After over 30 years of advances, multiphoton microscopy (MPM) is now instrumental in a wide range of in vivo biological imaging applications. However, it has, until recently, remained not achievable or affordable to meet the unmet need for fast monitoring of biological dynamics and large-scale examination of biological heterogeneity. Only within the past few years have new strategies emerged to empower MPM at a speed that was once inconceivable, notably at kilohertz two-dimensional (2D) frame rate, and 3D rate or beyond. This Review highlights the latest high-speed innovations and discusses the potential of their synergism with other advanced, but less speed-centric MPM toolboxes. Recognizing these prospects and challenges could inspire new approaches for reprioritizing imaging speed in future MPM developments.
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This work is supported by the Research Grants Council of the Hong Kong Special Administrative Region of China (grants 17208918, 17209017 and 17259316 to J.W. and K.K.T.; RFS2021-7S06 and C7047-16G to K.K.T.) and NIH BRAIN Initiative grants (1UF1NS107696 to J.W., N.J. and K.K.T.).
The authors declare no competing interests.
Peer review information Nature Photonics thanks Keisuke Goda and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Wu, J., Ji, N. & Tsia, K.K. Speed scaling in multiphoton fluorescence microscopy. Nat. Photon. 15, 800–812 (2021). https://doi.org/10.1038/s41566-021-00881-0
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