Green-to-Red Photoconvertible Dronpa Mutant for Multimodal Super-resolution Fluorescence Microscopy

Abstract

Advanced imaging techniques crucially depend on the labels used. In this work, we present the structure-guided design of a fluorescent protein that displays both reversibly photochromic and green-to-red photoconversion behavior. We first designed ffDronpa, a mutant of the photochromic fluorescent protein Dronpa that matures up to three times faster while retaining its interesting photochromic features. Using a combined evolutionary and structure-driven rational design strategy, we developed a green-to-red photoconvertible ffDronpa mutant, called pcDronpa, and explored different optimization strategies that resulted in its improved version, pcDronpa2. This fluorescent probe combines a high brightness with low photobleaching and photoblinking. We herein show that, despite its tetrameric nature, pcDronpa2 allows for multimodal subdiffraction imaging by sequentially imaging a given sample using both super-resolution fluctuation imaging and localization microscopy.

Novel uses of fluorescent proteins

“…Recent extension of this method promises higher (up to 5-fold) resolution improvement and extraction of additional useful information from the fluctuations [54], such as molecular brightness. SOFI has been shown to work with PAFP [55,56] even in 3D [57 ] in living cells. An improved reversibly switchable green FP Skylan-S was recently designed to provide robust fluctuations and low photofatigue for SOFI [58].…”

Optical Nanoscopy Tools for Biologists:Advancements of Fluorophores and Optics for High Resolution and Live Imaging

“…Many cellular events have been explored with high resolution using localization precision microscopy techniques. The localization precision for these methods can be calculated as described earlier using the following equation Absence of some actin fibres at the outer edges of the cell in the pcSOFI image is due to the increased z-sectioning in pcSOFI combined with imperfections of the 488 nm illumination, and also these structures being slightly out of focus 67 . [64][65][66] .…”

“…[64][65][66] . A few recently published studies show the potential of pcSOFI for fast, easy and live cell study well below the diffraction limit, using as few as 200-500 image frames for nanoscopic image reconstruction 65,67 . Combination of pcSOFI with reversibly photoswitchable fluorescent proteins (RSFPs) provides robust blinking desired for reconstruction of highresolution nanoscopic image of pcSOFI (Figure 4 b).…”

“…This group of FPs called reversibly photoswitchable fluorescent proteins (RSFPs) can be switched on/off repeatedly upon UV light irradiation ( Figure 5 c) 43,67 . Padron is unique among all RSFPs because of its positive-switching behaviour.…”

“…Dronpa and its variant were used in several super-resolution imaging studies, e.g. cellular dynamics 51 , dual-colour and 3D PALM imaging 55 and photochromic stochastic optical fluctuation imaging (pcSOFI) 65,67 . EGFP-based RSFPs (rsEGFP and rsEGFP2) were the first with fast switching speed and a large number of switching cycles 39,41 .…”