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.
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.
pH sensitive fluorescent proteins enabling chemical reactivation in resin are useful tools for fluorescence microimaging.
EYFP or EGFP improved from GFP in jellyfish are good for such applications. For simultaneous two-color imaging, a
suitable red fluorescent protein is of urgent need. Here a pH sensitive red fluorescent protein, pHuji, is selected and
verified to be compatible with hydrophobic resin embedding and thus may be promising for dual-colour chemical
reactivation imaging in conjunction with EGFP or EYFP.
We developed a novel reactive embedding resin that crosslinking with the biological tissue via the reaction of epoxy
group and amino group, which improves its compatibility with biological tissue and can be good to preserve
endogenous fluorescent protein and dyes.