Abstract
Fluorescent proteins serve as important biomarkers for visualizing both subcellular organelles in living cells and structural and functional details in large-volume tissues or organs. However, current techniques for plastic embedding are limited in their ability to preserve fluorescence while remaining suitable for micro-optical sectioning tomography of large-volume samples. In this study, we quantitatively evaluated the fluorescence preservation and penetration time of several commonly used resins in a Thy1-eYFP-H transgenic whole mouse brain, including glycol methacrylate (GMA), LR White, hydroxypropyl methacrylate (HPMA) and Unicryl. We found that HMPA embedding doubled the eYFP fluorescence intensity but required long durations of incubation for whole brain penetration. GMA, Unicryl and LR White each penetrated the brain rapidly but also led to variable quenching of eYFP fluorescence. Among the fast-penetrating resins, GMA preserved fluorescence better than LR White and Unicryl. We found that we could optimize the GMA formulation by reducing the polymerization temperature, removing 4-methoxyphenol and adjusting the pH of the resin solution to be alkaline. By optimizing the GMA formulation, we increased percentage of eYFP fluorescence preservation in GMA-embedded brains nearly two-fold. These results suggest that modified GMA is suitable for embedding large-volume tissues such as whole mouse brain and provide a novel approach for visualizing brain-wide networks.
Highlights
In recent years, plastic embedding has played an increasingly important role in three-dimensional, high-resolution imaging technologies [1,2,3,4]
We quantitatively examined the fluorescence preservation of four hydrophilic resins: glycol methacrylate (GMA), LR White, hydroxypropyl methacrylate (HPMA) and Unicryl
A Thy1eYFP-H transgenic mouse brain was fixed with 4% paraformaldehyde
Summary
Plastic embedding has played an increasingly important role in three-dimensional, high-resolution imaging technologies [1,2,3,4]. To image large-volume samples with submicron or nanometer resolution, several well-established optical microscopy and electron microscopy imaging techniques have been combined with serial thin-sectioning to improve imaging depth or axial resolution [5,6,7,8,9,10,11]. In these studies, the biological samples are typically embedded in resin to meet the hardness required for thin sectioning. C.elegans mouse brain section culture cells Zebrafish head C.elegans culture cells & yeast Zebrafish embryos Zebrafish embryos doi:10.1371/journal.pone.0060877.t001
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