A photoswitchable fluorescent protein for hours-time-lapse and sub-second-resolved super-resolution imaging

Abstract

Reversibly photoswitchable fluorescent proteins (RSFPs) are a class of fluorescent proteins whose fluorescence can be turned on and off by light irradiation. RSFPs have become essential tools for super-resolution (SR) imaging. Because most SR imaging techniques require high-power-density illumination, mitigating phototoxicity in cells due to intense light irradiation has been a challenge. Although we previously developed an RSFP named Kohinoor to achieve SR imaging with low phototoxicity, the photoproperties were insufficient to move a step further to explore the cellular dynamics by SR imaging. Here, we show an improved version of RSFP, Kohinoor2.0, which is suitable for SR imaging of cellular processes. Kohinoor2.0 shows a 2.6-fold higher fluorescence intensity, 2.5-fold faster chromophore maturation and 1.5-fold faster off-switching than Kohinoor. The analysis of the pH dependence of the visible absorption band revealed that Kohinoor2.0 and Kohinoor were in equilibria among multiple fluorescently bright and dark states, with the mutations introduced into Kohinoor2.0 bringing about a higher stabilization of the fluorescently bright states compared to Kohinoor. Using Kohinoor2.0 with our SR imaging technique, super-resolution polarization demodulation/on-state polarization angle narrowing, we conducted 4-h time-lapse SR imaging of an actin filament network in mammalian cells with a total acquisition time of 480 s without a noticeable indication of phototoxicity. Furthermore, we demonstrated the SR imaging of mitochondria dynamics at a time resolution of 0.5 s, in which the fusion and fission processes were clearly visualized. Thus, Kohinoor2.0 is shown to be an invaluable RSFP for the SR imaging of cellular dynamics.

Photoswitchable Fluorescent Proteins: Mechanisms on Ultrafast Timescales

“…Since the discovery of the first RSFP called asFP595 (tetrameric) in 2000 [ 15 ], many RSFPs with monomeric states, long emission wavelengths, high photostability, fast maturation, and high photoswitching quantum yield (PQY) have been developed [ 16 , 17 , 18 , 19 ]. In general, they can be classified into three categories: negative, positive, and decoupled types [ 20 ].…”

The Positive Switching Fluorescent Protein Padron2 Enables Live-Cell Reversible Saturable Optical Linear Fluorescence Transitions (RESOLFT) Nanoscopy without Sequential Illumination Steps

“…These characteristics were improved to some extent in Kohinoor, a variant based on Padron. The recently reported Kohinoor2.0 exhibits a 2.9-fold higher molecular brightness than Kohinoor, but its switching fatigue has not been investigated and it has not been used for RESOLFT nanoscopy . Kohinoor has been used for a demonstration of point-scanning RESOLFT nanoscopy, but we found that Kohinoor is still prone to switching fatigue.…”

“…The recently reported Kohinoor2.0 exhibits a 2.9-fold higher molecular brightness than Kohinoor, but its switching fatigue has not been investigated and it has not been used for RESOLFT nanoscopy. 28 Kohinoor has been used for a demonstration of point-scanning RESOLFT nanoscopy, but we found that Kohinoor is still prone to switching fatigue. However, resistance against switching fatigue is a key requirement for one-step RESOLFT imaging.…”

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

“…rsEGFP2 can be switched from a non-fluorescent "OFF" state to a green fluorescent "ON" state by irradiation with 405 nm, while 488 nm are used for fluorescence excitation and to simul-taneously switch rsEGFP2 back to the "OFF" state. 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].…”