Masafumi Yokoyama

Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy

Abstract. Two-photon excitation laser scanning microscopy has enabled the visualization of deep regions in a biospecimen. However, refractive-index mismatches in the optical path cause spherical aberrations that degrade spatial resolution and the fluorescence signal, especially during observation at deeper regions. Recently, we developed transmissive liquid-crystal devices for correcting spherical aberration without changing the basic design of the optical path in a conventional laser scanning microscope. In this study, the device was inserted in front of the objective lens and supplied with the appropriate voltage according to the observation depth. First, we evaluated the device by observing fluorescent beads in single- and two-photon excitation laser scanning microscopes. Using a 25× water-immersion objective lens with a numerical aperture of 1.1 and a sample with a refractive index of 1.38, the device recovered the spatial resolution and the fluorescence signal degraded within a depth of ±0.6  mm. Finally, we implemented the device for observation of a mouse brain slice in a two-photon excitation laser scanning microscope. An optical clearing reagent with a refractive index of 1.42 rendered the fixed mouse brain transparent. The device improved the spatial resolution and the yellow fluorescent protein signal within a depth of 0–0.54 mm.

Transmissive liquid-crystal device correcting primary coma aberration and astigmatism in laser scanning microscopy

Laser scanning microscopy allows 3D cross-sectional imaging inside biospecimens. However, certain aberrations produced can degrade the quality of the resulting images. We previously reported a transmissive liquid-crystal device that could compensate for the predominant spherical aberrations during the observations, particularly in deep regions of the samples. The device, inserted between the objective lens and the microscope revolver, improved the image quality of fixed-mouse-brain slices that were observed using two-photon excitation laser scanning microscopy, which was originally degraded by spherical aberration. In this study, we developed a transmissive device that corrects primary coma aberration and astigmatism, motivated by the fact that these asymmetric aberrations can also often considerably deteriorate image quality, even near the sample surface. The devices performance was evaluated by observing fluorescent beads using single-photon excitation laser scanning microscopy. The fluorescence intensity in the image of the bead under a cover slip tilted in the y-direction was increased by 1.5 times after correction by the device. Furthermore, the y- and z-widths of the imaged bead were reduced to 66% and 65%, respectively. On the other hand, for the imaged bead sucked into a glass capillary in the longitudinal x-direction, correction with the device increased the fluorescence intensity by 2.2 times compared to that of the aberrated image. In addition, the x-, y-, and z-widths of the bead image were reduced to 75%, 53%, and 40%, respectively. Our device successfully corrected several asymmetric aberrations to improve the fluorescent signal and spatial resolution, and might be useful for observing various biospecimens.

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

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.

Simple adaptive optic device for confocal laser scanning microscopy using liquid crystals

We have developed adaptive optic devices for CLSMs using liquid crystals. This device corrects aberration occurs due to thickness error of cover glasses or deep observations. We will report the design of the devices and experimental results.

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.