Label-free super-multiplex multiphoton imaging microscopy (Conference Presentation)

Multiphoton fluorescence microscopy is a powerful tool for revealing complex biological systems with high contrast, minimal out-of-focus bleaching, and deep penetration. However, clinical application faces challenges like the need for stable, tunable laser sources, comprehensive biological information, phototoxicity, and developing universal quality control tools. We developed a new generation label-free super-multiplex multiphoton imaging microscopy, enabling co-registered label-free 4-photon (tryptophan), 3-photon (NAD(P)H), 2-photon (FAD, porphyrin/lipofuscin), second harmonic (collagen), third harmonic (optical heterogeneity), and fluorescence lifetime imaging with single-shot, single-band excitation near 1110 nm. Our laser source features tunable wavelength (950-1150 nm), pulse repetition rate (1-10 MHz), and pulse width (40-400 fs), with stability over 2000 hours. We revealed phototoxicity mechanisms in nonlinear imaging, identified safe imaging parameters, and developed a universal tool for objective imaging performance comparison. This system maximizes fluorescence microscopy information while minimizing harm, paving the way for clinical translation.