Quantitative Assessment of Fluorescent Proteins for Expansion Microscopy

Super-resolution microscopy has become a vital tool in biologists' arsenal. Unlike hardware-based super-resolution microscopy techniques, Expansion microscopy (ExM) enables 3D nanoscale imaging under conventional diffraction-limited microscopes, which is achieved through physical magnification of biological samples embedded into swellable hydrogels. When combined with genetically encoded fluorescent proteins (FPs), ExM holds great potential for various biological applications. However, the sample preparation process does quench the intrinsic fluorescence of FPs to some extent. While some chemically stable FPs suitable for ExM have been reported, there is no comprehensive quantitative assessment of FPs for ExM, raising the question of how one should select an appropriate FP for ExM. Furthermore, a great diversity of available ExM protocols poses another challenge to the experimental design. To address this issue, we will conduct a quantitative assessment of the fluorescence retention after each treatment step for 37 widely used FPs in cultured mammalian cells using commonly employed ExM protocols. The top-performing FPs in each spectral class will be assessed for photostability after expansion and the oligomerization state in mammalian cells. The selected FPs with various emission spectra will be further validated for multiplexed imaging of organelles in cultured cells and mice. By providing a comprehensive evaluation, our work will empower researchers to make informed decisions, reduce experimental trial-and-error, and accelerate the adoption of ExM techniques across diverse biological applications.