3D-scanning microscopy with adaptive lenses and prisms for zebrafish studies

Point-scanning-based microscopy systems require combination of axial and lateral scanning to obtain three-dimensional (3D) data. Axial scanning was commonly achieved by mechanical displacement of the objective or the sample. To improve, various adaptive lens-based solutions have been reported to circumvent the need for mechanically moving parts. The lateral scanning is predominantly implemented using galvanometric mirrors. Although the performance of such devices is flawless, they require bulky, folded beam-paths that make their incorporation in compact hand-held devices challenging. Recently, we introduced an adaptive prism as a transmissive device that enables lateral scanning. We demonstrate the first all-adaptive 3D scanning in laser scanning microscopes employing a compact in-line transmission geometry without mechanically moving parts and beam folding, combining an adaptive lens and a novel adaptive prism. Characterization of the all-adaptive microscope performance shows a lateral tuning range of approximately X  =  Y  =  130  μm and an axial tuning range of about Z  =  500  μm. We successfully demonstrate 3D raster scanning of the fluorescence of a thyroid of a zebrafish embryo.