Nonlinear Optical Imaging Modalities in Biomedicine

Optical imaging techniques in biomedical research and clinical applications leverage electromagnetic radiation, often in the form of visible or infrared light, to directly visualize tissues and cells at the micrometer or submicrometer scale. These imaging technologies have allowed for minimally invasive or noninvasive means of qualitative and quantitative observation of biological processes. Nevertheless, conventional optical technologies, such as single photon fluorescence, are limited by their resolution, risk of photodamage, and limited capacity for live or three-dimensional imaging. In the last few decades, nonlinear optical (NLO) imaging has facilitated explorations beyond the capabilities of conventional imaging modalities, providing researchers with a wealth of information and enhancing translational research opportunities for clinical integration. Here we review multiple NLO imaging modalities, including multiphoton fluorescence, second harmonic generation, and Raman microscopy in molecular imaging, and their innovative integration into a multimodal platform for a robust, holistic characterization of metabolism at a subcellular scale.