Multiphoton Microscopy for Noninvasive and Label-Free Imaging of Human Skin and Oral Mucosa Equivalents

Abstract

Multiphoton microscopy has emerged as a powerful modality for noninvasive, spatial, and temporal imaging of biological tissues without the use of labels and/or dyes. It provides complimentary imaging modalities, which include two-photon excited fluorescence (2PEF) and second harmonic generation (SHG). 2PEF from endogenous chromophores such as nicotinamide adenine dinucleotides (NADH), flavins and keratin enable visualization of cellular and subcellular structures. SHG provides visualization of asymmetric macromolecular structures such as collagen. These modalities enable the visualization of biochemical and biological alterations within live tissues in their native state. Organotypic cultures of the skin and oral mucosa equivalents have been increasingly used across basic and translational research. However, assessment of the skin and oral mucosa equivalents is predominantly based on histological techniques which are not suited for real-time imaging and longitudinal studies of the tissues in their native state. 2PEF from endogenous chromophores and SHG from collagen can be effectively used as an imaging tool for noninvasive and label-free acquisition of cellular and matrix structures of live skin and oral mucosa cultures. In this chapter, the methods for noninvasive and label-free imaging of monolayer and organotypic cultures of the skin and oral mucosa using multiphoton microscopy are described.