Super-resolution fluorescence microscopy is a powerful tool to visualize biomolecules and cellular structures at the nanometer scale. Employing these techniques in living cells has opened up the possibility to study dynamic processes with unprecedented spatial and temporal resolution. Different physical approaches to super-resolution microscopy have been introduced over the last years. A bottleneck to apply these approaches for live-cell imaging has become the availability of appropriate fluorescent probes that can be specifically attached to biomolecules. In this Perspective, we discuss the role of small-molecule fluorescent probes for live-cell super-resolution microscopy and the challenges that need to be overcome for their generation. Recent trends in the development of labeling strategies are reviewed together with the required chemical and spectroscopic properties of the probes. Finally, selected examples of the use of small-molecule fluorescent probes in live-cell super-resolution microscopy are given.

Self-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications.

Resorcin[4]arene cavitands with four quinoxaline bridges are a family of macrocycles that adopt, at elevated temperature, a contracted, vase‐type conformation, capable of guest inclusion, whereas at low temperature they switch to an expanded, kite‐type conformation with a large flat surface. The present investigations lay the foundation for the use of such dynamic cavitands as miniaturized mechanical grippers for supramolecular construction at the single‐molecule level. New vase–kite switching modes, stimulated by pH changes or stoichiometric metal‐ion complexation, have been discovered and monitored by 1H NMR and optical absorption spectroscopy. The solid‐state geometries of the two states have been revealed by X‐ray crystallography, and the kinetics and thermodynamics of the switching processes in solution as well as their solvent dependency has been investigated in great detail. Monolayers of the cavitand in the vase form have been studied by scanning tunneling microscopy at molecular resolution; conformational switching is also observed in Langmuir monolayers at the air/water interface. Synthetic protocols have been developed for preparation of partially and asymmetrically bridged resorcin[4]arene cavitands, which are also shown to undergo conformational switching. These synthetic advances pave the way to new, dynamic molecular receptors for steroids, tetrathiofulvalene‐bridged grippers with the potential to undergo electrochemically induced conformational switching, and systems with greatly extended, rigid cavity walls functionalized at the termini by dipyrrometheneboron difluoride dyes. The latter cavitands are shown by fluorescence resonance energy transfer to undergo geometrically precisely defined motions between a contracted (≈ 7 Å linear extension) and a strongly expanded (≈ 7 nm linear extension) state.

The specific and covalent labeling of the protein HaloTag with fluorescent probes in living cells makes it a powerful tool for bioimaging. However, the irreversible attachment of the probe to HaloTag precludes imaging applications that require transient binding of the probe and comes with the risk of irreversible photobleaching. Here, we introduce exchangeable ligands for fluorescence labeling of HaloTag (xHTLs) that reversibly bind to HaloTag and that can be coupled to rhodamines of different colors. In stimulated emission depletion (STED) microscopy, probe exchange of xHTLs allows imaging with reduced photobleaching as compared to covalent HaloTag labeling. Transient binding of fluorogenic xHTLs to HaloTag fusion proteins enables points accumulation for imaging in nanoscale topography (PAINT) and MINFLUX microscopy. We furthermore introduce pairs of xHTLs and HaloTag mutants for dual-color PAINT and STED microscopy. xHTLs thus open up new possibilities in imaging across microscopy platforms for a widely used labeling approach.

Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Here we present a photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we create probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore’s outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.

The reversible switching from the C(4v)-symmetric vase to the C(2v)-symmetric kite conformation of an amphiphilic resorcin[4]arene cavitand was induced by Zn(II) ion coordination. Langmuir monolayers were obtained of both conformers with the area per molecule increasing dramatically from 120 A(2) for the vase to 270 A(2) for the kite form. (1)H NMR spectroscopy in chloroform solution supports the formation of a stoichiometric kite-cavitand[radical dot](Zn(II))(2) complex, with the metal ions coordinating to pairs of neighbouring quinoxaline N-atoms.

We report the synthesis of novel resorcin [4]arene-based cavitands featuring two extended bridges consisting of quinoxaline-fused TTF (tetrathiafulvalene) moieties. In the neutral form, these cavitands were expected to adopt the vase form, whereas, upon oxidation, the open kite geometry should be preferred due to Coulombic repulsion between the two TTF radical cations (Scheme 2). The key step in the preparation of these novel molecular switches was the P(OEt) 3 -mediated coupling between a macrocyclic bis(1,3-dithiol-2-thione) and 2 equiv. of a suitable 1,3-dithiol-2-one. Following the successful application of this strategy to the preparation of mono-TTF-cavitand 3 (Scheme 3), the synthesis of the bis-TTF derivatives 2 (Scheme 4) and 19 (Scheme 5) was pursued; however, the target compounds could not be isolated due to their insolubility. Upon decorating both the octol bowl and the TTF cavity rims with long alkyl chains, the soluble bis-TTF cavitand 23 was finally obtained, besides a minor amount of the novel cage compound 25a featuring a highly distorted TTF bridge (Scheme 6). In contrast to 25a, the deep cavitand 23 undergoes reversible vase ! kite switching upon lowering the temperature from 293 to 193 K (Fig. 1). Electrochemical studies by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) provided preliminary evidence for successful vase ! kite switching of 23 induced by the oxidation of the TTF cavity walls. The vase conformer is prevalent at room temperature at neutral pH, whereas the kite geometry is predominant at low temperatures ( 213 K) [1], upon protonation with acids such as CF 3 COOH (TFA) [4], or in the presence of Zn II ions [5]. At low temperature, solvation of the more extended surface stabilizes the kite geometry, whereas, at higher temperature, the entropic term TDS solv becomes unfavorable, and the vase conformation is dominant [1]. More recent investigations also showed that suitably sized solvent molecules (such as small benzene derivatives) favorably solvate (stabilize) the vase form and reduce the propensity for vase ! kite transition [6]. On the other hand, the kite conformation is additionally stabilized by solvents with substantial H-bonding acidity: weak H-bonding interactions between the mildly basic quinoxaline N-atoms, and solvent molecules are more efficient in the open kite than in the closed vase form [6b]. Acid-induced switching from the vase to the kite

Coenzyme A (CoA) is one of the central cofactors of metabolism, yet a method for measuring its concentration in living cells is missing. Here we introduce the first biosensor for measuring CoA levels in different organelles of mammalian cells. The semisynthetic biosensor is generated through the specific labeling of an engineered GFP–HaloTag fusion protein with a fluorescent ligand. Its readout is based on CoA-dependent changes in Förster resonance energy transfer efficiency between GFP and the fluorescent ligand. Using this biosensor, we probe the role of numerous proteins involved in CoA biosynthesis and transport in mammalian cells. On the basis of these studies, we propose a cellular map of CoA biosynthesis that suggests how pools of cytosolic and mitochondrial CoA are maintained.