In Vivo Two-Photon Imaging Techniques: Overcoming Technical Barriers to Longitudinal Diagnostics and Image-Guided Therapy

Two-photon microscopy overcomes the technical barriers of conventional optical imaging, such as limited tissue penetration and phototoxicity, and enables high-resolution, long-term tracking of biological processes in vivo, positioning it as a highly promising tool in precision medicine research. This review systematically surveys the engineering evolution of two-photon in vivo imaging, covering a complete technical framework from physical principles and instrumentation architecture to cross-species imaging windows. Building on this, key engineering advances are discussed, including adaptive optics, three-photon excitation, and miniaturized microscopes for freely behaving animals, along with how they extend the core performance boundaries of imaging depth, speed, and behavioral compatibility. Finally, typical applications of these continually refined in vivo imaging capabilities in neurodegenerative diseases, cerebrovascular disorders, and cancer research are introduced, encompassing longitudinal diagnostics and monitoring as well as image-guided precision therapy. This review aims to provide researchers with a structured guide that connects technical fundamentals with experimental design. With continued breakthroughs in miniaturization, multimodal integration, and artificial intelligence-assisted analysis, two-photon in vivo imaging is expected to gradually evolve from a laboratory tool to a clinically applicable platform for real-time diagnosis and treatment evaluation, thereby driving precision medicine toward in vivo, dynamic, and label-free paradigms.