Speed scaling in multiphoton fluorescence microscopy

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. This Review summarizes the latest state-of-the-art technologies for high-speed multiphoton (fluorescence) microscopy, especially at kilohertz 2D frame rate, and 3D video rate or beyond—a speed regime that was generally inconceivable until very recently, as well as the prospects and challenges of these emerging technologies.