Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release

The molecular organization of presynaptic active zones during calcium influx-triggered neurotransmitter release is the focus of intense investigation. The Drosophila coiled-coil domain protein Bruchpilot (BRP) was observed in donut-shaped structures centered at active zones of neuromuscular synapses by using subdiffraction resolution STED (stimulated emission depletion) fluorescence microscopy. At brp mutant active zones, electron-dense projections (T-bars) were entirely lost, Ca2+ channels were reduced in density, evoked vesicle release was depressed, and short-term plasticity was altered. BRP-like proteins seem to establish proximity between Ca2+ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity.     

Full three-dimensional photonic bandgap crystals at near-infrared wavelengths

An artificial crystal structure has been fabricated exhibiting a full three-dimensional photonic bandgap effect at optical communication wavelengths. The photonic crystal was constructed by stacking 0.7-micrometer period semiconductor stripes with the accuracy of 30 nanometers by advanced wafer-fusion technique. A bandgap effect of more than 40 decibels (which corresponds to 99.99% reflection) was successfully achieved. The result encourages us to create an ultra-small optical integrated circuit including a three-dimensional photonic crystal waveguide with a sharp bend.     

Red-Emitting Semiconductor Quantum Dot Lasers

Visible-stimulated emission in a semiconductor quantum dot (QD) laser structure has been demonstrated. Red-emitting, self-assembled QDs of highly strained InAlAs have been grown by molecular beam epitaxy on a GaAs substrate. Carriers injected electrically from the doped regions of a separate confinement heterostructure thermalized efficiently into the zero-dimensional QD states, and stimulated emission at approximately 707 nanometers was observed at 77 kelvin with a threshold current of 175 milliamperes for a 60-micrometer by 400-micrometer broad area laser. An external efficiency of approximately 8.5 percent at low temperature and a peak power greater than 200 milliwatts demonstrate the good size distribution and high gain in these high-quality QDs.     

Nanoscopy in a living mouse brain

We demonstrated superresolution optical microscopy in a living higher animal. Stimulated emission depletion (STED) fluorescence nanoscopy reveals neurons in the cerebral cortex of a mouse with <70-nanometer resolution. Dendritic spines and their subtle changes can be observed at their relevant scales over extended periods of time.     

Influence of synaptic vesicle position on release probability and exocytotic fusion mode

Neurotransmission depends on movements of transmitter-laden synaptic vesicles, but accurate, nanometer-scale monitoring of vesicle dynamics in presynaptic terminals has remained elusive. Here, we report three-dimensional, real-time tracking of quantum dot-loaded single synaptic vesicles with an accuracy of 20 to 30 nanometers, less than a vesicle diameter. Determination of the time, position, and mode of fusion, aided by trypan blue quenching of Qdot fluorescence, revealed that vesicles starting close to their ultimate fusion sites tended to fuse earlier than those positioned farther away. The mode of fusion depended on the prior motion of vesicles, with long-dwelling vesicles preferring kiss-and-run rather than full-collapse fusion. Kiss-and-run fusion events were concentrated near the center of the synapse, whereas full-collapse fusion events were broadly spread.     

Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy

Recent advances in far-field fluorescence microscopy have led to substantial improvements in image resolution, achieving a near-molecular resolution of 20 to 30 nanometers in the two lateral dimensions. Three-dimensional (3D) nanoscale-resolution imaging, however, remains a challenge. We demonstrated 3D stochastic optical reconstruction microscopy (STORM) by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photoswitchable probes enables high-precision 3D localization of each probe, and thus the construction of a 3D image, without scanning the sample. Using this approach, we achieved an image resolution of 20 to 30 nanometers in the lateral dimensions and 50 to 60 nanometers in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.     

High-Resolution Imaging by Fourier Transform X-ray Holography

Fourier transform x-ray holography has been used to image gold test objects with submicrometer structure, resolving features as small as 60 nanometers. The hologram-recording instrument uses coherent 3.4-nanometer radiation from the soft x-ray undulator beamline X1A at the National Synchrotron Light Source. The specimen to be imaged is placed near the first-order focal spot produced by a Fresnel zone plate; the other orders, chiefly the zeroth, illuminate the specimen. The wave scattered by the specimen interferes with the spherical reference wave from the focal spot, forming a hologram with fringes of low spatial frequency. The hologram is recorded in digital form by a charge-coupled device camera, and the specimen image is obtained by numerical reconstruction.     

Spectroscopic observations of Mercury's surface reflectance during MESSENGER's first Mercury flyby

During MESSENGER's first flyby of Mercury, the Mercury Atmospheric and Surface Composition Spectrometer made simultaneous mid-ultraviolet to near-infrared (wavelengths of 200 to 1300 nanometers) reflectance observations of the surface. An ultraviolet absorption (<280 nanometers) suggests that the ferrous oxide (Fe2+) content of silicates in average surface material is low (less than 2 to 3 weight percent). This result is supported by the lack of a detectable 1-micrometer Fe2+ absorption band in high-spatial-resolution spectra of mature surface materials as well as immature crater ejecta, which suggests that the ferrous iron content may be low both on the surface and at depth. Differences in absorption features and slope among the spectra are evidence for variations in composition and regolith maturation of Mercury's surface.     

Magnetic vortex core observation in circular dots of permalloy

Spin structures of nanoscale magnetic dots are the subject of increasing scientific effort, as the confinement of spins imposed by the geometrical restrictions makes these structures comparable to some internal characteristic length scales of the magnet. For a vortex (a ferromagnetic dot with a curling magnetic structure), a spot of perpendicular magnetization has been theoretically predicted to exist at the center of the vortex. Experimental evidence for this magnetization spot is provided by magnetic force microscopy imaging of circular dots of permalloy (Ni(80)Fe(20)) 0.3 to 1 micrometer in diameter and 50 nanometers thick.     

Preparation of photonic crystals made of air spheres in titania

Three-dimensional crystals of air spheres in titania (TiO2) with radii between 120 and 1000 nanometers were made by filling the voids in artificial opals by precipitation from a liquid-phase chemical reaction and subsequently removing the original opal material by calcination. These macroporous materials are a new class of photonic band gap crystals for the optical spectrum. Scanning electron microscopy, Raman spectroscopy, and optical microscopy confirm the quality of the samples, and optical reflectivity demonstrates that the crystals are strongly photonic and near that needed to exhibit band gap behavior.     

Observations of the liquid-crystal analog of the abrikosov phase

Freeze-fracture transmission electron micrographs of the smectic A(*) phase confirm the twist grain boundary model of Renn and Lubensky. The fracture surface has an undulating structure with a 0.5-micrometer helical pitch parallel to 4.1-nanometer smectic layers. The layers are disrupted by a lattice of screw dislocations oriented normal to the helical axis. Optical diffraction shows that rotation of smectic blocks occurs in discrete steps of about 17 degrees ; hence, the screw dislocations are 14 to 15 nanometers apart and the grain boundaries are 24 nanometers apart. These observations show that the SmA(*) phase is the liquid-crystal analog of the Abrikosov phase in superconductors.     

Combined analog and action potential coding in hippocampal mossy fibers

In the mammalian cortex, it is generally assumed that the output information of neurons is encoded in the number and the timing of action potentials. Here, we show, by using direct patchclamp recordings from presynaptic hippocampal mossy fiber boutons, that axons transmit analog signals in addition to action potentials. Excitatory presynaptic potentials result from subthreshold dendritic synaptic inputs, which propagate several hundreds of micrometers along the axon and modulate action potential-evoked transmitter release at the mossy fiber-CA3 synapse. This combined analog and action potential coding represents an additional mechanism for information transmission in a major hippocampal pathway.     

Action-potential modulation during axonal conduction

Once initiated near the soma, an action potential (AP) is thought to propagate autoregeneratively and distribute uniformly over axonal arbors. We challenge this classic view by showing that APs are subject to waveform modulation while they travel down axons. Using fluorescent patch-clamp pipettes, we recorded APs from axon branches of hippocampal CA3 pyramidal neurons ex vivo. The waveforms of axonal APs increased in width in response to the local application of glutamate and an adenosine A(1) receptor antagonist to the axon shafts, but not to other unrelated axon branches. Uncaging of calcium in periaxonal astrocytes caused AP broadening through ionotropic glutamate receptor activation. The broadened APs triggered larger calcium elevations in presynaptic boutons and facilitated synaptic transmission to postsynaptic neurons. This local AP modification may enable axonal computation through the geometry of axon wiring.     

High-resolution scanning x-ray diffraction microscopy

Coherent diffractive imaging (CDI) and scanning transmission x-ray microscopy (STXM) are two popular microscopy techniques that have evolved quite independently. CDI promises to reach resolutions below 10 nanometers, but the reconstruction procedures put stringent requirements on data quality and sample preparation. In contrast, STXM features straightforward data analysis, but its resolution is limited by the spot size on the specimen. We demonstrate a ptychographic imaging method that bridges the gap between CDI and STXM by measuring complete diffraction patterns at each point of a STXM scan. The high penetration power of x-rays in combination with the high spatial resolution will allow investigation of a wide range of complex mesoscopic life and material science specimens, such as embedded semiconductor devices or cellular networks.     

Target cell-dependent normalization of transmitter release at neocortical synapses

The efficacy and short-term modification of neocortical synaptic connections vary with the type of target neuron. We investigated presynaptic Ca2+ and release probability at single synaptic contacts between pairs of neurons in layer 2/3 of the rat neocortex. The amplitude of Ca2+ signals in boutons of pyramids contacting bitufted or multipolar interneurons or other pyramids was dependent on the target cell type. Optical quantal analysis at single synaptic contacts suggested that release probabilities are also target cell-specific. Both the Ca2+ signal and the release probability of different boutons of a pyramid contacting the same target cell varied little. We propose that the mechanisms that regulate the functional properties of boutons of a pyramid normalize the presynaptic Ca2+ influx and release probability for all those boutons that innervate the same target cell.     

A carbonate-rich, hydrated, interplanetary dust particle: possible residue from protostellar clouds

Transmission electron microscopy of a hydrated interplanetary dust particle (IDP) indicates that it contains abundant magnesium-iron carbonates, primarily breunnerite and magnesian siderite. This IDP displays a strong absorption band at 6.8 micrometers in its infrared spectrum, similar to that in certain protostellar spectra. The carbonates probably account for the 6.8-micrometer band in the IDP spectrum, suggesting that carbonate also may occur in interstellar dust and be the source of the controversial 6.8-micrometer feature from the protostellar spectra.     

Patterned condensation figures as optical diffraction gratings

Heterogeneous, patterned surfaces comprising well-defined hydrophobic and hydrophilic regions and having micrometer-scale periodicities were prepared by patterning the adsorption of omega-functionalized alkanethiolates in self-assembled monolayers (SAMs) on gold. Condensation of water on such surfaces resulted in drops that followed the patterns in the SAMs. These patterned condensation figures (CFs) acted as optical diffraction gratings for reflected (or transmitted) light from a helium-neon laser (wavelength of 632.8 nanometers). Under an atmosphere of constant relative humidity, the development of the condensation figure was monitored quantitatively, as the temperature of the surface was lowered, by following the change in intensity of a first-order diffraction spot. This experimental technique may be useful in the development of new types of optical sensors that respond to their environment by changing the reflectivity of patterned regions and for studying phenomena such as drop nucleation, contact angle hysteresis, and spontaneous dewetting and break-up of thin liquid films.     

Growth of diamond from atomic hydrogen and a supersonic free jet of methyl radicals

The growth of small ( approximately 10-micrometer) diamond particles (on 0.1-or 0.25-micrometer seed crystals) using an effusive glow discharge nozzle for H.and a separate supersonic pyrolysis jet for .CH(3) is reported. Laser micro-Raman, scanning electron microscopy, and x-ray photoelectron spectroscopy data are presented as evidence that well-crystallized diamond is indeed formed. Resonant multiphoton ionization spectroscopy is used as a diagnostic for the gas-phase chemistry indicating that the radical sources are clean and quantitative and that there is no detectable interconversion of .CH(3) to C(2)H(2) under the conditions of the experiment. Diamond growth is found at substrate temperatures greater than or equal to 650 degrees C with no marked increase in the rate of growth up to 850 degrees C. Acetylene does not give good quality diamond under similar conditions.     

Glutamate, the dominant excitatory transmitter in neuroendocrine regulation

Glutamate has been found to play an unexpectedly important role in neuroendocrine regulation in the hypothalamus, as revealed in converging experiments with ultrastructural immunocytochemistry, optical physiology with a calcium-sensitive dye, and intracellular electrical recording. There were large amounts of glutamate in boutons making synaptic contact with neuroendocrine neurons in the arcuate, paraventricular, and supraoptic nuclei. Almost all medial hypothalamic neurons responded to glutamate and to the glutamate agonists quisqualate and kainate with a consistent increase in intracellular calcium. In all magnocellular and parvocellular neurons of the paraventricular and arcuate nuclei tested, the non-NMDA (non-N-methyl-D-aspartate) glutamate antagonist CNQX (cyano-2,3-dihydroxy-7-nitroquinoxaline) reduced electrically stimulated and spontaneous excitatory postsynaptic potentials, suggesting that the endogenous neurotransmitter is an excitatory amino acid acting primarily on non-NMDA receptors. These results indicate that glutamate plays a major, widespread role in the control of neuroendocrine neurons.     

Equivalent effects of snake PLA2 neurotoxins and lysophospholipid-fatty acid mixtures

Snake presynaptic phospholipase A2 neurotoxins (SPANs) paralyze the neuromuscular junction (NMJ). Upon intoxication, the NMJ enlarges and has a reduced content of synaptic vesicles, and primary neuronal cultures show synaptic swelling with surface exposure of the lumenal domain of the synaptic vesicle protein synaptotagmin I. Concomitantly, these neurotoxins induce exocytosis of neurotransmitters. We found that an equimolar mixture of lysophospholipids and fatty acids closely mimics all of the biological effects of SPANs. These results draw attention to the possible role of local lipid changes in synaptic vesicle release and provide new tools for the study of exocytosis.