Abstract
We describe a high speed 3D Acousto-Optic Lens Microscope (AOLM) for femtosecond 2-photon imaging. By optimizing the design of the 4 AO Deflectors (AODs) and by deriving new control algorithms, we have developed a compact spherical AOL with a low temporal dispersion that enables 2-photon imaging at 10-fold lower power than previously reported. We show that the AOLM can perform high speed 2D raster-scan imaging (>150 Hz) without scan rate dependent astigmatism. It can deflect and focus a laser beam in a 3D random access sequence at 30 kHz and has an extended focusing range (>137 mum; 40X 0.8NA objective). These features are likely to make the AOLM a useful tool for studying fast physiological processes distributed in 3D space.
Highlights
Two photon microscopy is increasingly being used to study biological processes [1,2,3]
Current 2-photon microscopes, that use galvanometer mirrors to steer the laser beam and build up an image, are too slow to monitor many fast spatially distributed physiological processes, which occur on the 1-100 ms time scale, since they typically take more than 100 ms to form an image [2]
We describe the design of a high performance spherical Acousto-Optic Lens (AOL) that can be used for high speed 3D femtosecond based 2-photon imaging and random access point measurements
Summary
Two photon microscopy is increasingly being used to study biological processes [1,2,3]. Applications include imaging morphological structures, monitoring dynamic physiological processes with fluorescent reporters, triggering localized release of biologically active compounds with photolysis and controlling neuronal activity with genetically encoded light activated proteins. Current 2-photon microscopes, that use galvanometer mirrors to steer the laser beam and build up an image, are too slow to monitor many fast spatially distributed physiological processes, which occur on the 1-100 ms time scale, since they typically take more than 100 ms to form an image [2]. Most microscopes developed to date are optimized for imaging a single X-Y plane. These constraints are limiting for studying brain function, since information is encoded and transmitted as brief electrical impulses (~1 ms) in groups of neurons distributed in 3D space
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