Automated in-chamber specimen coating for serial block-face electron microscopy.

When imaging insulating specimens in a scanning electron microscope, negative charge accumulates locally ('sample charging'). The resulting electric fields distort signal amplitude, focus and image geometry, which can be avoided by coating the specimen with a conductive film prior to introducing it into the microscope chamber. This, however, is incompatible with serial block-face electron microscopy (SBEM), where imaging and surface removal cycles (by diamond knife or focused ion beam) alternate, with the sample remaining in place. Here we show that coating the sample after each cutting cycle with a 1-2 nm metallic film, using an electron beam evaporator that is integrated into the microscope chamber, eliminates charging effects for both backscattered (BSE) and secondary electron (SE) imaging. The reduction in signal-to-noise ratio (SNR) caused by the film is smaller than that caused by the widely used low-vacuum method. Sample surfaces as large as 12 mm across were coated and imaged without charging effects at beam currents as high as 25 nA. The coatings also enabled the use of beam deceleration for non-conducting samples, leading to substantial SNR gains for BSE contrast. We modified and automated the evaporator to enable the acquisition of SBEM stacks, and demonstrated the acquisition of stacks of over 1000 successive cut/coat/image cycles and of stacks using beam deceleration or SE contrast.

Controlling FIB-SBEM slice thickness by monitoring the transmitted ion beam.

Serial block-face electron microscopy with focused ion beam cutting suffers from cutting artefacts caused by changes in the relative position of beam and sample, which are, for example, inevitable when reconditioning the ion gun. The latter has to be done periodically, which limits the continuous stack-acquisition time to several days. Here, we describe a method for controlling the ion-beam position that is based on detecting that part of the ion beam that passes the sample (transmitted beam). We find that the transmitted-beam current decreases monotonically as the beam approaches the sample and can be used to determine the relative position of beam and sample to an accuracy of around one nanometre. By controlling the beam approach using this current as the feedback parameter, it is possible to ion-mill consecutive 5 nm slices without detectable variations in thickness even in the presence of substantial temperature fluctuations and to restart the acquisition of a stack seamlessly. In addition, the use of a silicon junction detector instead of the in-column detector is explored.

In vivo two-photon voltage-sensitive dye imaging reveals top-down control of cortical layers 1 and 2 during wakefulness.

Conventional methods of imaging membrane potential changes have limited spatial resolution, particularly along the axis perpendicular to the cortical surface. The laminar organization of the cortex suggests, however, that the distribution of activity in depth is not uniform. We developed a technique to resolve network activity of different cortical layers in vivo using two-photon microscopy of the voltage-sensitive dye (VSD) ANNINE-6. We imaged spontaneous voltage changes in the barrel field of the somatosensory cortex of head-restrained mice and analyzed their spatiotemporal correlations during anesthesia and wakefulness. EEG recordings always correlated more strongly with VSD signals in layer (L) 2 than in L1. Nearby (<200 mum) cortical areas were correlated with one another during anesthesia. Waking the mouse strongly desynchronized neighboring cortical areas in L1 in the 4- to 10-Hz frequency band. Wakefulness also slightly increased synchrony of neighboring territories in L2 in the 0.5- to 4.0-Hz range. Our observations are consistent with the idea that, in the awake animal, long-range inputs to L1 of the sensory cortex from various cortical and thalamic areas exert top-down control on sensory processing.

Direct measurement of coupling between dendritic spines and shafts.

Characterization of the diffusional and electrotonic coupling of spines to the dendritic shaft is crucial to understanding neuronal integration and synaptic plasticity. Two-photon photobleaching and photorelease of fluorescein dextran were used to generate concentration gradients between spines and shafts in rat CA1 pyramidal neurons. Diffusional reequilibration was monitored with two-photon fluorescence imaging. The time course of reequilibration was exponential, with time constants in the range of 20 to 100 milliseconds, demonstrating chemical compartmentalization on such time scales. These values imply that electrical spine neck resistances are unlikely to exceed 150 megohms and more likely range from 4 to 50 megohms.

Two-photon laser scanning fluorescence microscopy.

Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly photolytic release of caged effector molecules.

A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals.

Two-photon microscopy has enabled anatomical and functional fluorescence imaging in the intact brain of rats. Here, we extend two-photon imaging from anesthetized, head-stabilized to awake, freely moving animals by using a miniaturized head-mounted microscope. Excitation light is conducted to the microscope in a single-mode optical fiber, and images are scanned using vibrations of the fiber tip. Microscope performance was first characterized in the neocortex of anesthetized rats. We readily obtained images of vasculature filled with fluorescently labeled blood and of layer 2/3 pyramidal neurons filled with a calcium indicator. Capillary blood flow and dendritic calcium transients were measured with high time resolution using line scans. In awake, freely moving rats, stable imaging was possible except during sudden head movements.

Calcium imaging of single stereocilia in hair cells: localization of transduction channels at both ends of tip links.

Mechanically gated "transduction" channels in inner ear hair cells are thought to be connected to tip links stretched between adjacent stereocilia. To locate active channels, calcium-green fluorescence in single stereocilia was measured with two-photon laser scanning microscopy. Bundle deflection increased fluorescence in many but not all stereocilia; the increase was blocked by depolarization. The number of stereocilia responding was proportional to the transduction current, consistent with Ca2+ influx through transduction channels. Fluorescence rose first in the tips of stereocilia and then in the bases, in agreement with channel localization at the tips. Some of the shortest stereocilia in a bundle showed a fluorescence increase, as did some of the tallest, indicating that transduction channels can be at either or both ends of tip links.

Coincidence detection in single dendritic spines mediated by calcium release.

Cerebellar long-term depression (LTD) is a calcium-dependent process in which coincident activity of parallel fiber (PF) and climbing fiber (CF) synapses causes a long-lasting decrease in PF synaptic strength onto Purkinje cells. Here we show that pairing CF activation with bursts of PF activity triggers large (>10 microM) calcium signals in Purkinje cell dendrites. When PFs are densely activated, signals span whole dendritic branchlets and are mediated by voltage-dependent calcium entry. When PFs are sparsely activated, however, signals are restricted to single spines and blocked by metabotropic glutamate receptor antagonists. Single-spine signals and sparse-stimulation LTD are also blocked by thapsigargin, indicating that calcium must be released from stores. Single-spine signals and sparse-stimulation LTD are greatest when PF activation precedes the CF activation within 50-200 ms. This timing rule matches the properties of several forms of motor learning, providing a link between behavior and functional properties of cerebellar synaptic plasticity.

In vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons.

Dendritic Ca2+ action potentials in neocortical pyramidal neurons have been characterized in brain slices, but their presence and role in the intact neocortex remain unclear. Here we used two-photon microscopy to demonstrate Ca2+ electrogenesis in apical dendrites of deep-layer pyramidal neurons of rat barrel cortex in vivo. During whisker stimulation, complex spikes recorded intracellularly from distal dendrites and sharp waves in the electrocorticogram were accompanied by large dendritic [Ca2+ ] transients; these also occurred during bursts of action potentials recorded from somata of identified layer 5 neurons. The amplitude of the [Ca 2+] transients was largest proximal to the main bifurcation, where sodium action potentials produced little Ca2+ influx. In some cases, synaptic stimulation evoked [Ca2+] transients without a concomitant action potential burst, suggesting variable coupling between dendrite and soma.

Spread of dendritic excitation in layer 2/3 pyramidal neurons in rat barrel cortex in vivo.

In layer 2/3 pyramidal neurons of barrel cortex in vivo, calcium ion concentration ([Ca2+]) transients in apical dendrites evoked by sodium action potentials are limited to regions close to the soma. To study the mechanisms underlying this restricted pattern of calcium influx, we combined two-photon imaging of dendritic [Ca2+] dynamics with dendritic membrane potential measurements. We found that sodium action potentials attenuated and broadened rapidly with distance from the soma. However, dendrites of layer 2/3 cells were electrically excitable, and direct current injections could evoke large [Ca2+] transients. The restricted pattern of dendritic [Ca2+] transients is therefore due to a failure of sodium action-potential propagation into dendrites. Also, stimulating subcortical activating systems by tail pinch can enhance dendritic [Ca2+] influx induced by a sensory stimulus by increasing cellular excitability, consistent with the importance of these systems in plasticity and learning.

Dendritic processing.

Dendritic processing multiplies the computational power of a single neuron by enabling the processing of inputs in a spatio-temporally differentiated manner. Recently, the development of new and refined optical, electrophysiological and molecular-biological techniques has led to new insights into dendritic function and revealed an astonishing plethora of computational mechanisms.

Imaging calcium dynamics in dendritic spines.

Recent advances in optical imaging technology have enabled the measurement of Ca2+ dynamics in individual synaptic spines with high time resolution. Results from work using this new technology have confirmed the view that individual synaptic spines can act as functional chemical compartments with independent dynamics of second-messenger concentration. In particular, the ability of Ca2+ to directly mediate Hebbian coincidence detection has been confirmed.

Brain lipid peroxidation and hydroxy radical attack following the intravenous infusion of hydrogen peroxide in an infant.

Death following peroxide administration in humans has been reported repeatedly. Hydrogen peroxide, an odorless and clear solution is considered a harmless liquid and is in use for cleaning of superficial wounds. We describe the fatal infusion of this compound by mistake leading to oxygen embolism and, subsequently, to death as a warning for the clinician. Hydrogen peroxide is suggested a major substrate for the in vivo production of the potent oxidizing free radical species "hydroxy radical." No direct evidence for its in vivo production from hydrogen peroxide has been described so far. Using the principle of o-tyrosine determination we studied the formation of the hydroxy radical in the postmortem brain of the infant given intravenous hydrogen peroxide in comparison to postmortem brain samples from five infants. o-Tyrosine is formed by hydroxy radical attack on free and bound phenylalanine and was increased twofold in the brain of the infant given hydrogen peroxide. The significant increase of brain malondialdehyde, a major product and indicator of lipid peroxidation, paralleled the findings of hydroxy radical attack, suggesting that this reactive species has been leading to elevated lipid peroxidation. We propose that the generation of lipid peroxidation and the hydroxy radical from hydrogen peroxide can take place in humans.

Pulsing mercury arc lamps for uncaging and fast imaging.

It is shown that the light intensity from a mercury short arc light bulb can be boosted to about 100 times its steady-state value for a period of about 1 ms by superimposing a current pulse of up to 100 amperes in magnitude and 1-2 ms in duration on a simmer current of 3 amperes. The output spectrum changes in a remarkable way from a line to a broadband distribution. The radiance delivered during a 1-ms pulse is comparable to what can be obtained from a Xe flash bulb. The lack of a high voltage ignition pulse accounts for an extreme reduction of the electrical artifact seen for the pulsed Hg bulb as compared to a Xe flash lamp. Possible applications include the release of caged compounds, high speed imaging, and wavelength ratio-imaging without filter switching.

Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy.

Light scattering by brain tissue and phototoxicity are major obstacles to the use of high-resolution optical imaging and photo-activation ('uncaging') of bioactive compounds from inactive ('caged') precursors in intact and semi-intact nervous systems. Optical methods based on 2-photon excitation promise to reduce these obstacles (Denk, 1994; Denk et al., 1990, 1994). Here we show a range of imaging modes based on 2-photon laser scanning microscopy (TPLSM) as applicable to problems in neuroscience. Fluorescence images were taken of neurons labeled with ion-sensitive and voltage-sensitive dyes in invertebrate ganglia, mammalian brain slices, and from the intact mammalian brain. Scanning photochemical images with whole-cell current detection (Denk, 1994) show how the distribution of neurotransmitter receptors on the surface of specific cells can be mapped. All images show strong optical sectioning and usable images can be obtained at depths greater than 100 microns below the surface of the preparation.

Forward and reverse transduction at the limit of sensitivity studied by correlating electrical and mechanical fluctuations in frog saccular hair cells.

The spontaneous fluctuations of the intracellular voltage and the position of the sensory hairbundle were measured concurrently using intracellular microelectrodes and an optical differential micro interferometer. Magnitude and frequency distribution of the hair bundles' spontaneous motion suggest that it consists mostly of Brownian motion. The electrical noise, however, exceeds the value expected for thermal Johnson noise by several orders of magnitude, and its frequency distribution reflects the transduction tuning properties of the hair cells. Frequently, a strong correlation was observed between the fluctuations of the hair bundle position and the intracellular electrical noise. From the properties of the correlation and from experiments involving mechanical stimulation we conclude that in most cases mechano-electrical transduction of the bundles' Brownian motion causes this correlation. Small signal transduction sensitivities ranged from 18 to 500 microV/nm. Bundle motion that was observed in response to current injection in more than half of the cells suggests the existence of a fast reverse (electro-mechanical) transduction mechanism to be common in these cells. The sensitivities could be as high as 600 pm of bundle deflection per millivolt of membrane potential change. In a significant minority (4 in 44) of cells, all showing excess electrical noise, we found 'non-causal' components of the electro-mechanical correlation, and in two of those cells narrow-band bundle motion in excess of their thermal motion at frequencies coincident with peaks in the intracellular noise was observed.

Shots fired with silencers--a report on four cases and experimental testing.

Four cases of homicide, in which silenced firearms were used, are reported and supplemented by data from experimental investigations regarding wound features, marks, traces, and ballistic behavior. Wound features are largely determined by the construction of the silencer. In one case, even a muzzle imprint was produced by a silenced weapon fired at contact range. In general, silencers are likely to result in a decrease in bullet energy and accelerated energy release in the target (tissue). In terms of wound morphology, silencers produced a reduction in or even a lack of the contact ring (ring of dirt). In close-range and contact wounds, any features indicative of shots fired at close range were missing (such as soot deposit and powder tattooing). It is also worth mentioning that biological matter may get into the silencer in shots fired at contact range.

[Delayed epipleural hematoma as a fatal complication following blunt chest injury]. / Das zweizeitige epipleurale Hämatom als tödliche Komplikation nach stumpfem Thoraxtrauma.

Delayed epipleural haematoma was found on autopsy of a 92-year-old woman after a fall at her home. X-ray performed 3 hours after the blunt thoracic trauma showed fractures of 5 ribs on the right side without any indication of intrathoracic bleeding or masses. Dressing and moving the patient about one day later caused multiple injuries of intercostal arteries and subsequently led to epipleural haematoma and unexpected death. Origin and morphology of epipleural haematoma are discussed with reference to a pertinent report in the literature. Hospitalization, with utmost care in nursing attention, is recommended in the treatment of patients presenting with thoracic trauma.

[Fatal intracranial increase in pressure caused by a colloid cyst of the 3d ventricle not diagnosed intra vitam]. / Tödliche intrakranielle Drucksteigerung durch eine intra vitam nicht diagnostizierte Kolloidzyste des III. Ventrikels.

The case is reported of a 26 year-old man with a colloid cyst of the third ventricle (paraphyseal cyst; neuroepithelial cyst), which gave rise to a fatal increase in intracranial pressure. Despite repeated hospitalisation the diagnosis was not established prior to death. This report serves as a reminder that this rare disorder must be considered in the differential diagnosis of conditions causing increased intracranial pressure in view of the life-saving therapeutic management required to avert a fatal outcome.

Assault and homicide by chop blows: a series of 11 cases.

In a series of robberies, 11 elderly women living alone were attacked by an assailant (HS) by means of chop blows directed primarily at the throat, but also at the head, neck, and thorax. 5 died of their injuries. 1 died immediately after the assault. The other 4 victims survived for between 7-38 days. As a result of the injuries incurred, the women were confused, physically weakened and confined to bed, and eventually died from pneumonia. Their death was thus casually related to the violence sustained. Extensive haematomas of the scalp and cervical soft tissue were present. In 3 cases there were injuries to the laryngeal skeleton and adjacent soft tissue. In 2 cases there were conjunctival haemorrhages. The other 6 attacks were not fatal. The blows led to transient unconsciousness, which often resulted in a state of confusion, sometimes mimicking cerebrovascular accidents (CVA). Of other injuries, craniofacial haematomas were the most common. These cases are reviewed in this paper.