Multiplex imaging facilities biological studies in multicellular dynamics in living organisms due to its molecular specificity, 3D subcellular resolution, and deep tissue penetration. However, one major bottleneck is detecting and resolving multiplexed signals of weak fluorescence due to a tradeoff between signal throughput and spectral resolution. Here, we demonstrate high-speed, programmable, and broadband excitation encoding to enhance sensitivity without sacrificing signal throughput in multiplex multiphoton imaging. We utilize a 22-kHz programmable digital micromirror device to modulate the spectrum of a high-power broadband laser, achieving versatile excitation encoding schemes with a 750-nm bandwidth in the NIR regime. The proposed method will benefit applications that demand high-speed and high-content performance, including hyperspectral multiphoton microscopy and computational spectroscopy.

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Li-Yu Yu, Tong Qiu, Honghao Cao, Kunzan Liu, and Sixian You, "Tunable broadband fiber source for multiplexed nonlinear microscopy," Proc. SPIE PC12853, High-Speed Biomedical Imaging and Spectroscopy IX, PC1285308 (Presented at SPIE BiOS: January 27, 2024; Published: 13 March 2024); https://doi.org/10.1117/12.3002906.750b56d8-5726-ee11-a99a-b38e0bc17c27.