images up to 8 × 8 mm (VivaBlock ® ); a series of vertical stacks from the surface to the depth (VivaStack ® ) can also be acquired. VivaScope 1500 offers an integrated dermoscopic camera to allow navigation in the mosaic and immediate comparison with dermoscopy. The newest generation has a built-in camera for both clinical and dermoscopic pictures.
In our study, images sized 500 × 500 µm up to 200 µm in depth were collected using the VivaStack ® function with a commercially available reflectance confocal microscope (VivaScope ® 1500 and 3000, Mavig GmbH, Munich, Germany).
Further details on the device are described elsewhere. 13

| OCT
Three-dimensional OCT images sized 6 × 6 mm down to a skin depth of about 1.5 mm were acquired using the commercially dis-

| Subjects
The study was approved by the local ethics committee (Nr17-699), and informed consent was obtained from each subject. The study was conducted according to the principles of the declaration of Helsinki and international guidelines concerning human studies.
Two women aged 33 and 49 were enrolled in the study for measurements of healthy skin in three different locations. In addition, one woman aged 51 with an actinic keratosis on the nose, one man aged 89 with a nodular BCC on his right eyebrow and one man aged 78 with a malignant melanoma on his right thigh were examined in the study.

| Measurements
Healthy skin at different locations (right cheek, right volar forearm and right middle fingertip as well as exemplary neoplastic skin diseases were investigated. The following images were collected for each site: LC-OCT (en-face, vertical and 3D), OCT (vertical and dynamic en-face), RCM (VivaBlock ® and VivaStack fully described elsewhere 14 ). An adhesive ring marked the measuring position and guaranteed that exactly the same spot was measured with each method. Skin lesions except for healthy skin were compared with their standard histology in haematoxylin/eosin. Only good quality images were included into the study. Subjects were sitting or lying comfortably while the operator performed the measurements. On the test site, the skin was pre-treated with immersion oil to reduce light reflection from the surface for LC-OCT and RCM, whereas the OCT images were collected without pre-treatment.

| Healthy skin
The correlation between LC-OCT images and haematoxylineosin sections of normal skin can be seen in Figure 1A vs. B. In

| Cheek
As common on adnex-rich skin, the surface of the (thin) SC, EPI and DEJ in vertical mode appears to be slightly undulated (white arrows) in Figure 3A in OCT and in B in LC-OCT. Sebaceous glands appear as globular bright structures connected to the epidermis, sometimes with an opening into hair follicles (yellow area in Figure 3B). Some bright, roundish and homogeneous comedones, filled with amorphous material, can also be seen (yellow arrows in 4 d). Demodex mites as skin commensals are visible inside the hair follicles as hyperreflective globules in vertical mode and as large, grouped, hyperreflective structures in en-face mode (yellow arrow in Figure 3B, white arrows in Figure 4A and E). The DEJ is slightly more flattened in elderly skin (red arrows) in Figure 3C in LC-OCT and in Figure 3D in OCT. The dermis is brighter in the younger subject compared to the older one, probably due to the more compact structure of collagen fibres ( Figure 3B vs. C). The

F I G U R E 4
Comparison of en-face LC-OCT images and RCM images of the right cheek of a younger and an elderly woman. Some bright, roundish and homogeneous comedones, filled with amorphous material, can also be seen (yellow arrows in D). Demodex mites as skin commensals are visible inside the hair follicles as hyperreflective globules in vertical mode and as large, grouped, hyperreflective structures in en-face mode (yellow arrow in Figure 3B, white arrows in A Figure 4A and E. The dermis appears more rarefied and therefore darker in elderly skin ( Figure 4K in RCM and Figure 4L in LC-OCT) compared to the younger woman ( Figure 4E in LC-OCT and Figure 4F in RCM). Signs of sun damage, more represented in elderly skin, like mottled pigmentation (red areas in Figure 4J

| Forearm
In en-face mode, SC was slightly thicker than on the face, showing a more irregular surface (Figure 2 vs. Figure 4). Numerous hair follicles were visible, as well as dilated blood vessels in the PD ( Figure 2C, E, G, I in LC-OCT images, Figure 2F, H and J in RCM images).

| Fingertip
In the vertical mode, a thick SC characterizes the fingertip ( Figure 5A, B in vertical LC-OCT images and in OCT images) and, in general, the palmo-plantar skin. The entrance signal is very bright, and the surface has a characteristic wavy pattern ( Figure 5A, B in vertical LC-OCT images and in OCT images). Hyperreflective, coiled eccrine ducts can be seen in the epidermis in continuity with the skin surface (red arrows in Figure 5A, B in vertical In en-face mode, skin folds are very prominent dark furrows separating islands of polygonal keratinocytes (green arrows in Figure 5C, E in LC-OCT images and Figure 5D, F in RCM images).
Roundish hyperreflective structures with a dark centre are regularly distributed among ridges of the skin marks and correspond to eccrine sweat ducts (orange arrows in Figure 5C, E in LC-OCT images and Figure 5D, F in RCM images). Due to skin folds, the images at increasing depth show alternating, parallel layers of polygonal keratinocytes and junctional meshwork (corresponding to the meshwork junctional pattern in RCM terminology) interrupted by bright eccrine ducts.

| Basal cell carcinoma
In vertical mode in Figure 6, the BCC is clearly visible showing sharply demarcated tumour nests with palisading of the peripheral cells and surrounded by a dark cleft. This nodular BCC shows dark, cystic structures in the centre (yellow arrows in Figure 6D).

| Malignant melanoma (superficial spreading)
In vertical mode in Figure 7D, irregular conglomerates (red area) of bright ovoid cells (red arrows) corresponding to atypical melanocytes are visible at the DEJ, as an advantage compared to OCT, which resolution does not allow a morphological distinction of melanomas or display of single cells. In Figure 7E) OCT can however provide useful information on the lesion like the tumour thickness and also about vascularization with the dynamic function.
In en-face mode, an atypical honeycombed pattern with altered keratinocytes of different size and shape can be distinguished in both LC-OCT ( Figure 7G) and RCM ( Figure 7J)   Figure 6D, E). This nodular BCC shows dark, cystic structures in the centre (yellow arrows in Figure 6D). Deeper BCC nests, which can be seen in OCT, might not be identified in LC-OCT (red arrows in Figure 6E). In en-face mode, slightly altered epidermal architecture with enlarged spaces between keratinocytes shows an atypical honeycombed pattern as in RCM (yellow area with yellow arrows in Figure 6F). In the DEJ and the upper dermis, tumour nests (green areas in Figure 6G, I, J) appear both in LC-OCT and RCM as roundish or cord-like nests with peripheral palisading, surrounded by dark clefting and bright stromal reaction (green arrows in Figure 6G, I). Canalicular blood vessels are also visible (green asterisks in Figure 6G) The provided software is very intuitive in all three devices.

| Actinic keratosis
The authors found the LC-OCT software the most intuitive and quickest (less than one minute to register a patient´s record, le-sion´s location and launch the examination), followed by OCT (around a minute). There are however some software peculiarities for each device. LC-OCT is provided with additional 3D-slicer software, which reconstructs 3D cubes of an acquired stack of en-face images. OCT has extra software for an automated analysis of skin blood flow, skin texture and roughness (beta-version).
Images can be easily exported as TIFF or JPEG with all devices. A global export and a single patient/lesion export are possible. An online learning platform and training courses are already available for OCT and RCM and planned for LC-OCT.

| CON CLUS IONS
The first lesions we measured with all three methods suggest that LC-OCT combines the advantages of RCM and OCT while reducing their disadvantages. LC-OCT seems to have optimal parameters regarding resolution and penetration depth to diagnose all types of skin cancer.
Larger systematic studies are needed to further characterize the field of use of this device and assess its sensitivity and specificity compared to histology.

ACK N OWLED G M ENTS
The authors would like to thank DAMAE Medical for making the LC- OCT system available for this study.

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no conflicts of interest.
F I G U R E 7 Superficial spreading malignant melanoma, tumour thickness 0.8 mm, on the right thigh in en-face LC-OCT images and RCM images as well as vertical LC-OCT images and OCT images. (A, B) Clinical images of the malignant melanoma on the right thigh. (C) Dermoscopic image of malignant melanoma on the right thigh (DermoGenius 2, DermoScan GmbH, Regensburg, Germany). In vertical mode in Figure D, irregular conglomerates (red area) of bright ovoid cells (red arrows) corresponding to atypical melanocytes are visible at the DEJ. In Figure E, OCT enables tumour thickness determination since the lower border is detectable. An atypical honeycombed pattern with altered keratinocytes of different size and shape (yellow asterisks) can be distinguished in both LC-OCT (in Figure 7G) and RCM (in Figure 7J). The pagetoid spreading of malignant melanocytes is also visible, showing an invasion of large, hyperreflective round cells with big, dark nuclei and dendritic elements (in Figure 7F