Reflectance Confocal Microscopy: An Overview of Technology and Advances in Telepathology

RCM Image Acquisition and Interpretation

After patient and lesion history are obtained, visual and dermoscopic evaluation of the lesion is performed. Index fluid (mineral oil) is applied to the lesion and a metal ring with an optically clear, nonbirefringent polymer window is attached to the skin.³² A 5-megapixel dermoscopic-quality image is captured through this ring and window. A water-based immersion medium (ultrasound gel) for the objective lens is then placed on the window and the confocal microscope is magnetically attached to the ring. The index fluid has a refractive index similar to the stratum corneum and the window, allowing for optimal imaging down to the papillary dermis. The adhesive and magnetic attachments stabilize the skin lesion as the confocal microscope captures sequential 2-dimensional images. A mosaic is then created at the specified anatomic level. Levels of imaging are determined by the depth (in micrometers) from the stratum corneum and correspond to each anatomic layer. En face images also can be vertically stacked, generally in 3- to 5-μm increments.³²

The number of mosaics and stacks obtained are based on a preset standardized protocol. Once captured, images are saved and then sent over the telemedicine network to a remote confocal diagnostic reader for image interpretation, which can be done immediately or the images can be stored and sent (known as store and forward) at a later time.

Lesion evaluation begins with a review of patient and lesion history and dermoscopic images, followed by review of confocal images for additional information through visualization of cellular structures and architecture. Confocal interpretation commonly begins with examination of the mosaic at the level of the dermoepidermal junction, as it often provides the most diagnostic information. The papillary epidermal layers can then be used to confirm the working diagnosis, to further enhance the description, or to refine the diagnosis. Areas of interest may then be further examined in a vertical plane, which is achieved by observing a specific spot at different depths. Image interpretation can be approached in a variety of ways; however, the most critical aspect to any method is the recognition of skin morphology.

Confocal Images

Epidermal and dermal structures identified with in vivo confocal images are comparable to histology. The first consensus terminology glossary with illustrative images was published in 2007 with descriptions and definitions of image quality, normal skin morphology, lesional architecture, and cellular details of melanocytic lesions.³³ Figure 2 shows the normal structures that comprise the different layers of skin as seen on RCM.

^{Figure 2. Image correlation of normal skin including a histologic specimen (H&E, original magnification ×40) and corresponding reflectance confocal microscopy images with description of confocal architectural and cytologic features.}

Stratum Corneum

At a thickness of 0 to 15 μm, the stratum corneum is the first bright image seen on RCM.^1,2 The individual anucleate corneocytes often cannot be delineated; thus, sheets of cells appear as islands separated by dark furrows (wrinkles).^1,2

Stratum Granulosum

The first layer of viable cells is located 15 to 20 μm below the skin’s surface.^1,2 The large 25- to 35-μm polygonal structures (granular keratinocytes) contain bright border zones (cytoplasm), a central dark oval (nucleus), and a grainy appearance (keratohyalin granules, organelles, and melanosomes).^1-3 A honeycomb pattern is seen within the normal epidermis, whereas in darkly pigmented lesions where keratinocytes may contain some pigment, it has been described as cobblestone pattern.³

Stratum Spinosum

At a depth of 20 to 50 μm, the honeycomb pattern consists of smaller 15- to 25-μm polygonal structures (spinous keratinocytes) with thinner bright borders (cytoplasm) and a darker oval nucleus.^1-3

Stratum Basale

Below the spinous cells is a single layer of brighter round structures (basal keratinocytes), each 7 to 12 mm in diameter.^1,2 Due to the supranuclear melanin caps, the basal keratinocytes have increased reflectivity, appearing brighter than granular and spinous cells. The more abundant the melanin within the basal keratinocytes, the brighter the appearance.^1,2

Dermoepidermal Junction

Below the stratum corneum (50–150 μm) is the dermoepidermal junction.¹ The “peaks” of dermal papillae emerge as clusters of bright cells (basal keratinocytes). With deeper sectioning, the dark round-oval spaces rimmed by bright basal cells (dermal papillae) progressively enlarge. They continue to enlarge until neighboring papillae touch each other tangentially, corresponding to the valleys of rete ridges.¹

Papillary Dermis

At a depth of 60 to 80 μm, blood vessels and collagen fibers are seen.^1-3 Collagen and elastin fibers present as thin, delicately intertwined, highly reflective fibrillar structures (1–5 μm). Blood vessels appear as weakly reflective, round or canalicular structures within dermal papillae. Within the lumina, serum appears dark, but blood cells can be seen in real time as continually moving, weakly reflective or bright round structures corresponding to leukocytes, erythrocytes, and platelets. With real-time imaging, cells also can be identified based on their movement; leukocytes can fill or distend the lumen and roll slowly along vessel walls, whereas erythrocytes move rapidly within vessel lumina.^1-3