Transmissive liquid-crystal device for correcting primary coma aberration and astigmatism in biospecimen in two-photon excitation laser scanning microscopy.

Abstract

All aberrations produced inside a biospecimen can degrade the quality of a three-dimensional image in two-photon excitation laser scanning microscopy. Previously, we developed a transmissive liquid-crystal device to correct spherical aberrations that improved the image quality of a fixed-mouse-brain slice treated with an optical clearing reagent. In this study, we developed a transmissive device that corrects primary coma aberration and astigmatism. The motivation for this study is that asymmetric aberration can be induced by the shape of a biospecimen and/or by a complicated refractive-index distribution in a sample; this can considerably degrade optical performance even near the sample surface. The device’s performance was evaluated by observing fluorescence beads. The device was inserted between the objective lens and microscope revolver and succeeded in improving the spatial resolution and fluorescence signal of a bead image that was originally degraded by asymmetric aberration. Finally, we implemented the device for observing a fixed whole mouse brain with a sloping surface shape and complicated internal refractive-index distribution. The correction with the device improved the spatial resolution and increased the fluorescence signal by ?2.4×. The device can provide a simple approach to acquiring higher-quality images of biospecimens.