Two-dimensional electro-optical multiphoton microscopy.

Significance: The development of genetically encoded fluorescent indicators of neural activity with millisecond dynamics has generated demand for ever faster two-photon (2P) imaging systems, but acoustic and mechanical beam scanning technologies are approaching fundamental limits. We demonstrate that potassium tantalate niobate (KTN) electro-optical deflectors (EODs), which are not subject to the same fundamental limits, are capable of ultrafast two-dimensional (2D) 2P imaging in vivo.

Aim: To determine if KTN-EODs are suitable for 2P imaging, compatible with 2D scanning, and capable of ultrafast in vivo imaging of genetically encoded indicators with millisecond dynamics.

Approach: The performance of a commercially available KTN-EOD was characterized across a range of drive frequencies and laser parameters relevant to in vivo 2P microscopy. A second KTN-EOD was incorporated into a dual-axis scan module, and the system was validated by imaging signals in vivo from ASAP3, a genetically encoded voltage indicator.

Results: Optimal KTN-EOD deflection of laser light with a central wavelength of 960 nm was obtained up to the highest average powers and pulse intensities tested (power: 350 mW; pulse duration: 118 fs). Up to 32 resolvable spots per line at a 560 kHz line scan rate could be obtained with single-axis deflection. The complete dual-axis EO 2P microscope was capable of imaging a $13\mu m$ by $13\mu m$ field-of-view at over 10 kHz frame rate with $\sim 0.5\mu m$ lateral resolution. We demonstrate in vivo imaging of neurons expressing ASAP3 with high temporal resolution.

Conclusions: We demonstrate the suitability of KTN-EODs for ultrafast 2P cellular imaging in vivo, providing a foundation for future high-performance microscopes to incorporate emerging advances in KTN-based scanning technology.