Third Harmonic Generation Microscopy for Label-Free Imaging of Cells, Tissues, and the Tumor Microenvironment

Third harmonic generation (THG) microscopy is a label-free nonlinear imaging technique that visualizes refractive index boundaries within biological specimens, generating contrast at plasma membranes, lipid interfaces, vesicles, and extracellular matrix structures. Because THG does not require fluorescent labeling and induces minimal phototoxicity, it enables high-resolution imaging of living cells, tissues, and tumors in their native state. THG is particularly powerful in cancer research, where it reveals tumor cell morphology, vesicle-rich leading edges, stromal organization, adipocyte remodeling, vascular interfaces, and immune cell dynamics in vivo. This chapter provides practical guidance for implementing THG microscopy, including optical requirements, filter configurations for combined THG/SHG/fluorescence detection, sample preparation, and acquisition strategies. Detailed protocols are provided for (i) forward-detected THG imaging of tumor cells embedded in three-dimensional collagen matrices and (ii) backward-detected THG imaging of ex vivo skin and tumors growing in the dermis in vivo. Together, these workflows enable comprehensive, label-free visualization of cellular and microenvironmental features relevant to cancer invasion and tissue architecture.