Transmissive liquid crystal device correcting the spherical aberrations in laser scanning microscopy

Abstract

Two-photon excitation laser scanning microscopy has enabled us to visualize deep regions in a biospecimen. However, refractive-index mismatches in the optical path cause spherical aberrations, which degrade the spatial resolution and the fluorescent signal during observation, especially at deeper regions. Recently, we developed transmissive liquid crystal devices for correcting a certain spherical aberration without changing the basic design of the optical path in a conventional laser scanning microscope. The devices were inserted in front of the objective lens and supplied with appropriate voltages according to the observation depth. In our previous study, while the devices actually recovered the axial resolution and the fluorescent signal, which were degraded by artificially induced aberrations, those performances were not sufficient for practical use. In this paper, in order to improve the imaging performance of the devices and the objective lens, we first performed more precise numerical calculations. Next, we modified the design of the devices and evaluated these performances by observing fluorescent beads in a single-photon excitation laser scanning microscope. For a 25x water-immersion objective lens with a numerical aperture of 1.1 and a sample with a refractive index of 1.38, these modifications recovered the spatial resolution, and the fluorescent signal degraded within ±0.6 mm depth. Finally, we introduced these modified devices to a conventional two-photon excitation laser scanning microscope and succeeded in improving the spatial resolution; additionally, the fluorescent signal degraded in the same region. Therefore, our devices are expected to be useful for observing much deeper regions within a biospecimen.