Equations of Charge Distribution in the Environmental Scanning Electron Microscope (ESEM)

In the environmental scanning electron microscope (ESEM), the electron beam together with various signals emanating from the beam-specimen interaction ionize the gaseous medium in the specimen chamber. A detailed derivation of equations describing the charge density and current flow in the system is presented. It is shown that the various causes of ionization operate over distinct regions, which can be separated out by suitable electrode configuration. The electron probe retains a fraction of electrons with the original charge distribution; this is surrounded by a widespread electron skirt, which, in turn, is surrounded by charge created by the secondary electrons, beyond which extends the action of backscattered electrons

Environmental Scanning Electron Microscope: Some Critical Issues

In the environmental scanning electron microscope (ESEM), the gas flow around the main pressure limiting aperture establishes a density gradient through which the electron beam passes. Electron beam losses occur in this transition region and in the uniform gas layer above the specimen surface. In the oligo-scattering regime, the electron distribution consists of a widely scattered fraction of electrons surrounding an intact focussed probe. Secondary electrons are multiplied by means of gaseous ionization and detected both by the ionization current and the accompanying gaseous scintillation. The distribution of secondary electrons is governed by the applied external electric and magnetic fields and by electron diffusion in the gas. Backscattered electrons are detected both by means of the gaseous detection device and by solid scintillating detectors. Uncoated solid detectors offer the lowest signal-to-noise ratio especially under low beam accelerating voltages. The lowest pressure of operation with uncoated detectors has been expanded by the deliberate introduction of a gaseous discharge near the detector. Gaseous scintillation also offers the possibility of low noise detection and signal discrimination. The absorbed specimen current mode is re-examined in the conditions of ESEM. It is found that the current flowing through the specimen is not the contrast forming mechanism: it is all the electric carriers in motion that induce signals on the surrounding electrodes. The electric conductivity of the specimen may affect the contrast only indirectly, i.e., as a secondary, not a primary process. The ESEM can operate under any environment including high and low pressure, low or rough vacuum and high vacuum; it operates at both high and low beam accelerating voltage so that it may be considered as the universal instrument for virtually any application previously accessible or not to the conventional SEM

What environmental transmission electron microscopy measures and how this links to diffusivity: thermodynamics versus kinetics

Environmental or in situ electron microscopy means the observation of material in its native environment, which can be gaseous or liquid, as compared to more traditional post-mortem electron microscopy carried out under (ultra) high vacuum conditions. Experiments can be performed on bulk samples in scanning electron microscopes or on thinned samples in transmission (scanning) electron microscopes. In the latter, the movement, in real time and in situ, of nanoparticles, clusters or even single atoms on the surfaces of thinned material or within a liquid can be observed. It is argued here that due to the changes that a specimen typically undergoes during in situ observation, electron irradiation effects are difficult to evaluate and so thermodynamic parameters, such as activation energies for diffusion and segregation, which are governed by movements of only a minority of atoms in the specimen, cannot be reliably determined because of the potentially high energy transfer by the irradiating electron beam to some atoms in the sample. In order to measure diffusivities reliably, radiation effects and surface diffusion need to be excluded or kept minimal so as not to disturb the measurements, which can be checked by repeating experiments and comparing results as function of time and dose for the same position, at different positions or for different specimen thicknesses. Kinetic measurements of nucleation and growth phenomena, such as Ostwald ripening, are possibly influenced to a far lesser degree by irradiation effects, as a majority of atoms actively participate in these processes and if a small fraction of them will get extra energy from the irradiation process then their influence on the overall kinetics may be rather minor

Acousto-optical Scanning-Based High-Speed 3D Two-Photon Imaging In Vivo.

Recording of the concerted activity of neuronal assemblies and the dendritic and axonal signal integration of downstream neurons pose different challenges, preferably a single recording system should perform both operations. We present a three-dimensional (3D), high-resolution, fast, acousto-optic two-photon microscope with random-access and continuous trajectory scanning modes reaching a cubic millimeter scan range (now over 950 × 950 × 3000 μm3) which can be adapted to imaging different spatial scales. The resolution of the system allows simultaneous functional measurements in many fine neuronal processes, even in dendritic spines within a central core (>290 × 290 × 200 μm3) of the total scanned volume. Furthermore, the PSF size remained sufficiently low (PSFx < 1.9 μm, PSFz < 7.9 μm) to target individual neuronal somata in the whole scanning volume for simultaneous measurement of activity from hundreds of cells. The system contains new design concepts: it allows the acoustic frequency chirps in the deflectors to be adjusted dynamically to compensate for astigmatism and optical errors; it physically separates the z-dimension focusing and lateral scanning functions to optimize the lateral AO scanning range; it involves a custom angular compensation unit to diminish off-axis angular dispersion introduced by the AO deflectors, and it uses a high-NA, wide-field objective and high-bandwidth custom AO deflectors with large apertures. We demonstrate the use of the microscope at different spatial scales by first showing 3D optical recordings of action potential back propagation and dendritic Ca2+ spike forward propagation in long dendritic segments in vitro, at near-microsecond temporal resolution. Second, using the same microscope we show volumetric random-access Ca2+ imaging of spontaneous and visual stimulation-evoked activity from hundreds of cortical neurons in the visual cortex in vivo. The selection of active neurons in a volume that respond to a given stimulus was aided by the real-time data analysis and the 3D interactive visualization accelerated selection of regions of interest

Fabrication of a single sub-micron pore spanning a single crystal (100) diamond membrane and impact on particle translocation

The fabrication of sub-micron pores in single crystal diamond membranes, which span the entirety of the membrane, is described for the first time, and the translocation properties of polymeric particles through the pore investigated. The pores are produced using a combination of laser micromachining to form the membrane and electron beam induced etching to form the pore. Single crystal diamond as the membrane material, has the advantages of chemical stability and durability, does not hydrate and swell, has outstanding electrical properties that facilitate fast, low noise current-time measurements and is optically transparent for combined optical-conductance sensing. The resulting pores are characterized individually using both conductance measurements, employing a microcapillary electrochemical setup, and electron microscopy. Proof-of-concept experiments to sense charged polystyrene particles as they are electrophoretically driven through a single diamond pore are performed, and the impact of this new pore material on particle translocation is explored. These findings reveal the potential of diamond as a platform for pore-based sensing technologies and pave the way for the fabrication of single nanopores which span the entirety of a diamond membrane