Simultaneous two-plane, two-photon imaging based on spatial multiplexing.

The two-photon microscope is a powerful tool in life science. Conventional two-photon microscopy can image only a plane of a particular axial position at a time. Axial scanning can get the volumetric information, but it gets signals from different axial positions serially, which means that the exposure time at every plane is limited. Here we demonstrate a novel method, to the best of our knowledge, that can simultaneously record images from two planes at different xyz positions. The demultiplexing of the signal is realized using a confocal strategy. The experimental results show that it can be used for simultaneous two-photon imaging at two focal planes with little cross talk.

High-refractive index of acrylate embedding resin clarifies mouse brain tissue.

Biological tissue transparency combined with light-sheet fluorescence microscopy is a useful method for studying the neural structure of biological tissues. The development of light-sheet fluorescence microscopy also promotes progress in biological tissue clearing methods. The current clarifying methods mostly use liquid reagent to denature protein or remove lipids first, to eliminate or reduce the scattering index or refractive index of the biological tissue. However, denaturing protein and removing lipids require complex procedures or an extended time period. Therefore, here we have developed acrylate resin with a high refractive index, which causes clearing of biological tissue directly after polymerization. This method can improve endogenous fluorescence retention by adjusting the pH value of the resin monomer.

Development of a neutral embedding resin for optical imaging of fluorescently labeled biological tissue.

Plastic embedding is widely applied in light microscopy analyses. Previous studies have shown that embedding agents and related techniques can greatly affect the quality of biological tissue embedding and fluorescent imaging. Specifically, it is difficult to preserve endogenous fluorescence using currently available acidic commercial embedding resins and related embedding techniques directly. Here, we developed a neutral embedding resin that improved the green fluorescent protein (GFP), yellow fluorescent protein (YFP), and DsRed fluorescent intensity without adjusting the pH value of monomers or reactivating fluorescence in lye. The embedding resin had a high degree of polymerization, and its fluorescence preservation ratios for GFP, YFP, and DsRed were 126.5%, 155.8%, and 218.4%, respectively.