Electron diffraction patterns and images of three-dimensional (3D) objects are limited by the range of specimen tilt angles. This range is commonly limited by the specimen holder, but for angles >60°, the slab-like nature of most specimens is itself a fundamental limitation. At 60° the beam path is twice the thickness of the untilted specimen, at 70° it is three times, and at 90° it is infinite. For angles > ± 60°, the information is severely degraded or unattainable This causes serious problems for electron crystallography, and for electron microscopic tomography. For the former, it can result in series termination errors in the computation of electrostatic potential maps from the structure factor amplitudes, and for the latter, it results in reconstructions with anisotropic resolution. To overcome these limitations, we are developing specimen stages and preparation procedures based on a “cylindrical” geometry i.e. all dimensions transverse to the tilt axis are a few micrometers or less.
Confocal laser scanning microscopy (CLSM) has been used to correlate morphology and membrane physiology in cultured neurons, providing a model system for studying physiologic and pathologic conditions. Ion channels are studied by patch-clamp methods as a function of receptor stimulation and toxic excitatory amino acids, including those implicated in Alzheimer’s dementia. Glial cells are often closely associated with the neurons, and are difficult to detect in living cultures due to the relative sizes of glia and neurons (5-20 μm versus 125 μm), compounded with the fact that they are thick phase objects. Groups of glia can also be confused with neurons. Thus it is difficult to select appropriate cells and/or cell regions for patch-clamping. We are correlating physiology and conventional light microscopy with CLSM to determine the role of glia, and neuron surface geometry on the ability to establish Gigaohm membrane-micropipette seals. Morphology of the system as observed by CLSM is presented here.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.