Reversibly Switchable Fluorescent Proteins for RESOLFT Nanoscopy

Abstract

Diffraction-limited lens-based optical microscopy fails to discern fluorescent features closer than $$\sim $$ ∼ 200 nm. All super-resolution microscopy (nanoscopy) approaches that fundamentally overcome the diffraction barrier rely on fluorophores that can adopt different states, typically a fluorescent ‘on-’state and a dark, non-fluorescent ‘off-’state. In reversible saturable optical linear fluorescence transitions (RESOLFT) nanoscopy, light is applied to induce transitions between two states and to switch fluorophores on and off at defined spatial coordinates. RESOLFT nanoscopy relies on metastable reversibly switchable fluorophores. Thereby, it is particularly suited for live-cell imaging, because it requires relatively low light levels to overcome the diffraction barrier. Most implementations of RESOLFT nanoscopy utilize reversibly photoswitchable fluorescent proteins (RSFPs), which are derivatives of proteins from the green fluorescent protein (GFP) family. In recent years, analysis of the molecular mechanisms of the switching processes have paved the way to a rational design of new RSFPs with superior characteristics for super-resolution microscopy. In this chapter, we focus on the newly developed RSFPs, the light-driven switching mechanisms and the use of RSFPs for RESOLFT nanoscopy.

Application of Fluorescent Proteins for Functional Dissection of the Drosophila Visual System

“…Alternatively developed RSFPs come with high stability for long-term time-lapse image acquisition (Kohinoor2.0), far red-shifted wavelength properties (rsFusionRed3) or dual-color modes for pulse-chase setups and other more elaborate investigations (mIrisFP) [122][123][124]. A fairly extensive list of currently available RSFPs for RESOLFT microscopy has been published recently [125].…”

Multi-label in vivo STED microscopy by parallelized switching of reversibly switchable fluorescent proteins

“…RSFPs are key to RESOLFT (reversible saturable optical fluorescence transition) microscopy, a coordinate-targeted super-resolution technique that uses the cis-trans isomerization of RSFPs to switch off fluorescence and achieve super-resolution (Eggeling et al, 2015). RSFPs can be switched from a fluorescent on state to a fluorescent off state reversibly by light of different wavelength (Jensen et al, 2020). Faster than RESOLFT microscopy, we here use STED to read out a super-resolution image when the RSFP is in the on state.…”

Live‐cell RESOLFT nanoscopy of transgenic Arabidopsis thaliana

“…RESOLFT nanoscopy harnesses reversibly switchable fluorescent proteins (RSFPs), which can be repeatedly switched by light between a fluorescent on‐ and a nonfluorescent off‐state (Jensen, Jansen, Kamper, & Jakobs, 2020). RSFPs are closely related to conventional fluorescent proteins such as the green fluorescent protein (GFP).…”