Development of a green reversibly photoswitchable variant of Eos fluorescent protein with fixation resistance

Abstract

Super-resolution microscopy determines the localization of fluorescent proteins with high precision, beyond the diffraction limit of light. Super-resolution microscopic techniques include photoactivated localization microscopy (PALM), which can localize a single protein by the stochastic activation of its fluorescence. In the determination of single-molecule localization by PALM, the number of molecules that can be analyzed per image is limited. Thus, many images are required to reconstruct the localization of numerous molecules in the cell. However, most fluorescent proteins lose their fluorescence upon fixation. Here, we combined the amino acid substitutions of two Eos protein derivatives, Skylan-S and mEos4b, which are a green reversibly photoswitchable fluorescent protein (RSFP) and a fixation-resistant green-to-red photo-convertible fluorescent protein, respectively, resulting in the fixation-resistant Skylan-S (frSkylan-S), a green RSFP. The frSkylan-S protein is inactivated by excitation light and re-activated by irradiation with violet light, and retained more fluorescence after aldehyde fixation than Skylan-S. The qualities of the frSkylan-S fusion proteins were sufficiently high in PALM observations, as examined using α-tubulin and clathrin light chain. Furthermore, frSkylan-S can be combined with antibody staining for multicolor imaging. Therefore, frSkylan-S is a green fluorescent protein suitable for PALM imaging under aldehyde-fixation conditions.

In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants

“…The last decade brought forward advances in sample preparation for CLEM, which demonstrated the feasibility of ultrastructural analysis by EM while maintaining the fluorescence of endogenous proteins with the possibility of super-resolution imaging 1 – 3 or protocols that enable post-embedding on-section fluorescence labelling 4 . There has been considerable progress in endogenous labelling strategies, with different groups successfully developing SMLM-compatible, fixation-resistant fluorescent proteins, such as frSkylan_S 5 or mEos4b 6 . While the introduction of these constructs for stable expression in plants is time-consuming, there is an alternative for investigating structures in plant cells using exogenous labelling with low-molecular-weight compounds.…”