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.
In order to obtain 3D information of an object, laser-scanning techniques like confocal microscopy require a scan in three dimensions. The axial scan is commonly achieved by mechanical translation of the objective or the object. However, inertia is a problem, which limits the achievable scan rates and leads to motion artifacts. The usage of adaptive optical elements bears the potential to overcome these limitations. Adaptive lenses have been applied in different kinds of microscopes to perform the axial scan without the need for any mechanical translation. In this contribution, we introduce a novel bi-actor adaptive lens that enables to manipulate both the focus position and the specimen-induced spherical aberrations that occur in deep tissue applications as well as systematic scan induced aberrations. To achieve the desired lens behavior against environmental influences and hysteresis effects an in-situ monitoring based on digital holography or partitioned aperture wavefront sensing are applied. Experiments on Zebrafish and on phantom samples prove the capabilities of our approach Beside axial scanning adaptive lateral scanning is also addressed: Lateral scans are often realized using galvo scanner. Although this approach works well, it requires a folded beam path resulting in bulky setups. We introduce piezo-actuated adaptive prisms as a suitable alternative that enables an optical setup in in-line transmissive configuration instead. With this device wavefront tilts of up to ±7° can be induced, enabling lateral scanning. We show characterization measurements and first proof of concept applications on the adaptive prisms.