Confocal Scanning Laser Microscopy: Applications for Imaging Dynamic Processes in Skin In Vivo

Abstract

In vivo confocal scanning laser microscopy (CSLM) is a powerful imaging technique that enables physicians and researchers to evaluate skin dynamically and non-invasively at the cellular level. Traditionally, biopsy and histological processing are required to study the cellular details of skin. This involves tissue excision and staining, which may introduce artifacts to the sample. Staining and immunostaining have been used to identify specific proteins and organelles successfully. With CSLM, the examined areas are not damaged, removed, or processed, so imaging can be performed as often as desired. Studying dynamic changes in skin over an extended period of time or as a response to treatment is feasible. Unlike the vertical sections obtained by routine histology, en face (horizontal) images are captured with CSLM. During the last 20 years, advances in technology have resulted in images that are now near histological resolution and have enough detail for histological analysis (Pellacani et al., 2008; Scope et al., 2007; Rajadhyaksha et al., 1999). CSLM has been used in dermatology to assess skin lesions and skin conditions without biopsy. Both benign and malignant melanocytic skin tumors have been described and diagnosed, and proliferative and inflammatory skin diseases have been characterized (Gonzalez & Gilaberte-Calzada, 2008; Calzavara-Pinton et al., 2008). Margins of lesions are also identifiable from non-lesional skin. For physicians, CSLM provides real-time diagnostics and the chance to follow the evolution of skin lesions over time, which would not be feasible otherwise. Whereas the majority of CSLM skin research concentrates on dermatological conditions, this chapter focuses on capturing and studying non-diseased tissue, both healthy and wounded human tissue. We first assess the effect that the incident laser wavelength has on the images captured by comparing CSLM systems of three different wavelengths. Then, we specifically discuss two studies in which we use CSLM to study skin morphology and physiology: 1) a comparison of infant and adult skin morphology and 2) the wound healing response of micro-injuries in otherwise healthy human skin. The micro-injury study focuses on dynamic responses in skin over time. Ethically we are committed to minimize biopsy collection when studying healthy human skin. CSLM is our sole alternative to studying skin morphology and physiology at the cellular level non-