Light Sheet Fluorescence Microscopy (LSFM) is an ideal tool for imaging model organisms that are hundreds of microns in size, providing high resolution and fast frame rates. In order to extend LSFM to rapid 3D volumetric imaging, a popular method is to add an electrically tunable lens (ETL) in the detection path of the LSFM. But for larger fields of view and higher NA objectives, the ETL introduces aberrations in the system. Here, we developed an LSFM with adaptive optics and an ETL for rapid focusing. We demonstrate that the system enables imaging over a volume of 499 × 499 × 140 μm3 with a volumetric speed of 4 Hz. We apply the system to image neural activity in the zebrafish larvae to capture rare seizure events.
Light sheet fluorescence microscopy is an excellent tool for imaging larger and thicker model organisms such as zebrafish larvae. Samples can be imaged with high spatial and temporal resolution over large fields of view without photodamage. But light sheet microscopy still suffers from optical aberrations due to the thickness of the samples and the high resolution. We have previously demonstrated imaging of the zebrafish central nervous system and correction of aberrations using sensorless Adaptive Optics. Sensorless AO is slow because many images are required to achieve a correction. In this work, we demonstrate measuring the wavefront using a Shack-Hartmann wavefront sensor and a confocal spot as the guide star. This approach does not require special sample preparation or an additional laser. Here we demonstrate our approach by imaging fluorescent beads and inducing wavefront errors with the deformable mirror.
Light Sheet Microscopy has many advantages for imaging living model organisms. Its optical sectioning capability and high volumetric imaging speed over a large field of view make it especially favorable for recording highly dynamic biological events, such as neural signaling. The combination of an electrical tunable lens (ETL) and a scanning light sheet allows us to record image stacks at high speed without moving the sample or the detection objective. The performance of the light sheet microscope is affected by aberrations from the sample mounting and the sample itself as well as aberrations introduced by The ETL which limit the usable field of view and focusing range of the system. Here, we present the development of a light sheet microscope optimized for volumetric imaging of zebrafish larvae with adaptive optics correction for extended focusing range and increased image quality at a speed of 0.6Hz over 400 × 400 × 100μm3 using an electrical tunable lens.
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