Abstract
Removal of complex aberrations at millisecond time scales over millimeters in distance in multiphoton laser scanning microscopy limits the total spatiotemporal imaging throughput for deep tissue imaging. Using a single low resolution deformable mirror and time multiplexing (TM) adaptive optics, we demonstrate video rate aberration correction (5 ms update rate for a single wavefront mask) for a complex heterogeneous distribution of refractive index differences through a depth of up to 1.1 mm and an extended imaging FOV of up to 0.8 mm, with up to 167% recovery of fluorescence intensity 335 µm from the center of the FOV. The proposed approach, termed raster adaptive optics (RAO), integrates image-based aberration retrieval and video rate removal of arbitrarily defined regions of dominant, spatially varied wavefronts. The extended FOV was achieved by demonstrating rapid recovery of up to 50 distinct wavefront masks at 500 ms update rates that increased imaging throughput by 2.3-fold. Because RAO only requires a single deformable mirror with image-based aberration retrieval, it can be directly implemented on a standard laser scanning multiphoton microscope.
Highlights
Multiphoton microscopy provides high spatiotemporal information of living cells and associated biological processes volumetrically in a native microenvironment deep within the living host [1,2]
The overall results clearly demonstrate the effectiveness of raster adaptive optics (RAO) in imaging samples of varying distortion dependent on their axial refractive indexes with video rate correction speed (20 fps), a rate that has not been achieved with previous time multiplexing (TM) methods
Through digital segmentation and independent wavefront measurements, we demonstrate that RAO supports video rate imaging (20 fps) across the current imaging field of view (FOV) whilst removing optical and sample aberrations caused by objective lenses or thick samples with heterogenous refractive indexes
Summary
Multiphoton microscopy provides high spatiotemporal information of living cells and associated biological processes volumetrically in a native microenvironment deep within the living host [1,2]. Non-uniform and spatially varying aberrations, known as field-dependent spatial aberrations, place a fundamental limit on the amount of spatiotemporal information, which is defined by the optical space bandwidth product (SBP) [3] received by the imaging system at a given time interval [4,5,6]. Existing pupil-based adaptive optics (AO) in LSM use either time or spatial multiplexing to measure and correct spatially varying aberration masks across the imaging FOV. SM approaches achieve a large FOV at video rate but require specialized elements i.e. multi-angle prism [12] or non-pupil AO [13] arrangements, where the number of correctable field-dependent aberrations in a single acquisition is limited by either the size of a sub-element or number of actuators. TM can scale the number of aberration measurements and corrections across any FOV, albeit at the cost of increased acquisition time
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