Biology subjects

Hell, S. W.

Publications and source records attributed to Hell, S. W..

At least 19 recordsLinked to original sources

bioRxiv2026

Localization accuracy in single-molecule fluorescence microscopy

The two prevalent methods to determine the position of individual fluorophores in the focal plane of a microscope rely either on the centroid of the fluorescence pattern produced by the fluorophore on a camera or on targeting the fluorophore's coordinate with a spatially controlled illumination minimum, as realized in MINFLUX. In both methods, (sub)nanometer localization precisions have been reached, but little is known about their accuracy, i.e., the deviation of the obtained coordinate from its ground truth. Here we report a theoretical and experimental comparison of the accuracy provided by these two strategies. We quantify substantial systematic errors in camera-based localization due to the interplay of two difficult-to-control parameters: the orientation of the fluorophore in space and its axial deviation from the focal plane (defocus). For example, within an axial range of {+/-}120 nm around the focus, the median distance between two fluorophores on a nanobody measured by camera-based localization varies by 26.5 nm. Under the same conditions, a commercial MINFLUX system yields a median variation in distance of 1.4 nm, indicating an improvement in accuracy by an order of magnitude. The accuracy of MINFLUX localization can be further improved to (sub-)[a]ngstrom levels by reducing the average distance between the coordinates targeted by the illumination minimum and the fluorophore. As it delivers unrivaled precision and accuracy for mapping fluorophores and their distances, we consider coordinate-targeted localization essential for studying labeled cells at the sub-10 nm level and for exploring structural and conformational changes of labeled biomolecules.

biophysics↗

bioRxiv2026

3D-MINSTED nanoscopy and protein tracking in densely labelled living cells

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 {sigma} 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.

biophysics↗

bioRxiv2026

Nonstop nanometric resolution of randomly moving point scatterers with focused light

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.

biophysics↗

bioRxiv2025

Caging-group-free photoactivatable fluorophores with far-red emission

State of the art super-resolution microscopy methods profit 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 labelling, undergoing byproduct-free photoactivation to yield bright and photostable fluorophores that can be readily imaged by STED, PALM, or MINFLUX nanoscopy techniques. The labels derived from these photoactivatable dyes open up new possibilities for multiplexed imaging.

biochemistry↗

bioRxiv2024

MINFLUX fluorescence nanoscopy in biological tissue

Optical imaging access to nanometer-level protein distributions in intact tissue is a highly sought-after goal, as it would provide visualization in physiologically relevant contexts. Under the unfavorable signal-to-background conditions of increased absorption and scattering of the excitation and fluorescence light in the complex tissue medium, super-resolution fluorescence microscopy methods are severely challenged in attaining precise localization of molecules. We reasoned that the typical use of a confocal detection pinhole in MINFLUX nanoscopy, suppressing background and providing optical sectioning, should facilitate the detection and resolution of single fluorophores even amid scattering and optically challenging tissue environments. Here, we investigated the performance of MINFLUX imaging for different synaptic targets and fluorescent labels in tissue sections of the mouse brain. Single fluorophores were localized with a precision of < 5 nm at up to 80 {micro}m sample depth. MINFLUX imaging in two color channels allowed to probe PSD95 localization relative to the spine head morphology, while also visualizing presynaptic VGlut clustering and AMPA receptor clustering at the post-synapse. Our two-dimensional (2D) and three-dimensional (3D) two-color MINFLUX results in tissue, with < 10 nm 3D fluorophore localization, open up new avenues to investigate protein distributions on the single-synapse level in fixed and living brain slices.

biophysics↗

bioRxiv2024

MINFLUX Reveals Dynein Stepping in Live Neurons

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 whereby dynein consumes one adenosine 5-triphosphate (ATP) per step. Our study underscores the power of MINFLUX localization to elucidate the spatiotemporal changes underlying protein function in living cells.

biophysics↗

bioRxiv2024

Dual-color MINFLUX: Kinesin-1 takes Chasse-Inchworm steps

Withdrawal statementThe authors have withdrawn this manuscript because refined experiments with a remodeled two-color MINFLUX setup featuring improved accuracy and precision of the two-color registration, do not support the reported Chasse-Inchworm stepping mechanism of kinesin-1. The Chasse-Inchworm mechanism was postulated based on recurrent observations of separations of 0 and 16 nanometers between a green-absorbing and a red-absorbing fluorophore, each respectively attached to the two motor domains of kinesin-1. However, the same experiments performed with the improved two-color MINFLUX setup revealed merely separations of 0 and 8 nanometers, aligning with the widely accepted hand-over-hand mechanism. Since the authors no longer stand behind their initial conclusions, they do not wish this work to be cited as a reference. In case of questions, please contact the corresponding author.

biophysics↗

bioRxiv2024

Diffraction minima resolve point scatterers at tiny fractions (1/80) of the wavelength

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 naive 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.

biophysics↗

bioRxiv2023

Uncovering kinesin dynamics in neurites with MINFLUX

Neurons grow neurites of several tens of micrometers in length, necessitating active transport from the cell body by motor proteins. By tracking fluorophores as minimally invasive labels, MINFLUX is able to quantify the motion of those proteins with nanometer/millisecond resolution. Here we study the substeps of a truncated kinesin-1 mutant in primary rat hippocampal neurons, which have so far been mainly observed on microtubules polymerized on glass coverslips. A gentle fixation protocol largely maintains the structure and surface modifications of the microtubules in the cell. By analyzing the time between the substeps, we identify the ATP-binding state of kinesin-1 and observe the associated rotation of the kinesin-1 head in neurites. We also observed kinesin-1 switching microtubules mid-walk, highlighting the potential of MINFLUX to study the details of active cellular transport.

biophysics↗

bioRxiv2023

4Pi MINFLUX arrangement maximizes spatio-temporal localization precision of fluorescence emitter

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 (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.

biophysics↗

bioRxiv2023

MINSTED tracking of single biomolecules

We show that MINSTED localization, a method whereby the position of a fluorophore is identified with precisely controlled beams of a STED microscope, tracks fluorophores and hence labeled biomolecules with nanometer/millisecond spatio-temporal precision. By updating the position for each detected photon, MINSTED recognizes fluorophore steps of 16 nm within < 250 microseconds using about 13 photons. The power of MINSTED tracking is demonstrated by quantifying side-steps of the motor protein kinesin-1 walking on microtubules and switching protofilaments.

biophysics↗

bioRxiv2023

Direct optical measurement of intra-molecular distances down to the Angstrom scale

Optical investigations of nanometer distances between proteins, their subunits, or other biomolecules have been the exclusive prerogative of Forster Resonance Energy Transfer (FRET) microscopy for decades. Here we show that MINFLUX fluorescence nanoscopy measures intra-molecular distances down to 1 nm - and in planar projections down to 1 Angstrom - directly, linearly, and with Angstrom precision. Our method is validated by quantifying well-characterized 1-to-10 nm distances in polypeptides and proteins. Moreover, we visualize the orientations of immunoglobulin subunits and reveal specific configurations of a histidine kinase domain dimer. Our results open the door for examining proximities and interactions of macromolecules under physiological conditions.

biophysics↗

bioRxiv2023

Bioorthogonal caging-group-free photoactivatable probes for minimal-linkage-error nanoscopy

Here we describe highly compact, click compatible, and photoactivatable dyes for super-resolution fluorescence microscopy (nanoscopy). By combining the photoactivatable xanthone (PaX) core with a tetrazine group, we achieve minimally sized and highly sensitive molecular dyads for selective labeling of unnatural amino acids introduced by genetic code expansion. We exploit the excited state quenching properties of the tetrazine group to attenuate the photoactivation rates of the PaX, and further reduce the overall fluorescence emission of the photogenerated fluorophore, providing two mechanisms of selectivity to reduce off-target signal. Coupled with MINFLUX nanoscopy, we demonstrate our dyads in the minimal-linkage-error imaging of vimentin filaments, demonstrating the molecular scale precision in fluorophore positioning.

biochemistry↗

bioRxiv2022

Photoactivatable large Stokes shift fluorophores for multicolor nanoscopy

We designed caging-group-free photoactivatable live-cell permeant dyes with red fluorescence emission and [~]100 nm Stokes shifts based on a 1-vinyl-10-silaxanthone imine core structure. The proposed fluorophores undergo byproduct-free one- and two-photon activation, are suitable for multicolor fluorescence microscopy in fixed and living cells and are compatible with super-resolution techniques such as STED (stimulated emission depletion) and PALM (photoactivated localization microscopy). Use of photoactivatable labels for strain-promoted azide-alkyne cycloaddition and self-labeling protein tags (HaloTag, SNAP-tag), and duplexing of imaging channel with another large Stokes shift dye have been demonstrated.

biochemistry↗

bioRxiv2022

Spontaneously blinking fluorophores for accelerated MINFLUX nanoscopy

Spontaneously blinking fluorophores, a class of molecules switching rapidly between a dark and a brightly emitting state, have emerged as a popular core to build fluorescent markers for super-resolution microscopy. With typical on-times in the order of tens of milliseconds, they are most suitable for STORM and related nanoscopy methods. Recent MINFLUX nanoscopy, however, can localize molecules even within a millisecond and achieve an up to ten times higher localization precision. Here, we present a series of spontaneous blinkers with short on-times (1-3 ms) matching MINFLUX recording time-scales. Our design builds upon a silicon rhodamine fluorescent core with a modified thiophene- or a benzothiophene-fused spirolactam fragment, which shifts the spirocyclization equilibrium toward the dark closed form at physiological conditions, imparting cell permeability. Concurrently, we obtain a highly photostable, short-lived open form with bright red emission. Characterizing the blinking behavior of single fluorophores bound to three different protein tags (antibodies, nanobodies, and HaloTag self-labeling enzyme) allowed us to select the best candidate for MINFLUX microscopy. The short on-times speed up MINFLUX localization by up to 30-fold.

biophysics↗

bioRxiv2022

MINFLUX dissects the unimpeded walking of kinesin-1

We report on an interferometric MINFLUX microscope that records protein movements with down to 1.7 nm precision within less than 1 ms. While such spatio-temporal resolution has so far required linking a strongly scattering 30-500 nm diameter bead to the much smaller protein, MINFLUX localization requires the detection of only down to 20 photons from an ~1-nm sized fluorophore. Harnessing this resolution, we dissect the unhindered stepping of the motor protein kinesin-1 on microtubules at up to physiological ATP concentrations. By attaching the fluorophore to different kinesin-1 sites and resolving steps and substeps of these protein constructs, we uncover a three-dimensional orientation change of the unbound kinesin head. We also find that kinesin-1 takes up ATP while only one head is bound, whereas hydrolysis of ATP occurs with both heads bound to the microtubule, resolving a long-standing conundrum of its mechanochemical cycle. Our results establish MINFLUX as a non-invasive tool for tracking protein movements and probing submillisecond structural rearrangements with nanometer resolution.

biophysics↗

bioRxiv2022

Assessment of 3D MINFLUX data for quantitative structural biology in cells revisited

Prakash and Curd provide a re-analysis1 of individual datasets taken from our report2 demonstrating MINFLUX 3D imaging in cells. Their evaluation confirms the unique localization precision provided by MINFLUX2,3 featuring a standard deviation of {sigma} = 1-3 nm. We appreciate their confirmation and also welcome the opportunity to clarify their remaining points. The hitherto almost unconceivable 3D localization precision attained by MINFLUX is likely to hold the key to an all-optical dynamical structural biology.

biophysics↗

bioRxiv2022

MINSTED nanoscopy enters the Angstrom localization range

We report all-optical, room-temperature localization of fluorophores with precision in the [A]ngstrom range. These precisions are attained in a STED microscope, by encircling the fluorophore with the low-intensity edge of the STED donut beam, while constantly increasing the absolute donut power. Individual fluorophores bound to a DNA strand are localized with{sigma} = 4.7 [A], corresponding to a fraction of the fluorophore size, with only 2,000 detected photons. MINSTED fluorescence nanoscopy with single-digit nanometer resolution is exemplified by imaging nuclear pore complexes and the distribution of nuclear lamin in mammalian cells labeled by transient DNA hybridization. Since our experiments yield a localization precision{sigma} = 2.3 [A], estimated for 10,000 detected photons, we anticipate that MINSTED will open up entirely new areas of application in the study of macromolecular complexes in cells.

biophysics↗