In established super-resolution fluorescence microscopy, resolving multiple fluorescent molecules at sub-diffraction distances requires the molecules to emit sequentially so that they become discernible from their neighbors one after another. Simultaneous tracking of multiple fluorophores that are only a few nanometers apart is thus conceptually and practically impossible. We have recently shown that probing a sub-diffraction region with an excitation beam featuring an intensity zero, i.e., MINFLUX, super-resolves and tracks closely packed fluorophores without interruption. Here, we provide a conceptual framework for resolving and tracking constantly emitting fluorophores – more generally, point scatterers – that undergo random changes in position. In particular, we show that the detection rates available in fluorescence microscopy are sufficient to track sub-10 nm distance changes within micro-to milliseconds. By using a DNA origami construct with a fixed and a movable fluorophore as a proxy, we prove the concept that thermally driven conformational changes of biomolecules are continuously detectable with visible light. Conformational changes of the DNA nanostructure leading to random jumps in distance of about 10 nm between two labels are registered within about a millisecond. Our work paves the way towards super-resolving complex conformational transitions of individual biomolecules with focused light. ### Competing Interest Statement The Max Planck Society owns patents on MINFLUX with S.W.H. as inventor, covering aspects of this method. A further application has been filed with S.W.H. and T.A.H. as inventors. S.W.H. consults and owns shares of Abberior Instruments GmbH, a manufacturer of MINFLUX microscopes. The other authors declare no competing interests. Bundesministerium für Forschung, Technologie und Raumfahrt (BMBF) Max Planck Society, https://ror.org/01hhn8329 Fraunhofer Society, https://ror.org/05hkkdn48

Benzo[b]thiophene 1,1-dioxide is a relatively underrepresented sulfone-embedded heterocycle with niche uses in material science (OLED emitters), pharmaceutical research (an electrophilic warhead in targeted covalent inhibitors), and as a structural unit in reversibly switchable diarylethene fluorophores. Benzo[b]thiophene 1,1-dioxides are most commonly prepared by the oxidation of the corresponding benzothiophenes. Here, we propose an alternative approach based on Pd-catalyzed sulfinylation of ortho-carbonyl-substituted aryl triflates followed by S-alkylation and Knoevenagel condensation. This methodology allows for an expedient access to diversely substituted benzo[b]thiophene 1,1-dioxides, including 6-dialkylamino "push-pull" type fluorophores possessing large (>140 nm) Stokes shifts. Fluorescent labels derived from this core structure are compatible with live-cell imaging using self-labeling protein tags (HaloTag, SNAP-tag, and CLIP-tag). Together with the established live-cell fluorescent labels and orthogonal self-labeling tags, they allow for simultaneous observation of up to three intracellular targets below the diffraction limit using fluorescence microscopy with three excitation lasers (485, 561, and 640 nm) and stimulated emission depletion (STED) at 775 nm.

Photoactivatable (PA) fluorescent dyes with a reactive group are designated markers used in bioimaging techniques, tracking cellular processes, and observing the nanoscale organization of biological specimens with high spatiotemporal resolution. Conventional (non-PA) phenoxazine dyes are widely used in fluorescence microscopy owing to their high brightness, fluorescence emission in the far-red, and outstanding photostability, which allow their detection down to single molecules. So far, there has been no general synthetic route to PA oxazines with various emission colors and a reactive group. By applying metal-catalyzed C-H activation to symmetric and easily available N10-acetylphenoxazines, we demonstrated that this approach represents a powerful tool for the design of PA dyes. Versatile and relatively short syntheses directly involved 3,7-disubstituted-10-acetylphenoxazines or commercial Resazurin (7-hydroxy-10-oxyphenoxazin-3-one) as starting materials. These underwent site-selective Rh- or Ru-catalyzed C1-H activation followed by olefination with alkyl acrylates. The presence of the acrylate C═C bond attached to C1 in the N10-acetylphenoxazine scaffold results in a red shift (ca. 50 nm) in the absorption spectra, provides a site trapping the acetyl group cleaved off upon irradiation, and enables PA above 400 nm. Red-emitting PA oxazines having a CH═CHCONHR group were prepared and delivered to living and fixed cells. The PA probes incorporating HaloTag or BG-PEG amine (for labeling of Halo- or SNAP-Tags) were found to be cell-permeable and provided good images in (two-color) fluorescence microscopy and nanoscopy techniques, such as PALM (Photoactivation Localization Microscopy) and MINFLUX (MINimal FLUXes), based on the activation and detection of single molecules.

The near-infrared (NIR) spectral region is attractive for live-cell imaging, due to low autofluorescence and reduced phototoxicity. Some phytochrome-derived fluorescent proteins absorb and emit fluorescence in the NIR, but have short fluorescence lifetimes and relatively low quantum yields, requiring higher laser powers thus limiting their usefulness for live-cell superresolution microscopy. Using the bacterial phytochrome miRFP703 as a template, we screened for variants with longer fluorescence lifetimes, because the quantum yield and fluorescence lifetime are linked. We identified the bright monomeric fluorescent protein elite-niRFP704, which has a longer lifetime (1.12 ns) and a correspondingly higher quantum yield (0.21) than its template, absorbing and emitting completely in the NIR spectral region. elite-niRFP704 was used to tag proteins in living cells and facilitated extended stimulated emission depletion (STED) microscopy on cell lines stably expressing a fusion protein. Finally, elite-niRFP704 and miRFP703 could be separated based on their significantly different lifetimes, enabling two-channel NIR STED microscopy of living mammalian cells.

State-of-the-art super-resolution microscopy methods benefit greatly when combined with photoactivatable or photoswitchable fluorophores with far-red emission, but the majority of these fluorophores require specialized buffers or rely on photolabile protecting groups that limit their biocompatibility. We report here a caging-group-free strategy for photoactivatable dyes based on 1-vinyl-10-silaxanthone derivatives containing 9-acylimino or 9-(alkoxycarbonyl)imino groups, enabling minimally sized photoactivatable dyes with >= 680 nm emission. The 9-(alkoxycarbonyl)imino silaxanthones are particularly suitable for live-cell labeling, undergoing byproduct-free photoactivation to yield bright and photostable fluorophores that can be readily imaged by STED (stimulated emission depletion), PALM (photoactivated localization microscopy), or MINFLUX (minimal photon fluxes) nanoscopy techniques. The labels derived from these photoactivatable dyes open up new possibilities for multiplexed imaging.

Photoactivatable (PA) dyes with symmetric structures and two caging groups, rhodamines and carbo- and silicon-rhodamines, have been widely applied in super-resolution microscopy of subcellular structures with optical resolution far below the diffraction limit. The presence of two "heavy" caging groups reduces the solubility of a probe and, eventually, makes it less biocompatible. The photocleavage in two steps prolongs the photolysis time required for complete PA; it may cause excessive bleaching and secondary photoreactions. Here, we introduce "monocaged" PA fluorescent dyes based on xanthene cores with two heteroatoms (N, O, or N, N). In contrast to standard approaches, we protected only one heteroatom (either N or O) with a photocleavable (4,5-dimethoxy-2-nitrobenzyl) group and demonstrate that it is sufficient to mask the fluorescence of carbo-rhodol and carbo-rhodamine dyes. The monocaged PA probes have significantly lower molecular masses than their analogs with two bulky caging groups. The probes with a reactive group (COOH) provide facile labeling and undergo irreversible single-step photoactivation toward products emitting yellow or orange light. For carborhodol with a free hydroxyl and the protected N-methyl group, the reversible increase in emission was found at pH > 7 (as an activation tool, orthogonal to photolysis). Carboxamides incorporating the HaloTag amine (O2) ligand were applied for targeting and imaging of the HaloTag self-labeling enzyme fused with a protein of interest (vimentin). The utility and imaging performance of the probes with two heteroatoms belonging to a fluorophore, but only one caging group, were demonstrated in live cell labeling, conventional (confocal) microscopy, Minimal Photon Fluxes (MINFLUX) nanoscopy, and single-molecule localization methods (SMLM).

In established superresolution fluorescence microscopy or nanoscopy, resolving identical fluorescent molecules at subdiffraction distances requires the molecules to emit sequentially so that they briefly become discernible from their neighbors one after another. Simultaneous tracking of multiple fluorophores that are only a few nanometers apart is thus conceptually and practically impossible. We have recently shown that probing a subdiffraction region with an excitation beam featuring an intensity zero, i.e., MINFLUX superresolution, localizes and resolves closely packed identical fluorophores without interruption. Here, we provide a conceptual framework for resolving and tracking constantly emitting identical fluorophores-more generally, point scatterers-that undergo random changes in position. In particular, we show that the detection rates available in fluorescence microscopy are sufficient to track sub-10 nm distance changes within micro- to milliseconds. By using a DNA origami construct with a fixed and a movable fluorophore as a proxy, we provide a proof-of-concept that thermally driven conformational changes of macromolecules are continuously detectable with visible light. Conformational changes of the DNA nanostructure leading to random jumps in distance of about 10 nm between two labels are registered within ~1 ms. Our work paves the way toward superresolving complex conformational transitions of individual biomolecules with focused light.

Abstract Investigating the movements and conformational changes of proteins in living cells is essential for understanding their function. The recently introduced fluorescence nanoscopy method called MINSTED has successfully tracked single fluorophore-labelled proteins, albeit in fixed cells and two dimensions only. Here we introduce a MINSTED setup for live-cell tracking of individual proteins in three dimensions (3D) with a localization precision σ down to < 1 nm. Applied to the motor protein kinesin-1, our MINSTED nanoscope follows single proteins in 3D as they process along microtubules in 16 nm steps. Individual steps are resolved amid substantial intracellular background. The unique capability of the 3D stimulated emission depletion (STED) beam to carve out the signal of the protein label of interest enables efficient single-molecule investigations at hitherto unpractically high concentrations of labelled proteins and thus in crowded live-cell environments.

Resolving two or more constantly scattering identical point sources using freely propagating waves is limited by diffraction. Here we show that, by illuminating with a diffraction minimum, a given number of point scatterers can be resolved at distances of small fractions of the wavelength. Specifically, we identify an 8 nm distance, which corresponds to 1/80 of the employed 640 nm wavelength, between two constantly emitting fluorescent molecules in the focal plane of an optical microscope. We also measure 22 nm side length for a quadratic array of four molecules. Moreover, we show that the measurement precision improves with decreasing distance and with increased scatterer density. This work opens up the prospect of resolving individual scatterers in clusters that are far smaller than the wavelength.

Here, we present a fluorogenic supramolecular host-guest system comprising cucurbit[7]uril (CB7) and a rationally designed rosamine fluorescent dye (P-ARose) tailored for super-resolution imaging applications. By strategically designing the meso-aryl moiety of the guest, we concomitantly quenched the dye's emission in its free form and achieved strong binding with the host. The formation of the complex suppresses quenching and encompasses a large fluorescence turn-ON effect. Experimental and theoretical studies revealed that CB7 complexation significantly improved the photophysical properties of P-ARose, with a 6.4-fold fluorescence increase and 4.2-fold enhanced emission quantum yield. Further functionalization of P-ARose with a HaloTag ligand or an NHS reactive group rendered it suitable for live-cell and immunofluorescence labeling, yielding specificity, negligible background, and a minimal fluorescence signal in the unbound state. The addition of CB7 drastically increased fluorescence, enabling conventional and stimulated emission depletion (STED) imaging with subdiffraction resolution. Furthermore, the turn-ON ability of the host-guest complex facilitated pseudo two-color sequential imaging of different protein combinations within the same acquisition channel. These results demonstrate the potential of this supramolecular system to enable an additional super-resolution imaging multiplexing modality through noncovalent chemistry.

REversible Saturable Optical Linear Fluorescence Transitions (RESOLFT) superresolution microscopy fundamentally overcomes the diffraction barrier in far-field fluorescence microscopy. It relies on reversibly switchable fluorescent proteins (RSFPs) that allow repeated light-induced transitions between fluorescent on- and nonfluorescent off-states. Because these transitions are induced by low-light intensities, RESOLFT superresolution microscopy is particularly suitable for live-cell imaging. So far, RESOLFT imaging has only been performed in the visible range of the electromagnetic spectrum. To expand the RESOLFT concept into the near-infrared (NIR) region, which is characterized by reduced autofluorescence, lower scattering and decreased phototoxicity, we developed the photostable NIR reversibly switchable fluorescent protein (PENELOPE), which is the first RSFP applicable in the NIR window. PENELOPE was generated by mutagenesis of the chromophore-binding domain of the Deinococcus radiodurans bacteriophytochrome. This NIR-RSFP exhibits high photostability and high ensemble switching contrast at low-light intensities. It also undergoes an unusually fast thermal fluorescence recovery from the dark state into an on-state. This was exploited for low-light intensity RESOLFT imaging with only a single wavelength, as the same light wavelength (660 nm) is used for off-switching and fluorescence readout, while the on-switching occurs in the absence of illumination. We demonstrate RESOLFT recordings both in chemically fixed and in living human cells using PENELOPE as a fusion protein.

The use of photoactivatable fluorescent diarylethenes (fDAEs) in biology-related light microscopy has been restricted by the lack of probes having freely variable structural and spectral properties. Facing these challenges, we synthesized "turn-on" fDAEs with four hydroxyl groups and a single functionality in the core structure (HO-fDAEs). HO-fDAEs emit green light; they were used for the preparation of cell-permeant mitochondrion-selective probes and bioconjugation with proteins (via a single COOH group). HO-fDAEs were combined in bio-imaging with a thiophene-substituted fDAE (Th-fDAE) possessing red-shifted absorption and emission bands of the open- and closed-ring isomers. Th-fDAE undergoes full on-switching with 405 nm and excitation with green light (< 560 nm). The large Stokes shifts of HO-and Th-fDAE (80 and 110 nm, respectively) and different excitation wavelengths permitted color separation in optical microscopy. Photoactivation of HO-fDAEs and transient on-off switching (blinking) of Th-fDAE made it possible to acquire two-color images of cellular structures with optical superresolution.

Controlled photoactivation is an auspicious and emerging approach in super-resolution microscopy, offering virtually zero background signal from the marker prior to activation. Pyronins are well-established fluorophores, but due to their inherent intercalating tendency towards nucleic acids, their use has been mostly avoided in super-resolution microscopy. Here, we describe a new class of diaryl ether and diaryl silane molecules that upon photoactivation close into fluorescent (silicon-)pyronins and term them Pyronin Upon Light Irradiation (PULI). This concept exploits the outstanding photophysical properties of pyronins (bright, photostable, and optimal spectral features for standard microscopes), while overcoming their major drawback (intrinsic affinity of accumulating in the nucleus and around RNA) for the design of fluorescent markers for imaging applications. Furthermore, we also demonstrate that this approach is applicable to their Si-bridged analogues, extending this family of photoactivatable molecules to the far-red regime. The versatility of our approach was also highlighted by tagging diverse biological targets in cells and visualizing them using advanced super-resolution microscopy techniques, such as PALM, STED, and MINFLUX.

In a search for dyes photoactivatable with visible light, fluorenes with substituents at positions 2 and 7 were prepared, and their absorption and emission spectra were studied. In particular, the synthesis route to 9-diazofluorenes with 2-(N,N-dialkylamino) and N-modified 7-(4-pyridyl) substituents was established. These compounds are initially non-fluorescent, undergo photolysis with UV or blue light, and—in non-polar media—provide orange- to red-emitting products with a large separation between absorption and emission bands. Irradiation of non-fluorescent 9-diazoderivative 20 in dioxane with the light of 365 nm or 470 nm was accompanied by strong fluorescence gain (10 to 20 times), orange–red emission, and a large Stokes shift of photoproducts, which structurally relate to fluorescent betaine 13 (model compound without diazo group). Photolysis of 20 in protic solvents (ROH = MeOH, H2O) provided clean transformation to C9-OR derivatives, though the emission gain in protic solvents was low.

Discerning two or more identical and constantly scattering point sources using freely propagating waves is thought to be limited by diffraction. Here we show both theoretically and experimentally that by employing a diffraction minimum rather than a maximum for resolution, a given number of point scatterers can be discerned at tiny fractions of the employed wavelength. Specifically, we identify an 8 nm distance between two constantly emitting (non-blinking, non-switchable) fluorescent molecules, corresponding to 1/80 of the wavelength. Moreover, we show that contrary to naïve expectations, the measurement precision improves with decreasing distance between the scatterers and with increased scatterer density, thus opening up the prospect of resolving clusters of (optical) point scatterers at tiny fractions of the wavelength.

We introduce MINFLUX localization with interferometric illumination through opposing objective lenses for maximizing the attainable precision in 3D-localization of single inelastic scatterers, such as fluorophores. Our 4Pi optical configuration employs three sequentially tilted counter-propagating beam pairs for illumination, each providing a narrow interference minimum of illumination intensity at the focal point. The localization precision is additionally improved by adding the inelastically scattered or fluorescence photons collected through both objective lenses. Our 4Pi configuration yields the currently highest precision per detected photon among all localization schemes. Tracking gold nanoparticles as non-blinking inelastic scatterers rendered a position uncertainty <0.4 nm3 in volume at a localization frequency of 2.9 kHz. We harnessed the record spatio-temporal precision of our 4Pi MINFLUX approach to examine the diffusion of single fluorophores and fluorescent nanobeads in solutions of sucrose in water, revealing local heterogeneities at the nanoscale. Our results show the applicability of 4Pi MINFLUX to study molecular nano-environments of diffusion and its potential for quantifying rapid movements of molecules in cells and other material composites.

Optical investigations of nanometer distances between proteins, their subunits, or other biomolecules have been the exclusive prerogative of Förster resonance energy transfer (FRET) microscopy for decades. In this work, we show that MINFLUX fluorescence nanoscopy measures intramolecular distances down to 1 nanometer—and in planar projections down to 1 angstrom—directly, linearly, and with angstrom precision. Our method was validated by quantifying well-characterized 1- to 10-nanometer distances in polypeptides and proteins. Moreover, we visualized the orientations of immunoglobulin subunits, applied the method in human cells, and revealed specific configurations of a histidine kinase PAS domain dimer. Our results open the door for examining proximities and interactions by direct position measurements at the intramacromolecular scale.

The single-molecule localization concept MINFLUX has triggered a reevaluation of the features of fluorophores for attaining nanometer-scale resolution. MINFLUX nanoscopy benefits from temporally controlled fluorescence ("on"/"off") photoswitching. Combined with an irreversible switching behavior, the localization process is expected to turn highly efficient and quantitative data analysis simple. The potential in the recently reported photoactivable xanthone (PaX) dyes is recognized to extend the list of molecular switches used for MINFLUX with 561 nm excitation beyond the fluorescent protein mMaple. The MINFLUX localization success rates of PaX560, PaX+560, and mMaple are quantitatively compared by analyzing the effective labeling efficiency of endogenously tagged nuclear pore complexes. The PaX dyes prove to be superior to mMaple and on par with the best reversible molecular switches routinely used in single-molecule localization microscopy. Moreover, the rationally designed PaX595 is introduced for complementing PaX560 in dual color 561 nm MINFLUX imaging based on spectral classification and the deterministic, irreversible, and additive-independent nature of PaX photoactivation is showcased in fast live-cell MINFLUX imaging. The PaX dyes meet the demands of MINFLUX for a robust readout of each label position and fill the void of reliable fluorophores dedicated to 561 nm MINFLUX imaging.

Dynein is the primary molecular motor responsible for retrograde intracellular transport of a variety of cargoes, performing successive nanometer-sized steps within milliseconds. Due to the limited spatiotemporal precision of established methods for molecular tracking, current knowledge of dynein stepping is essentially limited to slowed-down measurements in vitro. Here, we use MINFLUX fluorophore localization to directly track CRISPR/Cas9-tagged endogenous dynein with nanometer/millisecond precision in living primary neurons. We show that endogenous dynein primarily takes 8 nm steps, including frequent sideways steps but few backward steps. Strikingly, the majority of direction reversals between retrograde and anterograde movement occurred on the time scale of single steps (16 ms), suggesting a rapid regulatory reversal mechanism. Tug-of-war-like behavior during pauses or reversals was unexpectedly rare. By analyzing the dwell time between steps, we concluded that a single rate-limiting process underlies the dynein stepping mechanism, likely arising from just one adenosine 5′-triphosphate hydrolysis event being required during each step. Our study underscores the power of MINFLUX localization to elucidate the spatiotemporal changes underlying protein function in living cells.

New photostable and bright supramolecular complexes based on cucurbit[7]uril (CB7) host and diketopyrrolopyrole (DPP) guest dyes having two positively charged 4-(trimethylammonio)phenyl groups were prepared and characterized. The dye core displays large Stokes shift (in H2O, abs./emission max. 480/550 nm; ϵ~19 000, τfl>4 ns), strong binding with the host (~560 nM Kd) and a linker affording fluorescence detection of bioconjugates with antibody and nanobody. Combination of protein-functionalized DPP dye with CB7 improves photostability and affords up to 12-fold emission gain. Two-color confocal and stimulated emission depletion (STED) microscopy with 595 nm or 655 nm STED depletion lasers shows that the presence of CB7 not only leads to improved brightness and image quality, but also results in DPP becoming cell-permeable.