Chapter 32: Reflectance Confocal Microscopy: the Era of Noninvasive Imaging

BASIC PRINCIPLES OF REFLECTANCE CONFOCAL MICROSCOPY

History

• The confocal microscope was invented by Marvin Minsky in 1957

• The reflectance confocal microscope (RCM) was later modified and commercialized with the use of a laser light source and spinning polygon mirror

Utility

• In vivo RCM produces real-time optical sections, parallel to the skin surface, that are less than 5 μm in thickness, with a lateral resolution of 0.5 to 1.0 μm

• The cellular resolution allows for in vivo visualization of histological manifestations in the epidermis and superficial dermis such as architecture, cell size, shape, and occasionally organelles

Optical Principles

• The RCM consists of: a point source of light; condenser and objective lenses, scanning optomechanical components (polygon and galvanometric mirrors) and a point detector

• Creating an image

  • A 30x water immersion objective lens microscope is coupled with water to the skin such that the refractive index remains consistent (water, n = 1.33; epidermis, n = 1.34) and that aberrations are minimized

  • The device utilizes 830-nm coherent laser light (5–10 mW) to focus on diffraction-limited tissue of 0.5 μm in diameter

  • When an image is initiated, the laser is deflected off of a polygon scan mirror and the focused light is scanned in the tissue

  • The light then travels sequentially back through the optics to a conjugate focal plane within the microscope that contains a pinhole detector

  • The image by the objective lens is sequentially translated into 2-dimensional images of 500 μm, which are further composited into mosaics of 8 × 8 images, visualizing an entirety of a 4 × 4 mm area of tissue

  • Total imaging depth of RCM in normal skin is 200 to 300 μm, that is, the level of papillary dermis and upper reticular dermis

• Contrast and reflectance

  • Contrast is endogenous

  • The mechanism of reflectance involves detection of backscattering of light

    • – Structures that are bright (highly reflective) contain: (1) components with high refractive index (n) compared with surroundings, or (2) are similar in size to the wavelength of light

    • – Highly reflective components of the skin include

      • Melanin (n = 1.72)

      • Hydrated collagen (n = 1.43)

      • Keratin (n = 1.51)

  • Cells containing increased amounts of granules such as cells in a hypermetabolic state (i.e., neoplastic cells, inflammatory cells) will also present with high reflectivity when compared to surrounding nonpigmented cells and structures

Keratinocytic Tumors

• Seborrheic keratosis

  • Very common, benign epidermal tumors typically occurring after the third decade of life

  • These neoplasms are usually found on the trunk and extremities; anatomic regions subjected to greater amounts of friction over time tend to develop more seborrheic keratoses

  • Clinical features

    • – Morphology ranges from skin-colored flat-topped papules to dark brown plaques

    • – The neoplasms tend to have a "stuck-on" appearance and are characteristically verrucoid, with our ...