GMars-T Enabling Multimodal Subdiffraction Structural and Functional Fluorescence Imaging in Live Cells

Abstract

Fluorescent probes with multimodal and multilevel imaging capabilities are highly valuable as imaging with such probes not only can obtain new layers of information but also enable cross-validation of results under different experimental conditions. In recent years, the development of genetically encoded reversibly photoswitchable fluorescent proteins (RSFPs) has greatly promoted the application of various kinds of live-cell nanoscopy approaches, including reversible saturable optical fluorescence transitions (RESOLFT) and stochastic optical fluctuation imaging (SOFI). However, these two classes of live-cell nanoscopy approaches require different optical characteristics of specific RSFPs. In this work, we developed GMars-T, a monomeric bright green RSFP which can satisfy both RESOLFT and photochromic SOFI (pcSOFI) imaging in live cells. We further generated biosensor based on bimolecular fluorescence complementation (BiFC) of GMars-T which offers high specificity and sensitivity in detecting and visualizing various protein-protein interactions (PPIs) in different subcellular compartments under physiological conditions (e.g., 37 °C) in live mammalian cells. Thus, the newly developed GMars-T can serve as both structural imaging probe with multimodal super-resolution imaging capability and functional imaging probe for reporting PPIs with high specificity and sensitivity based on its derived biosensor.

Split Green Fluorescent Proteins: Scope, Limitations, and Outlook

“…To extend the scope of the technique to include the potential for multicolor imaging, FP variants spanning a wide spectral range from the visible to the near infrared were engineered as split reporters and implemented in BiFC assays (Table 1), and several resources outline design principles and best practices to guide those interested in developing a new split FP (42, 63, 67, 74, 89). One particularly exciting advancement in BiFC is the extension of split reporters to FPs with unique photophysical properties such as photoactivation, photoconversion, and photoswitching (Figure 3 b ), which allows for the combination of super-resolution imaging and PPI detection in live cells (17, 33, 41, 75, 80, 92, 148, 149, 157). The current diversity and continual innovation of split FPs is a testament to the utility of BiFC as a biochemical tool, the robustness of the conserved FP β-barrel, and the advances in molecular biology and protein engineering technologies over the past two decades.…”

Optimizing the fluorescent protein toolbox and its use

“…Besides new and improved non-phototransformable FPs, the selection of photochromic FPs has also been expanded recently. The newly developed GMars variants are green RSFPs derived from the green-to-red photoconvertible mMaple3 [25,26,27 ]. Although the GMars variants differ in only one amino acid, they have very distinct properties.…”

“…Both beneficial properties can be assigned to efficient shelving of GMars-Q proteins in dark states [28], which unfortunately also introduces additional complexity in the photophysical behavior and limits the amount of detectable fluorescence after repeated photoswitching. GMars-T as a second example was found to have beneficial spectroscopic and photochromic properties for multimodal super resolution imaging, realizing a significant resolution improvement in both super resolution optical fluctuation imaging (SOFI) and RESOLFT experiments [26].…”

Between Life and Death: Reducing Phototoxicity in Super-Resolution Microscopy

“…This overcomes a substantial barrier in SIM, in that conventional SIM reconstruc-tion algorithms perform poorly on low-illumination datasets, leading to artefacts within the resulting images. Approaches have also been proposed for low-power STED microscopy based on reconstructing images with knowledge of fluorescence lifetime changes induced by the STED beam (75,164).…”

“…Other ML-based techniques have also allowed for prediction of enhanced resolution images from low illumination diffraction-limited images (Fig. 6c), for example: converting confocal to Airyscan-type or STED-type images (75,169); or widefield to SIM-type images (75).…”