Graded arc beam in light needle microscopy for axially resolved, rapid volumetric imaging without nonlinear processes.

High-speed three-dimensional (3D) imaging is essential for revealing the structure and functions of biological specimens. Confocal laser scanning microscopy has been widely employed for this purpose. However, it requires a time-consuming image-stacking procedure. As a solution, we previously developed light needle microscopy using a Bessel beam with a wavefront-engineered approach [Biomed. Opt. Express13, 1702 (2022)10.1364/BOE.449329]. However, this method applies only to multiphoton excitation microscopy because of the requirement to reduce the sidelobes of the Bessel beam. Here, we introduce a beam that produces a needle spot while eluding the intractable artifacts due to the sidelobes. This beam can be adopted even in one-photon excitation fluorescence 3D imaging. The proposed method can achieve real-time, rapid 3D observation of 200-nm particles in water at a rate of over 50 volumes per second. In addition, fine structures, such as the spines of neurons in fixed mouse brain tissue, can be visualized in 3D from a single raster scan of the needle spot. The proposed method can be applied to various modalities in biological imaging, enabling rapid 3D image acquisition.

Signal Improved ultra-Fast Light-sheet Microscope (SIFT) for large tissue imaging.

Light-sheet fluorescence microscopy (LSFM) in conjunction with tissue clearing techniques enables morphological investigation of large tissues faster and with excellent optical sectioning. Recently, cleared tissue axially swept light-sheet microscope (ctASLM) demonstrated three-dimensional isotropic resolution in millimeter-scaled tissues. But ASLM based microscopes suffer from low detection signal and slow imaging speed. Here we report a simple and efficient imaging platform that employs precise control of two fixed distant light-sheet foci to carry out ASLM. This allowed us to carry out full field of view (FOV) imaging at 40 frames per second (fps) which is a four-fold improvement compared to the current state-of-the-art. In addition, in a particular frame rate, our method doubles the signal compared to the current ASLM technique. To augment the overall imaging performance, we also developed a deep learning based tissue information classifier that enables faster determination of tissue boundary. We demonstrated the performance of our imaging platform on various cleared tissue samples and demonstrated its robustness over a wide range of clearing protocols.

Efficient visible/NIR light-driven uncaging of hydroxylated thiazole orange-based caged compounds in aqueous media.

In photoactivation strategies with bioactive molecules, one-photon visible or two-photon near-infrared light-sensitive caged compounds are desirable tools for biological applications because they offer reduced phototoxicity and deep tissue penetration. However, visible-light-sensitive photoremovable protecting groups (PPGs) reported so far have displayed high hydrophobicity and low uncaging cross sections (ÎµΦ < 50) in aqueous media, which can obstruct the control of bioactivity with high spatial and temporal precision. In this study, we developed hydroxylated thiazole orange (HTO) derivatives as visible-light-sensitive PPGs with high uncaging cross sections (ÎµΦ ≈ 370) in aqueous solution. In addition, 2PE photolysis reactions of HTO-caged glutamate were achieved using a NIR laser (940 nm). Moreover, HTO-caged glutamate can activate N-methyl-d-aspartic acid receptors in Xenopus oocytes and mammalian cells with green-light illumination, thus allowing optical control of biological functions.