A detailed review of genetically encodable RFPs and far-RFPs and their applications in advanced super-resolution imaging techniques.

The red fluorescent proteins (RFPs) and far-red fluorescent proteins (far-RFPs) that are encoded genetically can emit fluorescence within the spectral ranges of 580-680 nm when exposed to the light of appropriate wavelengths. Unlike many RFPs derived from coral species, numerous far-RFPs are optimized synthetic constructs engineered from different orange or red-emitting progenitors. Various categories have been established for the available RFPs and far-red fluorescent proteins based on their photo-chemical profile, fluorescence mechanism, and switching kinetics. Fluorescent probes (FPs) derived from these classes are extensively utilized for labelling and visualizing in vivo and in vitro specimens. Traditional optical microscopy methods generate diffraction-limited, indistinct images owing to the restricted resolution capability of light ranging from 200 to 300 nm. Since 2005, super-resolution microscopy has been a topic of great interest due to its ability to achieve imaging at spatial resolutions of less than 100 nm. The 2014 Nobel Prize in Chemistry was awarded to Eric Betzig, Stefan Hell, and William E. Moerner for their contributions to demonstrating the effectiveness of genetically encodable fluorescent proteins in visualizing biological systems through super-resolution fluorescence microscopy. This review provides a concise overview of RFPs and far-RFPs, including the involvement of surrounding residues in chromophore intactness, stability, autocatalytic maturation and switching kinetics. All the chemical pathways proposed for photoactivation, photoconversion and photoswitching mechanisms are concisely reviewed. Subsequently, a comprehensive summary was provided regarding the various types of super-resolution techniques that are commonly employed, elucidating their underlying principles of operation, as well as the potential future applications of RFPs/far-RFPs in the field of biological imaging.