Evaluating the far-red emitting fluorescence-activating and absorption-shifting tag (frFAST) chemogenetic labeling...

Fluorogen-activating proteins (FAPs) provide renewable fluorescence signals through continuous fluorogen exchange, resulting in imaging that is virtually resistant to photobleaching, an especially appealing feature for deterministic super-resolution microscopy (SRM) techniques. Furthermore, careful adjustment of the fluorogen concentration may lead to reversible, exchange-driven stochastic blinking, allowing the use of FAPs in stochastic SRM methods. The green-light-excitable, far-red-emitting fluorescence-activating and absorption-shifting tag (frFAST) is particularly well suited for biological imaging due to its favorable spectral properties and live-cell compatibility, however, its performance across different SRM modalities has not been evaluated. Here, we assess frFAST for single-molecule localization microscopy (SMLM), fluctuation-based computational approaches (super-resolution optical fluctuation imaging (SOFI) and extended super-resolution radial fluctuations (eSRRF)), and stimulated emission depletion (STED) microscopy. By tuning fluorogen concentration, we induced reversible stochastic blinking in fixed and live cells, enabling SMLM imaging of cytoskeletal structures and filopodia in mammalian and neuroblastoma cells, without the need for harsh reducing agents. Additionally, we successfully labeled and imaged mitochondrial outer membrane, microtubules, cytoskeleton and histone proteins in live cell STED microscopy. While the renewable nature of the frFAST:HPAR-3OM interaction supports extended live-cell imaging for up to 40 min with limited photobleaching, we found that its performance is strongly modality dependent. In particular, intrinsic photophysical properties limit its suitability for classical SMLM, whereas fluctuation-based methods and live-cell STED microscopy are more compatible with frFAST.