Three-dimensional deep tissue multiphoton frequency-domain uorescence lifetime imaging microscopy via phase multiplexing and adaptive optics

摘要

We propose and demonstrate a novel multiphoton frequency-domain uorescence lifetime imaging microscopy(MPM-FD-FLIM) system that is able to generate 3D lifetime images in deep scattering tissues. The imagingspeed of FD-FLIM is improved using phase multiplexing, where the uorescence signal is split and mixed withthe reference signal from the laser in a multiplexing manner. The system allows for easy generation of phasorplots, which not only address multi-exponential decay problems but also clearly represent the dynamics of theuorophores being investigated. Lastly, a sensorless adaptive optics setup is used for FLIM imaging in deepscattering tissues. The capability of the system is demonstrated in xed and living animal models, includingmice and zebrash.