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

Reflectance confocal microscopy (RCM) creates an image by detecting backscattered light from illuminated tissue and displaying it on a monitor at high resolution and contrast. The grayscale images, oriented in a horizontal (en face) plane, reveal cellular and morphologic architecture in progressive depths from the epidermis to the papillary dermis.^1,2 Analyses of confocal features have shown good correlation with histologic and dermoscopic findings, allowing key features of normal skin topography as well as cutaneous lesions to be delineated.^1-15 Most research has focused on differentiating benign and malignant lesions, but RCM also has proven utility in presurgical mapping and in monitoring therapeutic efficacy of topical treatments of malignancies.^16-19 Most recently, this US Food and Drug Administration 510(k)-cleared tool for imaging in vivo unstained epithelium (including blood, collagen, and pigment) has an added adjunct: a telepathology network dedicated to the transfer of confocal images from a private practice to a remote confocal diagnostic reader for image interpretation. As a noninvasive technique, RCM is a promising tool, not only in the field of dermatology but also in primary care.

Comparison of Diagnostic Modalities

Historically, diagnostic modalities have included visual and histopathologic examination; however, basing a diagnosis solely on clinical grounds may not be reliable, and obtaining a tissue specimen may not be feasible or practical. Thus, noninvasive modalities as adjuncts for evaluation were developed, including high-frequency ultrasound, high-definition optical coherence tomography, dermoscopy, and in vivo RCM. Sonography was not reliable in clinical practice due to poor echogenicity and insufficient resolution, and although high-definition optical coherence tomography is emerging as an important tool for the evaluation of lesions with high clinical suspicion for nonmelanoma skin cancers (eg, basal cell carcinoma [BCC]), resolution is still insufficient for definitive diagnosis; therefore, these devices cannot be reliably used on pigmented lesions suspicious for melanoma.^20-23

Reflectance confocal microscopy has many properties similar to both dermoscopy and histology (Table).^1-3,24-28 Dermoscopy and RCM are used by physicians to noninvasively analyze lesions en face in real time; both modalities operate through optical magnification and liquid immersion without exogenous contrast agents and can be used to monitor lesion progression over time.^25,29 However, when comparing these modalities for melanoma identification among equivocal melanocytic lesions, they revealed statistically similar sensitivities (dermoscopy, 88%; RCM, 91%) but notably different specificities with RCM achieving more than double the specificity (dermoscopy, 32%; RCM, 68%).³⁰

Similar to histology images, RCM images provide high axial and lateral resolution, delineating cellular and morphologic architecture in both vertical and horizontal planes.^29,31 Unlike histology, RCM does not require tissue removal and processing, thus the images are immediately available for analysis. Although RCM is noninvasive similar to dermoscopy and has resolutions comparable to histology, it uniquely demonstrates the dynamic processes of living skin in real time.^1-3

Technical Properties of RCM

There are 7 components to the microscope: a laser light source, scanning elements, a relay telescope, a beam splitter, a pinhole aperture, an objective lens, and a detector (Figure 1).^1,2,32 A low-power laser beam illuminates a point on or within the skin. The scattered light reflected back into the optical system is imaged on a detector. A pinhole aperture in front of this detector filters out the scattered light and allows only the light from the image plane (a thin, in-focus plane in the tissue) to pass through, creating a high-resolution image (3–5 μm horizontal optical sections) of the target lesion. The optical parameters include an 830-nm laser with an operating power of 22 mW and an immersion objective lens with a 0.9 numerical aperture. Each image has a 500-μm field of view with approximately ×30 magnification. A larger 2-dimensional image is created when the laser rapidly scans across the plane of the skin lesion, sequentially capturing multiple images. These individual images are stitched together to create a mosaic with a field of up to 8×8 mm.^1,2,32

^{Figure 1. Components of the reflectance confocal microscope. A low-power laser beam illuminates a point on or within the skin. Light reflects back onto the optical system and is imaged on a detector. The pinhole aperture in front of this detector filters out the scattered light and allows only the light from the image plane (a thin, in-focus plane in the tissue) to pass through, creating a high-resolution image of the target lesion.}

The maximum imaging depth extends into the papillary dermis (up to 350 mm, depending on tissue thickness).^1,2,27,32 Depth of light penetration is limited by wavelength and intensity to maximize resolution of discernible structures and to avoid tissue damage. Contrast is dependent on light scattering, which is generated in 2 ways: (1) by differences between the refractive index of water and tissue constituents, and (2) by diffraction of light by structures similar in size to the illuminating light wavelength. Thus, highly reflective or diffractive structures such as melanin, keratin, hydrated collagen, and melanosomes produce backscattered light that appears bright (white) compared to their surroundings.^1,2,27,32