Abstract
Versatile, sterically accessible imaging systems capable of in vivo rapid volumetric functional and structural imaging deep in the brain continue to be a limiting factor in neuroscience research. Towards overcoming this obstacle, we present integrated one- and two-photon scanned oblique plane illumination (SOPi, /sōpī/) microscopy which uses a single front-facing microscope objective to provide light-sheet scanning based rapid volumetric imaging capability at subcellular resolution. Our planar scan-mirror based optimized light-sheet architecture allows for non-distorted scanning of volume samples, simplifying accurate reconstruction of the imaged volume. Integration of both one-photon (1P) and two-photon (2P) light-sheet microscopy in the same system allows for easy selection between rapid volumetric imaging and higher resolution imaging in scattering media. Using SOPi, we demonstrate deep, large volume imaging capability inside scattering mouse brain sections and rapid imaging speeds up to 10 volumes per second in zebrafish larvae expressing genetically encoded fluorescent proteins GFP or GCaMP6s. SOPi's flexibility and steric access makes it adaptable for numerous imaging applications and broadly compatible with orthogonal techniques for actuating or interrogating neuronal structure and activity.
Highlights
The field of biological imaging is driven by the pressing need for new techniques that offer higher resolution, faster acquisition speed, and deeper imaging capabilities
While super-resolution microscopy approaches STED [2], stochastic optical reconstruction microscopy (STORM) [3], PALM [4], and SIM [5] provide the greatest resolution, they compromise on imaging speed, limiting many functional imaging applications
While 1P scanned oblique plane illumination (SOPi) allows for rapid volumetric imaging exceeding 10 volumes per second, 2P imaging has better imaging capability for imaging light-scattering samples
Summary
The field of biological imaging is driven by the pressing need for new techniques that offer higher resolution, faster acquisition speed, and deeper imaging capabilities. A modified form of axial plane optical microscopy [24], called OS-2P-LSFM [23] has been proposed as an approach to overcome scan position dependent tilt in single front facing objective based oblique plane light-sheet microscopy. This approach utilizes a refractive glass window as a scanning element to help maintain constant light-sheet sweep angle, but it is limited to low-tilt angle (nearly axial) light-sheet architecture. We use our system for volumetric imaging in mouse brain sections and for in vivo structural and functional imaging of behaving zebrafish larvae
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