In this work, we customized a multi-photon spinning disc unit with a tunable high-power femtosecond laser to demonstrate the first three-photon spinning disk microscopy, achieving ~100Hz frame rate. We demonstrate its pioneering applications on green fluorescent beads, in vivo Drosophila brains, and fixed mouse brains. Furthermore, our findings from brain tissue imaging reveal that three-photon spinning disk imaging exhibits a significantly lower attenuation rate when compared to its two-photon spinning disk counterpart, paving the way of utilizing three-photon excitation on high-speed deep tissue imaging.
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