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 sample, superresolution 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.

Direct optical measurement of intramolecular distances with angstrom precision.

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.

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.

Bleaching protection and axial sectioning in fluorescence nanoscopy through two-photon activation at 515 nm.

Activation of caged fluorophores in microscopy has mostly relied on the absorption of a single ultraviolet (UV) photon of ≲400 nm wavelength or on the simultaneous absorption of two near-infrared (NIR) photons >700 nm. Here, we show that two green photons (515 nm) can substitute for a single photon (~260 nm) to activate popular silicon-rhodamine (Si-R) dyes. Activation in the green range eliminates the chromatic aberrations that plague activation by UV or NIR light.

β-Galactosidase- and Photo-Activatable Fluorescent Probes for Protein Labeling and Super-Resolution STED Microscopy in Living Cells.

We report on the synthesis of two fluorescent probes which can be activated by β-Galactosidase (β-Gal) enzymes and/or light. The probes contained 2-nitro-4-oxybenzyl and 3-nitro-4-oxybenzyl fragments, with β-Gal residues linked to C-4. We performed the enzymatic and photoactivation of the probes in a cuvette and compared them, prior to the labeling of fusion protein in live cells with overexpressed β-galactosidase.

Supramolecular Complex of Cucurbit[7]uril with Diketopyrrolopyrole Dye: Fluorescence Boost, Biolabeling and Optical Microscopy.

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 HO, abs./emission max.

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 polymerized microtubules deposited onto glass coverslips.

Photoactivatable Xanthone (PaX) Dyes Enable Quantitative, Dual Color, and Live-Cell MINFLUX Nanoscopy.

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.

MINSTED tracking of single biomolecules.

Here 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 spatiotemporal precision. By updating the position for each detected photon, MINSTED recognizes fluorophore steps of 16 nm within <250 μs using about 13 photons. The power of MINSTED tracking is demonstrated by resolving the stepping of the motor protein kinesin-1 walking on microtubules and switching protofilaments.

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 or fluorescence photons collected through both objective lenses.

Synthesis of Thioxanthone 10,10-Dioxides and Sulfone-Fluoresceins via Pd-Catalyzed Sulfonylative Homocoupling.

Our report describes the facile and scalable preparation of 9-thioxanthen-9-one 10,10-dioxides via Pd-catalyzed sulfonylative homocoupling of the appropriately substituted benzophenones. This transformation provides a straightforward route to previously unreported sulfone-fluoresceins and -fluorones. Several examples of these red fluorescent dyes have been prepared, characterized, and evaluated as live-cell permeant labels compatible with super-resolution fluorescence microscopy with 775 nm stimulated emission depletion.

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 the 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 the off-target signal.

Photoactivatable Carbo- and Silicon-Rhodamines and Their Application in MINFLUX Nanoscopy.

New photoactivatable fluorescent dyes (rhodamine, carbo- and silicon-rhodamines [SiR]) with emission ranging from green to far red have been prepared, and their photophysical properties studied. The photocleavable 2-nitrobenzyloxycarbonyl unit with an alpha-carboxyl group as a branching point and additional functionality was attached to a polycyclic and lipophilic fluorescent dye. The photoactivatable probes having the HaloTagTM amine (O2) ligand bound with a dye core were obtained and applied for live-cell staining in stable cell lines incorporating Vimentin (VIM) or Nuclear Pore Complex Protein NUP96 fused with the HaloTag.

MINFLUX dissects the unimpeded walking of kinesin-1.

We introduce an interferometric MINFLUX microscope that records protein movements with up to 1.7 nanometer per millisecond spatiotemporal precision. Such precision has previously required attaching disproportionately large beads to the protein, but MINFLUX requires the detection of only about 20 photons from an approximately 1-nanometer-sized fluorophore.

Accelerated MINFLUX Nanoscopy, through Spontaneously Fast-Blinking Fluorophores.

The introduction of MINFLUX nanoscopy allows single molecules to be localized with one nanometer precision in as little as one millisecond. However, current applications have so far focused on increasing this precision by optimizing photon collection, rather than minimizing the localization time. Concurrently, commonly used fluorescent switches are specifically designed for stochastic methods (e.

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 tetrazine ligation and self-labeling protein tags (HaloTag, SNAP-tag), and duplexing of an imaging channel with another large Stokes shift dye have been demonstrated.

Calibration of Deformable Mirrors for Open-Loop Control.

Deformable mirrors enable the control of wave fronts for the compensation of aberrations in optical systems and/or for beam scanning. Manufacturers of deformable mirrors typically provide calibration data that encode for the fabrication tolerances among the actuators and mirror segments to support open-loop control with high wave front fidelity and accuracy. We report a calibration method that enables users of the deformable mirrors to measure the response of the mirror itself to validate and improve the calibration data.

MINSTED nanoscopy enters the Ångström localization range.

Super-resolution techniques have achieved localization precisions in the nanometer regime. Here we report all-optical, room temperature localization of fluorophores with precision in the Ångström range. We built on the concept of MINSTED nanoscopy where precision is increased by encircling the fluorophore with the low-intensity central region of a stimulated emission depletion (STED) donut beam while constantly increasing the absolute donut power.

Designing chromatic optical retarder stacks for segmented next-generation easySTED phase plates.

Fluorescence nanoscopy methods based on the RESOLFT principle, such as beam-scanning STED nanoscopy, require the co-alignment of optical beams for molecular state (on/off) switching and fluorescence excitation. The complexity and stability of the beam alignment can be drastically simplified and improved by using a single-mode fibre as the sole light source for all required laser beams. This in turn then requires a chromatic optical element for shaping the off-switching beam into a focal-plane donut while simultaneously leaving the focal intensity distributions at other wavelengths shaped as regular focal spots.

DNA-PAINT MINFLUX nanoscopy.

MINimal fluorescence photon FLUXes (MINFLUX) nanoscopy, providing photon-efficient fluorophore localizations, has brought about three-dimensional resolution at nanometer scales. However, by using an intrinsic on-off switching process for single fluorophore separation, initial MINFLUX implementations have been limited to two color channels. Here we show that MINFLUX can be effectively combined with sequentially multiplexed DNA-based labeling (DNA-PAINT), expanding MINFLUX nanoscopy to multiple molecular targets.

-Cyanorhodamines: cell-permeant, photostable and bathochromically shifted analogues of fluoresceins.

Fluorescein and its analogues have found only limited use in biological imaging because of the poor photostability and cell membrane impermeability of their -unprotected forms. Herein, we report rationally designed -cyanorhodamines as orange- to red-emitting, photostable and cell-permeant fluorescent labels negatively charged at physiological pH values and thus devoid of off-targeting artifacts often observed for cationic fluorophores. In combination with well-established fluorescent labels, self-labelling protein (HaloTag, SNAP-tag) ligands derived from -cyanorhodamines permit up to four-colour confocal and super-resolution STED imaging in living cells.

Supramolecular Complex of Photochromic Diarylethene and Cucurbit[7]uril: Fluorescent Photoswitching System for Biolabeling and Imaging.

Photoswitchable fluorophores─proteins and synthetic dyes─whose emission is reversibly switched on and off upon illumination, are powerful probes for bioimaging, protein tracking, and super-resolution microscopy. Compared to proteins, synthetic dyes are smaller and brighter, but their photostability and the number of achievable switching cycles in aqueous solutions are lower. Inspired by the robust photoswitching system of natural proteins, we designed a supramolecular system based on a fluorescent diarylethene () and cucurbit[7]uril (CB7) (denoted as @CB7).

A general design of caging-group-free photoactivatable fluorophores for live-cell nanoscopy.

The controlled switching of fluorophores between non-fluorescent and fluorescent states is central to every super-resolution fluorescence microscopy (nanoscopy) technique, and the exploration of radically new switching mechanisms remains critical to boosting the performance of established, as well as emerging super-resolution methods. Photoactivatable dyes offer substantial improvements to many of these techniques, but often rely on photolabile protecting groups that limit their applications. Here we describe a general method to transform 3,6-diaminoxanthones into caging-group-free photoactivatable fluorophores.