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.
Adaptive lenses enable compact, fast and quasi-motionless scanning in optical microscopy . One drawback, however, is that the elements of an imaging system are usually optimized for a certain design-focal-length of the adaptive lens. In particular, spherical aberrations negatively influence the axial and lateral resolution as well as the signal strength in a confocal microscope. We address this problem using a novel fluid-membrane lens that is based on a piezo-glass composite membrane, where an ultrathin glass membrane is sandwiched between two piezo rings. With their two degrees of freedom, they can bend and buckle the membrane, enabling different rotated conic-section-like surfaces. An iterative control algorithm enables the simultaneous, independent tuning of the focal length and the induced spherical aberrations. We apply our adaptive lens in a confocal microscope that is extended with an additional phase measurement system to enable a wavefront-based control of the adaptive lens. Applying the aberration correction to a confocal measurement of a phantom yields an enhancement of
the axial resolution improvement compared to the uncorrected measurement. To investigate the usability of the system for biological specimen, we show confocal measurements at zebrafish embryos with reporter gene-driven fluorescence in the thyroid gland.
 Katrin Philipp, André Smolarski, Nektarios Koukourakis, Andreas Fischer, Moritz Stürmer, Ulrike Wallrabe, and Jürgen W Czarske, Volumetric HiLo microscopy employing an electrically tunable lens, Optics Express Vol. 24, Issue 13, pp. 15029-15041 (2016)
We present a fluid-membrane lens with two piezoelectric actuators that offer versatile, circular symmetric lens surface shaping. A wavefront-measurement-based control system ensures robustness against creeping and hysteresis effects of the piezoelectric actuators. We apply the adaptive lens to correct synthetic aberrations induced by a deformable mirror. The results suggest that the lens is able to correct spherical aberrations with standard Zernike coefficients between 0 μm and 1 μm, while operating at refractive powers up to about 4m-1. We apply the adaptive lens in a custom-built confocal microscope to allow simultaneous axial scanning and spherical aberration tuning. The confocal microscope is extended by an additional phase measurement system to include the control algorithm. To verify our approach, we use the maximum intensity and the axial FWHM of the overall confocal point spread function as figures of merit. We further discuss the ability of the adaptive lens to correct specimen-induced aberrations in a confocal microscope.
Deformable mirrors are the standard adaptive optical elements for aberration correction in confocal microscopy. Their usage leads to increased contrast and resolution. However, these improvements are achieved at the cost of bulky optical setups. Since spherical aberrations are the dominating aberrations in confocal microscopy, it is not required to employ all degrees of freedom commonly offered by deformable mirrors. In this contribution, we present an alternative approach for aberration correction in confocal microscopy based on a novel adaptive lens with two degrees of freedom. These lenses enable both axial scanning and aberration correction, keeping the setup simple and compact. Using digital holography, we characterize the tuning range of the focal length and the spherical aberration correction ability of the adaptive lens. The operation at fixed trajectories in terms of focal length and spherical aberrations is demonstrated and investigated in terms of reproducibility. First results indicate that such adaptive lenses are a promising approach towards high-resolution, high-speed three-dimensional microscopy.