SignificanceThe 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.AimTo 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.ApproachThe 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.ResultsOptimal 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 μm by 13 μm field-of-view at over 10 kHz frame rate with ∼0.5 μm lateral resolution. We demonstrate in vivo imaging of neurons expressing ASAP3 with high temporal resolution.ConclusionsWe 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.
To visualize neuronal structure and function in the physiological context, optical microscopy that is non-invasive and capable of resolving sub-cellular structures has become the method of choice. Structured illumination microscopy (SIM) is a widefield fluorescence imaging technique that optically sections 3D samples, but its applications have been usually limited to in vitro samples. To apply SIM to in vivo imaging, we modified optical-sectioning SIM reconstruction algorithm and incorporated adaptive optics. We demonstrated fast, high-resolution in vivo imaging with optical sectioning for structural and functional interrogations of the brain in vivo.
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