Dual-objective two-photon microscope for volumetric imaging of dense scattering biological samples by bidirectional excitation and collection

Full view observation throughout entire specimens over a prolonged period is crucial when exploring the physiological functions and system-level behaviors. Multi-photon microscopy (MPM) has been widely employed for such purposes owing to its deep penetration ability. However, the current MPM struggles with balancing the imaging depth and quality while avoiding photodamage for the exponential increasement of excitation power with the imaging depth. Here, we present a dual-objective two-photon microscope (Duo-2P), characterized by bidirectional two-photon excitation and fluorescence collection, for long-duration volumetric imaging of dense scattering samples. Duo-2P effectively doubles the imaging depth, reduces the total excitation energy by an order of magnitude for samples with a thickness five times the scattering length, and enhances the signal-to-noise ratio up to 1.4 times. Leveraging these advantages, we acquired volumetric images of a 380-μm suprachiasmatic nucleus slice for continuous 4-h recording at a rate of 1.67 s/volume, visualized the calcium activities over 4000 neurons, and uncovered their state-switching behavior. We conclude that Duo-2P provides an elegant and powerful means to overcome the fundamental depth limit while mitigating photodamages for deep tissue volumetric imaging.