Super-resolution expansion microscopy in plant roots.

Super-resolution methods provide far better spatial resolution than the optical diffraction limit of about half the wavelength of light (∼200-300 nm). Nevertheless, they have yet to attain widespread use in plants, largely due to plants' challenging optical properties. Expansion microscopy improves effective resolution by isotropically increasing the physical distances between sample structures while preserving relative spatial arrangements and clearing the sample.

Human hippocampal CA3 uses specific functional connectivity rules for efficient associative memory.

Our brain has remarkable computational power, generating sophisticated behaviors, storing memories over an individual's lifetime, and producing higher cognitive functions. However, little of our neuroscience knowledge covers the human brain. Is this organ truly unique, or is it a scaled version of the extensively studied rodent brain? Combining multicellular patch-clamp recording with expansion-based superresolution microscopy and full-scale modeling, we determined the cellular and microcircuit properties of the human hippocampal CA3 region, a fundamental circuit for memory storage.

Chiral and nematic phases of flexible active filaments.

The emergence of large-scale order in self-organized systems relies on local interactions between individual components. During bacterial cell division, FtsZ-a prokaryotic homologue of the eukaryotic protein tubulin-polymerizes into treadmilling filaments that further organize into a cytoskeletal ring. In vitro, FtsZ filaments can form dynamic chiral assemblies.

Imaging brain tissue architecture across millimeter to nanometer scales.

Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanometer synaptic scales in diverse chemically fixed brain preparations, including rodent and human.

Dense 4D nanoscale reconstruction of living brain tissue.

Three-dimensional (3D) reconstruction of living brain tissue down to an individual synapse level would create opportunities for decoding the dynamics and structure-function relationships of the brain's complex and dense information processing network; however, this has been hindered by insufficient 3D resolution, inadequate signal-to-noise ratio and prohibitive light burden in optical imaging, whereas electron microscopy is inherently static. Here we solved these challenges by developing an integrated optical/machine-learning technology, LIONESS (live information-optimized nanoscopy enabling saturated segmentation). This leverages optical modifications to stimulated emission depletion microscopy in comprehensively, extracellularly labeled tissue and previous information on sample structure via machine learning to simultaneously achieve isotropic super-resolution, high signal-to-noise ratio and compatibility with living tissue.

A direct excitatory projection from entorhinal layer 6b neurons to the hippocampus contributes to spatial coding and memory.

The mammalian hippocampal formation (HF) plays a key role in several higher brain functions, such as spatial coding, learning and memory. Its simple circuit architecture is often viewed as a trisynaptic loop, processing input originating from the superficial layers of the entorhinal cortex (EC) and sending it back to its deeper layers. Here, we show that excitatory neurons in layer 6b of the mouse EC project to all sub-regions comprising the HF and receive input from the CA1, thalamus and claustrum.

CHD8 haploinsufficiency links autism to transient alterations in excitatory and inhibitory trajectories.

Mutations in the chromodomain helicase DNA-binding 8 (CHD8) gene are a frequent cause of autism spectrum disorder (ASD). While its phenotypic spectrum often encompasses macrocephaly, implicating cortical abnormalities, how CHD8 haploinsufficiency affects neurodevelopmental is unclear. Here, employing human cerebral organoids, we find that CHD8 haploinsufficiency disrupted neurodevelopmental trajectories with an accelerated and delayed generation of, respectively, inhibitory and excitatory neurons that yields, at days 60 and 120, symmetrically opposite expansions in their proportions.

Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development.

De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 (CUL3) lead to autism spectrum disorder (ASD). In mouse, constitutive Cul3 haploinsufficiency leads to motor coordination deficits as well as ASD-relevant social and cognitive impairments. However, induction of Cul3 haploinsufficiency later in life does not lead to ASD-relevant behaviors, pointing to an important role of Cul3 during a critical developmental window.

Advantages of Acute Brain Slices Prepared at Physiological Temperature in the Characterization of Synaptic Functions.

Acute brain slice preparation is a powerful experimental model for investigating the characteristics of synaptic function in the brain. Although brain tissue is usually cut at ice-cold temperature (CT) to facilitate slicing and avoid neuronal damage, exposure to CT causes molecular and architectural changes of synapses. To address these issues, we investigated ultrastructural and electrophysiological features of synapses in mouse acute cerebellar slices prepared at ice-cold and physiological temperature (PT).

Strategies to maximize performance in STimulated Emission Depletion (STED) nanoscopy of biological specimens.

Super-resolution fluorescence microscopy has become an important catalyst for discovery in the life sciences. In STimulated Emission Depletion (STED) microscopy, a pattern of light drives fluorophores from a signal-emitting on-state to a non-signalling off-state. Only emitters residing in a sub-diffraction volume around an intensity minimum are allowed to fluoresce, rendering them distinguishable from the nearby, but dark fluorophores.

A practical guide to optimization in X10 expansion microscopy.

Expansion microscopy is a relatively new approach to super-resolution imaging that uses expandable hydrogels to isotropically increase the physical distance between fluorophores in biological samples such as cell cultures or tissue slices. The classic gel recipe results in an expansion factor of ~4×, with a resolution of 60-80 nm. We have recently developed X10 microscopy, which uses a gel that achieves an expansion factor of ~10×, with a resolution of ~25 nm.

A Fast and Simple Contact Printing Approach to Generate 2D Protein Nanopatterns.

Protein micropatterning has become an important tool for many biomedical applications as well as in academic research. Current techniques that allow to reduce the feature size of patterns below 1 μm are, however, often costly and require sophisticated equipment. We present here a straightforward and convenient method to generate highly condensed nanopatterns of proteins without the need for clean room facilities or expensive equipment.

Optical control of L-type Ca channels using a diltiazem photoswitch.

L-type Ca channels (LTCCs) play a crucial role in excitation-contraction coupling and release of hormones from secretory cells. They are targets of antihypertensive and antiarrhythmic drugs such as diltiazem. Here, we present a photoswitchable diltiazem, FHU-779, which can be used to reversibly block endogenous LTCCs by light.

Novel reversibly switchable fluorescent proteins for RESOLFT and STED nanoscopy engineered from the bacterial photoreceptor YtvA.

The reversibly switchable fluorescent proteins (RSFPs) commonly used for RESOLFT nanoscopy have been developed from fluorescent proteins of the GFP superfamily. These proteins are bright, but exhibit several drawbacks such as relatively large size, oxygen-dependence, sensitivity to low pH, and limited switching speed. Therefore, RSFPs from other origins with improved properties need to be explored.

Multicolour Multilevel STED nanoscopy of Actin/Spectrin Organization at Synapses.

Superresolution fluorescence microscopy of multiple fluorophores still requires development. Here we present simultaneous three-colour stimulated emission depletion (STED) nanoscopy relying on a single STED beam at 620 nm. Toggling the STED beam between two or more power levels ("multilevelSTED") optimizes resolution and contrast in all colour channels, which are intrinsically co-aligned and well separated.

Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super-Resolution STED Microscopy in Living Cells.

A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500-630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 μm solutions of the dye-ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure-property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one- and two-color images of living cells with an optical resolution of 40-60 nm.

Coordinate-targeted and coordinate-stochastic super-resolution microscopy with the reversibly switchable fluorescent protein Dreiklang.

Diffraction-unlimited far-field super-resolution fluorescence (nanoscopy) methods typically rely on transiently transferring fluorophores between two states, whereby this transfer is usually laid out as a switch. However, depending on whether this is induced in a spatially controlled manner using a pattern of light (coordinate-targeted) or stochastically on a single-molecule basis, specific requirements on the fluorophores are imposed. Therefore, the fluorophores are usually utilized just for one class of methods only.

Inducing transport in a dissipation-free lattice with super Bloch oscillations.

Particles in a perfect lattice potential perform Bloch oscillations when subject to a constant force, leading to localization and preventing conductivity. For a weakly interacting Bose-Einstein condensate of Cs atoms, we observe giant center-of-mass oscillations in position space with a displacement across hundreds of lattice sites when we add a periodic modulation to the force near the Bloch frequency. We study the dependence of these "super" Bloch oscillations on lattice depth, modulation amplitude, and modulation frequency and show that they provide a means to induce linear transport in a dissipation-free lattice.

Pinning quantum phase transition for a Luttinger liquid of strongly interacting bosons.

Quantum many-body systems can have phase transitions even at zero temperature; fluctuations arising from Heisenberg's uncertainty principle, as opposed to thermal effects, drive the system from one phase to another. Typically, during the transition the relative strength of two competing terms in the system's Hamiltonian changes across a finite critical value. A well-known example is the Mott-Hubbard quantum phase transition from a superfluid to an insulating phase, which has been observed for weakly interacting bosonic atomic gases.

Confinement-induced resonances in low-dimensional quantum systems.

We report on the observation of confinement-induced resonances in strongly interacting quantum-gas systems with tunable interactions for one- and two-dimensional geometry. Atom-atom scattering is substantially modified when the s-wave scattering length approaches the length scale associated with the tight transversal confinement, leading to characteristic loss and heating signatures. Upon introducing an anisotropy for the transversal confinement we observe a splitting of the confinement-induced resonance.

Precision molecular spectroscopy for ground state transfer of molecular quantum gases.

One possibility for the creation of ultracold, high phase space density quantum gases of molecules in the rovibronic ground state relies on first associating weakly-bound molecules from quantum-degenerate atomic gases on a Feshbach resonance and then transferring the molecules via several steps of coherent two-photon stimulated Raman adiabatic passage (STIRAP) into the rovibronic ground state. Here, in ultracold samples of Cs2 Feshbach molecules produced out of ultracold samples of Cs atoms, we observe several optical transitions to deeply-bound rovibrational levels of the excited 0(u)+ molecular potentials with high resolution. At least one of these transitions, although rather weak, allows efficient STIRAP transfer into the deeply-bound vibrational level (see text for symbols)v = 73 > of the singlet X1 sigma(g)+ ground state potential, as recently demonstrated (J.

Realization of an excited, strongly correlated quantum gas phase.

Ultracold atomic physics offers myriad possibilities to study strongly correlated many-body systems in lower dimensions. Typically, only ground-state phases are accessible. Using a tunable quantum gas of bosonic cesium atoms, we realized and controlled in one-dimensional geometry a highly excited quantum phase that is stabilized in the presence of attractive interactions by maintaining and strengthening quantum correlations across a confinement-induced resonance.

Quantum gas of deeply bound ground state molecules.

Molecular cooling techniques face the hurdle of dissipating translational as well as internal energy in the presence of a rich electronic, vibrational, and rotational energy spectrum. In our experiment, we create a translationally ultracold, dense quantum gas of molecules bound by more than 1000 wave numbers in the electronic ground state. Specifically, we stimulate with 80% efficiency, a two-photon transfer of molecules associated on a Feshbach resonance from a Bose-Einstein condensate of cesium atoms.