Long-working-distance high-collection-efficiency three-photon microscopy for long-term imaging of zebrafish and organoids.

Zebrafish and organoids, crucial for complex biological studies, necessitate an imaging system with deep tissue penetration, sample protection from environmental interference, and ample operational space. Traditional three-photon microscopy is constrained by short-working-distance objectives and falls short. Our long-working-distance high-collection-efficiency three-photon microscopy (LH-3PM) addresses these challenges, achieving a 58% fluorescence collection efficiency at a 20 mm working distance. LH-3PM significantly outperforms existing three-photon systems equipped with the same long working distance objective, enhancing fluorescence collection and dramatically reducing phototoxicity and photobleaching. These improvements facilitate accurate capture of neuronal activity and an enhanced detection of activity spikes, which are vital for comprehensive, long-term imaging. LH-3PM's imaging of epileptic zebrafish not only showed sustained neuron activity over an hour but also highlighted increased neural synchronization and spike numbers, marking a notable shift in neural coding mechanisms. This breakthrough paves the way for new explorations of biological phenomena in small model organisms.