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.
Highlights
Confocal laser scanning microscopy (LSM) has enabled impressive three-dimensional fluorescence imaging
This device, which is inserted between the objective lens and revolver of a microscope for correcting spherical aberrations, improved the quality of fluorescence images of deep regions in a fixed-mouse-brain slice treated by an optical clearing reagent in 2P-LSM
We developed a transmissive liquid-crystal device that can compensate for four low-order asymmetric aberrations: astigmatism in the 0-deg and 45-deg directions, and primary coma aberration in the 0-deg and 90-deg directions [Astig(0), Astig(45), Coma(0), and Coma(90), respectively]
Summary
Confocal laser scanning microscopy (LSM) has enabled impressive three-dimensional fluorescence imaging. We previously reported a transmissive liquid-crystal device that can compensate for spherical aberrations.[13,14] Spherical aberrations are predominant aberrations produced during observations of deep regions in samples and are usually caused by refractive-index differences between a sample and immersion medium.[15] This device, which is inserted between the objective lens and revolver of a microscope for correcting spherical aberrations, improved the quality of fluorescence images of deep regions in a fixed-mouse-brain slice treated by an optical clearing reagent in 2P-LSM. Asymmetric aberrations can be induced by the shape of a biospecimen and/or a complicated refractive-index distribution in the sample; they can degrade optical performance considerably, even near the sample surface.[16] Aberrations in biospecimens and their effects have been experimentally investigated by several authors.[17,18,19] Further, wavefront aberrations can be described using Zernike polynomials.[20] Low-order. We experimentally demonstrated that the device enhanced the image quality of the biospecimen with a sloping surface shape and a complicated internal refractive-index distribution
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
