Mouse Heterochromatin Adopts Digital Compaction States without Showing Hallmarks of HP1-Driven Liquid-Liquid Phase Separation.

The formation of silenced and condensed heterochromatin foci involves enrichment of heterochromatin protein 1 (HP1). HP1 can bridge chromatin segments and form liquid droplets, but the biophysical principles underlying heterochromatin compartmentalization in the cell nucleus are elusive. Here, we assess mechanistically relevant features of pericentric heterochromatin compaction in mouse fibroblasts. We find that (1) HP1 has only a weak capacity to form liquid droplets in living cells; (2) the size, global accessibility, and compaction of heterochromatin foci are independent of HP1; (3) heterochromatin foci lack a separated liquid HP1 pool; and (4) heterochromatin compaction can toggle between two "digital" states depending on the presence of a strong transcriptional activator. These findings indicate that heterochromatin foci resemble collapsed polymer globules that are percolated with the same nucleoplasmic liquid as the surrounding euchromatin, which has implications for our understanding of chromatin compartmentalization and its functional consequences.

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. 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.

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, 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.

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. Here we describe novel designs of such so-called 'easySTED phase plates' and provide a rationale how to find the desired spectral signature for combinations of multiple wavelengths.

STED nanoscopy of the centrosome linker reveals a CEP68-organized, periodic rootletin network anchored to a C-Nap1 ring at centrioles.

The centrosome linker proteins C-Nap1, rootletin, and CEP68 connect the two centrosomes of a cell during interphase into one microtubule-organizing center. This coupling is important for cell migration, cilia formation, and timing of mitotic spindle formation. Very little is known about the structure of the centrosome linker. Here, we used stimulated emission depletion (STED) microscopy to show that each C-Nap1 ring at the proximal end of the two centrioles organizes a rootletin ring and, in addition, multiple rootletin/CEP68 fibers. Rootletin/CEP68 fibers originating from the two centrosomes form a web-like, interdigitating network, explaining the flexible nature of the centrosome linker. The rootletin/CEP68 filaments are repetitive and highly ordered. Staggered rootletin molecules (N-to-N and C-to-C) within the filaments are 75 nm apart. Rootletin binds CEP68 via its C-terminal spectrin repeat-containing region in 75-nm intervals. The N-to-C distance of two rootletin molecules is ∼35 to 40 nm, leading to an estimated minimal rootletin length of ∼110 nm. CEP68 is important in forming rootletin filaments that branch off centrioles and to modulate the thickness of rootletin fibers. Thus, the centrosome linker consists of a vast network of repeating rootletin units with C-Nap1 as ring organizer and CEP68 as filament modulator.

Robust nanoscopy of a synaptic protein in living mice by organic-fluorophore labeling.

Extending superresolution fluorescence microscopy to living animals has remained a challenging frontier ever since the first demonstration of STED (stimulated emission depletion) nanoscopy in the mouse visual cortex. The use of fluorescent proteins (FPs) in in vivo STED analyses has been limiting available fluorescence photon budgets and attainable image contrasts, in particular for far-red FPs. This has so far precluded the definition of subtle details in protein arrangements at sufficient signal-to-noise ratio. Furthermore, imaging with longer wavelengths holds promise for reducing photostress. Here, we demonstrate that a strategy based on enzymatic self-labeling of the HaloTag fusion protein by high-performance synthetic fluorophore labels provides a robust avenue to superior in vivo analysis with STED nanoscopy in the far-red spectral range. We illustrate our approach by mapping the nanoscale distributions of the abundant scaffolding protein PSD95 at the postsynaptic membrane of excitatory synapses in living mice. With silicon-rhodamine as the reporter fluorophore, we present imaging with high contrast and low background down to ∼70-nm lateral resolution in the visual cortex at ≤25-µm depth. This approach allowed us to identify and characterize the diversity of PSD95 scaffolds in vivo. Besides small round/ovoid shapes, a substantial fraction of scaffolds exhibited a much more complex spatial organization. This highly inhomogeneous, spatially extended PSD95 distribution within the disk-like postsynaptic density, featuring intricate perforations, has not been highlighted in cell- or tissue-culture experiments. Importantly, covisualization of the corresponding spine morphologies enabled us to contextualize the diverse PSD95 patterns within synapses of different orientations and sizes.

Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores.

Bax is a key regulator of apoptosis that, under cell stress, accumulates at mitochondria, where it oligomerizes to mediate the permeabilization of the mitochondrial outer membrane leading to cytochrome c release and cell death. However, the underlying mechanism behind Bax function remains poorly understood. Here, we studied the spatial organization of Bax in apoptotic cells using dual-color single-molecule localization-based super-resolution microscopy. We show that active Bax clustered into a broad distribution of distinct architectures, including full rings, as well as linear and arc-shaped oligomeric assemblies that localized in discrete foci along mitochondria. Remarkably, both rings and arcs assemblies of Bax perforated the membrane, as revealed by atomic force microscopy in lipid bilayers. Our data identify the supramolecular organization of Bax during apoptosis and support a molecular mechanism in which Bax fully or partially delineates pores of different sizes to permeabilize the mitochondrial outer membrane.

Breaking the diffraction limit of light-sheet fluorescence microscopy by RESOLFT.

We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent molecular state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biological specimens with low light dose and axial resolution far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5-12-fold compared with their conventional diffraction-limited LS analogs.

Ultrafast, temporally stochastic STED nanoscopy of millisecond dynamics.

Electro-optical scanning (>1,000 frames/s) with pixel dwell times on the order of the lifetime of the fluorescent molecular state renders stimulated emission depletion (STED) nanoscopy temporally stochastic. Photon detection from a molecule occurs stochastically in one of several scanning frames, and the spatial origin of the photon is known with subdiffraction precision. Images are built up by binning consecutive frames, making the time resolution freely adjustable. We demonstrated nanoscopy of vesicle motions in living Drosophila larvae and the cellular uptake of viral particles with 5- to 10-ms temporal resolution.

Hydroxylated Fluorescent Dyes for Live-Cell Labeling: Synthesis, Spectra and Super-Resolution STED.

Hydroxylated rhodamines, carbopyronines, silico- and germanorhodamines with absorption maxima in the range of 530-640 nm were prepared and applied in specific labeling of living cells. The direct and high-yielding entry to germa- and silaxanthones tolerates the presence of protected heteroatoms and may be considered for the syntheses of various sila- and germafluoresceins, as well as -rhodols. Application in stimulated emission depletion (STED) fluorescence microscopy revealed a resolution of 50-75 nm in one- and two-color imaging of vimentin-HaloTag fused protein and native tubulin. The established structure-property relationships allow for prediction of the spectral properties and the positions of spirolactone/zwitterion equilibria for the new analogues of rhodamines, carbo-, silico-, and germanorhodamines using simple additive schemes.

2000-fold parallelized dual-color STED fluorescence nanoscopy.

Stimulated Emission Depletion (STED) nanoscopy enables multi-color fluorescence imaging at the nanometer scale. Its typical single-point scanning implementation can lead to long acquisition times. In order to unleash the full spatiotemporal resolution potential of STED nanoscopy, parallelized scanning is mandatory. Here we present a dual-color STED nanoscope utilizing two orthogonally crossed standing light waves as a fluorescence switch-off pattern, and providing a resolving power down to 30 nm. We demonstrate the imaging capabilities in a biological context for immunostained vimentin fibers in a circular field of view of 20 µm diameter at 2000-fold parallelization (i.e. 2000 "intensity minima"). The technical feasibility of massively parallelizing STED without significant compromises in resolution heralds video-rate STED nanoscopy of large fields of view, pending the availability of suitable high-speed detectors.

isoSTED nanoscopy with intrinsic beam alignment.

Despite the need for isotropic optical resolution in a growing number of applications, the majority of super-resolution fluorescence microscopy setups still do not attain an axial resolution comparable to that in the lateral dimensions. Three-dimensional (3D) nanoscopy implementations that employ only a single objective lens typically feature a trade-off between axial and lateral resolution. 4Pi arrangements, in which the sample is illuminated coherently through two opposing lenses, have proven their potential for rendering the resolution isotropic. However, instrument complexity due to a large number of alignment parameters has so far thwarted the dissemination of this approach. Here, we present a 4Pi-STED setup combination, also called isoSTED nanoscope, where the STED and excitation beams are intrinsically co-aligned. A highly robust and convenient 4Pi cavity allows easy handling without the need for readjustments during imaging experiments.

Sharper low-power STED nanoscopy by time gating.

Applying pulsed excitation together with time-gated detection improves the fluorescence on-off contrast in continuous-wave stimulated emission depletion (CW-STED) microscopy, thus revealing finer details in fixed and living cells using moderate light intensities. This method also enables super-resolution fluorescence correlation spectroscopy with CW-STED beams, as demonstrated by quantifying the dynamics of labeled lipid molecules in the plasma membrane of living cells.

Reversible chemical reactions for single-color multiplexing microscopy.

Recent developments in biology demand an increasing number of simultaneously imaged structures with standard fluorescence microscopy. However, the number of multiplexed channels is limited for most multiplexing modalities, such as spectral multiplexing or fluorescence-lifetime imaging. We propose extending the number of imaging channels by using chemical reactions, controlling the emissive state of fluorescent dyes. As proof of concept, we reversibly switch a fluorescent copper sensor to enable successive imaging of two different structures in the same spectral channel. We also show that this chemical multiplexing is orthogonal to existing methods. By using two different dyes, we combine chemical with spectral multiplexing for the simultaneous imaging of four different structures with only two spectrally different channels. We characterize and discuss the approach and provide perspectives for extending imaging modalities in stimulated emission depletion microscopy, for which spectral multiplexing is technically demanding.

Distinct subsets of Syt-IV/BDNF vesicles are sorted to axons versus dendrites and recruited to synapses by activity.

BDNF plays a critical role in the regulation of synaptic strength and is essential for long-term potentiation, a phenomenon that underlies learning and memory. However, whether BDNF acts in a diffuse manner or is targeted to specific neuronal subcompartments or synaptic sites to affect circuit function remains unknown. Here, using photoactivation of BDNF or syt-IV (a regulator of exocytosis present on BDNF-containing vesicles) in transfected rat hippocampal neurons, we discovered that distinct subsets of BDNF vesicles are targeted to axons versus dendrites and are not shared between these compartments. Moreover, syt-IV- and BDNF-harboring vesicles are recruited to both presynaptic and postsynaptic sites in response to increased neuronal activity. Finally, using syt-IV knockout mouse neurons, we found that syt-IV is necessary for both presynaptic and postsynaptic scaling of synaptic strength in response to changes in network activity. These findings demonstrate that BDNF-containing vesicles can be targeted to specific sites in neurons and suggest that syt-IV-regulated BDNF secretion is subject to spatial control to regulate synaptic function in a site-specific manner.

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. Therefore, we were able to study the stepping of the motor protein kinesin-1 on microtubules at up to physiological adenosine-5'-triphosphate (ATP) concentrations. We uncovered rotations of the stalk and the heads of load-free kinesin during stepping and showed that ATP is taken up with a single head bound to the microtubule and that ATP hydrolysis occurs when both heads are bound. Our results show that MINFLUX quantifies (sub)millisecond conformational changes of proteins with minimal disturbance.

Three-dimensional organization of promyelocytic leukemia nuclear bodies.

Promyelocytic leukemia nuclear bodies (PML-NBs) are mobile subnuclear organelles formed by PML and Sp100 protein. They have been reported to have a role in transcription, DNA replication and repair, telomere lengthening, cell cycle control and tumor suppression. We have conducted high-resolution 4Pi fluorescence laser-scanning microscopy studies complemented with correlative electron microscopy and investigations of the accessibility of the PML-NB subcompartment. During interphase PML-NBs adopt a spherical organization characterized by the assembly of PML and Sp100 proteins into patches within a 50- to 100-nm-thick shell. This spherical shell of PML and Sp100 imposes little constraint to the exchange of components between the PML-NB interior and the nucleoplasm. Post-translational SUMO modifications, telomere repeats and heterochromatin protein 1 were found to localize in characteristic patterns with respect to PML and Sp100. From our findings, we derived a model that explains how the three-dimensional organization of PML-NBs serves to concentrate different biological activities while allowing for an efficient exchange of components.

Quantum dot blueing and blinking enables fluorescence nanoscopy.

We demonstrate superresolution fluorescence imaging of cells using bioconjugated CdSe/ZnS quantum dot markers. Fluorescence blueing of quantum dot cores facilitates separation of blinking markers residing closer than the diffraction barrier. The high number of successively emitted photons enables ground state depletion microscopy followed by individual marker return with a resolving power of the size of a single dot (∼12 nm). Nanoscale imaging is feasible with a simple webcam.

Molecular orientation affects localization accuracy in superresolution far-field fluorescence microscopy.

We investigate the cooperative effect of molecular tilt and defocus on fluorophore localization by centroid calculation in far-field superresolution microscopy based on stochastic single molecule switching. If tilt angle and defocus are unknown, the localization contains systematic errors up to about ±125 nm. When imaging rotation-impaired fluorophores of unknown random orientation, the average localization accuracy in three-dimensional samples is typically limited to about ±32 nm, restricting the attainable resolution accordingly.