Confocal Microscopy of Skin Cancer: Ch. 5 in Advances in Optical Imaging

Although Marvin Minsky invented the confocal microscope in 1957, the instrumentation and applications for the in vivo evaluation, diagnosis, and management of cutaneous tumors has only evolved during the past decade. Reflectance confocal microscopy (RCM) consists of a point source of light, produced by a laser beam, which passes through an objective lens and illuminates a probe volume in skin. The light backscattered from the probe volume passes through an optically conjugate point aperture, consisting of a pinhole, and is then captured by a detector to produce a pixel. Scanning the probe volume in two dimensions permits the operator to capture a corresponding two-dimensional array of pixels, which produces an image of a thin optical section within thick tissue. Imaging of thin optical sections is noninvasive, in contrast to conventional histology, which requires physical sectioning of tissue.

RCM enables real-time visualization of nuclear and cellular morphology in vivo. The ability to observe nuclear and cellular details clearly sets this imaging modality apart from other noninvasive imaging instruments, such as magnetic resonance imaging [1], optical coherence tomography [2], and high-frequency ultrasound [3]. The lateral resolution of RCM is typically 0.2 to 1.0 μm, and the thickness of optical sectioning is 1 to 3 μm [4,5], which is analogous to viewing histology at high magnification power and high resolution. As a result, RCM is being developed as a portable bedside tool for diagnosis of melanoma [6] and nonmelanoma [7] skin cancers.

At present, there is a commercially available reflectance confocal microscope (VivaScope 1500, Lucid Inc., Rochester, New York), consisting of a scanning unit that is mounted on an articulating arm for positioning on the patient. A metal ring fixture with an adhesive window is applied to the skin. The microscope is then coupled to the metal ring, thereby creating a stable contact between the skin and the objective lens. A gel with a refractive index close to that of water (i.e., 1.33) is placed between the objective lens and the tissue. With a 30× lens, the field of view in the tissue is 0.5 × 0.5 mm. However, larger fields of view are achievable by “stitching” together, via computer software, sequentially acquired adjacent images to create and display a mosaic. The current version of this software allows up to 16 × 16 images to be “stitched” together, thereby creating and displaying up to an 8 × 8 mm field of view. This is analogous to a low magnifying power of 2×. Imaging more than 16 × 16 images is currently impractical, due to the long acquisition times required for imaging such a large field. Use of a near-infrared wavelength of 830 nm permits imaging to depths of 100 to 200 μm in normal skin. This allows for visualization of the epidermis and superficial dermis. Although 830 nm is the wavelength of choice for most clinical applications, longer wavelengths such as 1064 nm, as well as shorter visible wavelengths of 488 to 700 nm, have been used. While shorter wavelengths provide higher resolution and thinner optical sectioning, longer near-infrared wavelengths allow deeper imaging, due to reduced scattering.

Figure 5.1 (A) RCM image of skin and (B) corresponding histology. Melanocytes appear in the black-and-white RCM images as bright cells against a dark background. The roundish dark structures within the outline of some of the cells correspond to the nucleus. In contrast, melanocytes in the corresponding H& E-stained histopathology section show purple nuclei and pink cytoplasm on a light pink background. The melanocytes shown are those of a melanoma; they are abnormal because they display variability in the size, shape, and refractivity of cells on RCM and variability in the size and shape of nuclei on H& E histopathology. The scale pertains to both images.

Although RCM is able to image at a resolution comparable to histology, there are some important differences between RCM and conventional histopathology. First, RCM images appear in black and white as opposed to the purple and pink colors seen in hematoxylin and eosin (H&E)–stained histology. Second, there is a contrast inversion with RCM. In routine histological staining with H&E, structures that absorb the stain appear dark in contrast to the nonstained bright background (Figure 5.1). In other words, H& E histology represents brightfield imaging. However, in RCM, the background tissue appears dark, while cells with higher reflectance appear brighter (Figure 5.1). This is equivalent to dark-field imaging. Third, although the nominal resolution and optical sectioning of RCM is similar to that of histopathology, the RCM image quality degrades with increasing depth in the tissue. At deeper dermis levels, strong scattering and aberrations at the dermal–epidermal junction (DEJ) and superficial dermis cause loss of optical sectioning, loss of resolution, and loss of contrast. Since structure-specific stains are not being applied in vivo to living human skin, we must rely solely on endogenous reflectance contrast for imaging. Unfortunately, image quality does degrade with loss of endogenous contrast. By comparison, observation of physically prepared thin histopathologic sections is superior to RCM images obtained in vivo because there is no overlying tissue to degrade the image and because of the benefit of stains to enhance contrasts. This fact, in turn, explains the reason for why ex vivo RCM imaging of tissue can produce an image quality that closely approaches that of histopathology.

Melanin provides the best source of endogenous contrast by strongly backscattering light. Cells that contain melanin, including melanocytes (Figure 5.1), keratinocytes, and dermal melanophages, appear bright on RCM. In general, the greater the melanin content and concentration within cells, the brighter the RCM images will appear. Some organelles and cytoplasmic granules, such as Iftimia c05.tex V1 - 07/31/2010 2:27pm Page 166 166 CONFOCAL MICROSCOPY OF SKIN CANCER keratohyaline granules in keratinocytes and Birbeck granules in Langerhans cells, also provide contrast, thus allowing them to be visualized as well [5].