Microscope specimens are 3-dimensional objects, but the images from conventional light microscopes are flat - they only show two dimensions. Multiple oblique illumination is a novel lighting technique for transmitted light microscopes that produces multiple (i.e. two or more) high definition 3-dimensional images using conventional microscope objective lenses. We have previously described its use in transmitted light. The same optical theory has now been expanded to include reflection microscopy. In the present paper, we describe a new development which will make this approach more widely available. It is a retrofit illumination system that will produce true 3-dimensional images directly through the eyepieces of conventional microscopes.

By seeing z-axis information in real-time and in the context of a specimen's entire thickness, researchers can gain additional, unambiguous information about the interrelationships between structures, whereas critical information about 3-dimensional structures can be obscured, lost or misinterpreted when using 2-dimensional instruments. The importance of accurate z-axis information has popularized methods such as deconvolution and confocal microscopy.

Cytosolic and organellar free Ca2+ concentrations are very dynamic; they are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations.

Green fluorescent protein (GFP) is a spontaneously fluorescent protein from the jelly fish Aequorea victoria. Its cDNA can be concatenated with those encoding many other proteins, and the resulting fusion proteins are usually fluorescent and often preserve the biochemical functions and cellular localizations of the partner proteins. Mutagenesis has produced GFP mutants with shifted wavelengths of excitation or emission that can serve as donors and acceptors for fluorescence resonance energy transfer (FRET).

Our new indicators consist of tandem fusions of a blue- or cyan-emitting mutant of GFP, calmodulin (CaM), the calmodulin-binding peptide Ml3, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the Ml3 domain, increasing the FRET between the flanking GFPs (Fig. 1).

Previously, we showed rapid mitochondrial Ca2+ transients in adult rabbit cardiac myocytes during the contractile cycle. Ruthenium red, an inhibitor of mitochondrial Ca2+ uptake by the Ca2+ uniporter, inhibits mitochondrial Ca2+ transients in adult rabbit cardiac myocytes during electrical stimulation. Here, we extend this finding to show that ruthenium red inhibition is specific for mitochondrial Ca2+ transients and not cytosolic Ca2+ transients.

Ca 2+-tolerant adult rabbit cardiac myocytes were isolated by collagenase and hyaluronidase digestion and loaded in the cold with 2.5 μM Rhod 2-AM for 30 minutes. Subsequently, the cells were incubated in nutrient medium at 37°C for 4-6 hours during which time cytosolic but not mitochondrial Rhod 2 was lost. Subsequently, the myocytes were loaded with 10 μM Fluo 3-AM for 15 minutes at 37°C, conditions that lead to predominantly cytosolic localization of Fluo 3. In sequential scans, the red fluorescence of Rhod 2 and the green fluorescence of Fluo 3 were imaged using a Zeiss LSM 410 laser scanning confocal microscope.

Synthesis and assembly of gap junction proteins into intercellular channels is an important biological problem with direct bearing on embryonic development. In particular, transgenic mice Cx43 gap junction protein knock-outs 1 and mutations in Cx43 sequence in human Viscera Hyper Taxia patients result in severely deformed hearts.

NRK cells are one of the best studied systems for gap junction synthesis, transport and assembly. Gap junctions contain numerous cell-cell channels which are composed of two paired oligomers (called a connexon or hemichannel) each contributed from the plasma membrane of apposing cells. Although biochemical studies have shown indirectly that unpaired Cx43 connexons are in the plasma membrane, direct hemichannel visualization has not been demonstrated. Musil and Goodenough have shown that oligomerization occurs in the trans-Golgi network and the connexon is transported to the plasma membrane.

The use of animation can significantly enhance the visualization of three-dimensional (3D) structures. It can present a focused train of thought, or it can be used to systematically scan through previously unfathomable quantities of data to examine for unknown features and consistencies. In order to establish a modern animation facility requires a variety of technical, psychological and artistic skills, in addition to advanced computer graphics and related equipment. 3D graphics combined with animation has proven to be a very effective tool in storytelling. The dynamic visual image is uniquely suited for thought experiments, simulations and traversing vast quantities of data otherwise incomprehensible to the human mind. Sometimes animation is the only method that allows a clear presentation of complex empirical or theoretical information in many dimensions. Scientific visualization is by its nature an exploratory process. And frequently animations are iteratively refined and polished to enhance comprehensibility for the researchers and their peers.

The structure of tubulin, the major protein component of microtubules, has been obtained by electron crystallography of zinc-induced 2-D crystals. The atomic model of the tubulin dimer, built into a 3.7 Å density map, shows that α- and β-tubulin have basically identical structures. Each monomer is very compact and is formed by two interacting beta sheets surrounded by helices. The structure can be divided into three sequential domains that are functionally distinctive. The N-terminal domain forms a Rossmann fold that binds the nucleotide. Connected to the nucleotide-binding domain by the core helix is a second domain that in β-tubulin contains the binding site of taxol. The C-terminal region is formed by two long helices that constitute most of the interactive, outside surface of the microtubule. The nucleotide-binding and second domains are common with FtsZ, a bacterial homologue of tubulin essential for cell division. The bacterial protein, which lacks the C-terminal helices of tubulin, has generally shorter loops and contains an extra helix at the N-terminus.

Molecular motors belonging to the myosin and kinesin superfamilies utilize ATP to move along their respective F-actin and microtubule tracks. The track-motor complexes have not been amenable to crystallization, so x-ray crystallographic investigations have focused on structure determinations of the individual proteins. Although providing detailed descriptions of the structure of each protein, this approach cannot reveal the geometry of interaction of the proteins nor the conformational changes which occur during the mechanochemical cycle. To obtain this information, we use cryoelectron microscopy and image analysis to calculate three dimensional maps of the track-motor complexes at moderate resolution (15-30A) and combine these data with the high resolution x-ray structures to provide near-atomic models of the working assemblies.

We have so far built models of the rigor (nucleotide-free) and ADP actomyosin complexes. In smooth muscle myosin II (a collaboration with H.L. Sweeney, U. Perm.) and brush border myosin I (BBMI), the motor domain of the myosin head is similar in both biochemical states.

A superfamily of actin crosslinking proteins, the calponin-homology domain (CH) family is implicated in directing the assembly and controlling the organization of the cytoskeleton through a highly conserved actin binding domain (ABD). This domain, composed of a tandem pair of CH motifs, is found in actin crosslinking proteins such as fimbrin, alpha-actinin, spectrin, and dystrophin. In addition to its role in crosslinking actin filaments, a putative homologous ABD in intermediate filament-binding proteins, plectin, dystonin, and BPAGlnl, suggests a possible role in crosslinks between desmosomal and vimentin intermediate filaments and actin. Thus, the CH domain could integrate the functions of the actin and IF cytoskeletons by physically linking these structural systems. We present an atomic model for the complex of the CH domain and actin.

In crosslinking proteins that organize actin into two and three dimensional networks or gels the ABDs lie at the ends of long, flexible molecules.

The acrosomal process is an intracellular quasi-crystalline organelle in the head of the sperm of the horseshoe crab Limulus polyphemus. It consists of 100 - 130 actin-scruin filaments packed together in a pseudo-hexagonal lattice2 and is up to 60 (im long with a diameter of 0.1 μm. Scruin-scruin interactions are responsible for cross-linking the actin filaments together in the bundle. The goal of the current study is to reveal interfilament interactions in the bundle. We have taken tilt series images in the electron microscope3 to reconstruct its three-dimensional structure without assuming helical symmetry.

The acrosomal process was purified as described.34 Bundles were embedded in vitreous ice over holes on a holey carbon film on copper grids. The specimen was kept at -167°C in a JEOL 4000EX electron microscope operating at 400 kV. Straight 6-10 (im long bundles were found using a TV-rate CCD camera in defocused diffraction mode.

The calcium pump (Ca2+-ATPase) from sarcoplasmic reticulum (SR) is a prominent member of the large family of ATP-dependent cation pumps, which include Na+ /K+-ATPase, H+/K+-ATPase from the stomach, H+-ATPase from yeast and Neurospora, and various detoxifying pumps for Cd+, Cu+ and other metals. In muscle, calcium is stored inside the SR and contraction is initiated by regulated release through specific calcium channels; Ca2+ -ATPase is responsible for relaxation by pumping calcium back into the SR lumen. Many techniques (chemical modification, site mutagenesis, reaction kinetics) have been used to correlate Ca2+-ATPase sequence with function, but no high resolution three-dimensional structure of Ca2+-ATPase, or any P-type pump, has yet been determined. In the current work, we have determined the structure from helical crystals at 8 A resolution and thus revealed the alpha-helical architecture of the transmembrane domain. In addition, a specific inhibitor of Ca2+-ATPase, thapsigargin, was used to promote crystallization and we have characterized the structural consequences of its inhibition.

Gap junction membrane channels mediate the electrical and metabolic coupling between cells. The channels are formed by the end-to-end docking of two hemichannels (connexons), each of which is formed by a hexameric cluster of protein subunits (connexins). The principal gap junction protein in the heart, α1 connexin (also designated Cx43), mediates action potential propagation between cells in order to synchronize cardiac contraction.

We recently utilized electron cryo-microscopy and image analysis to examine frozen-hydrated two-dimensional (2D) crystals of a recombinant, truncated α1- connexin (α1Cx263T). The projection map at 7Å resolution revealed that each 30kD connexin subunit has a transmembrane α-helix that lines the aqueous pore and a second α-helix in close contact with the membrane lipids [Nature Struct. Biol. 4: 39-43 (1997)]. The distribution of densities allowed us to propose a model in which the two apposing connexons that form the channel are staggered by ∼30°. Furthermore, apparent non-crystallographic twofold axes predicted that the two apposing connexons adopt identical conformations.

Potassium channels are ubiquitous ion channel proteins which play a crucial role in a broad spectrum of important cell functions. KcsA is a potassium channel found in the bacterium, Streptomyces lividan, and is believed to have only two transmembrane helices.

The KcsA protein has been cloned, overexpressed and purified to homogeneity, and reconstituted with phospholipids to form two-dimensional (2-D) crystals. Crystal samples were embedded in trehalose and examined in a cryo-holder (-170 °C) using a JEOL 4000EX electron microscope operated at 400kV in low dose mode. Images were recorded in spot scan-mode at a magnification of 60,000X, then digitized on a Perkin Elmer PDS flat bed microdensitometer and processed by using the MRC program suite. Processed images were brought to a common phase origin, and the amplitudes and phases from individual images were vectorially combined.

New state-of-the-art analytical equipment is becoming more specialized and more expensive. With the availability of federal funding decreasing over the years, it has become increasingly difficult for University laboratories to maintain and upgrade existing analytical equipment, or purchase new analytical equipment. There are also instances where large industrial laboratories may only need a particular piece of analytical equipment on a part-time basis, or where small companies cannot afford to purchase a particular technique. An obvious solution to these problems is to merge industry and university into a viable working collaboration whereby resources are shared among partners.

The University of Central Florida and Cirent Semiconductor (a joint venture of Lucent Technologies and Cirrus Logic) have entered into such a collaboration with the development of the UCF/Cirent Materials Characterization facility (MCF). The MCF is a shared user facility that is available to (i) UCF and other State University System (SUS) Florida University students, faculty, and staff, and (ii) MCF industrial affiliates and others requiring specialized instrumentation and expertise.

The Shared Research Equipment (SHaRE) User Facility and Program at Oak Ridge National Laboratory (ORNL) provides microanalytical facilities for studies within the materials sciences. Available instrumentation includes advanced analytical electron microscopes, atom probe field ion microscopes, and nanoindentation facilities. Through SHaRE, researchers from U.S. universities, industries, and government laboratories may collaborate with Facility scientists to perform research not possible at their home institutions. International collaborations are also possible. Most SHaRE projects seek correlations at the microscopic or atomic scale between structure and properties in a wide range of metallic, ceramic, and other structural materials. Typical research projects include studies of magnetic materials, advanced alloys, catalysts, semiconductor device materials, high Tc superconductors, and surface-modified polymers. Projects usually involve one or more external researchers visiting the SHaRE Facility for up to three weeks during the fiscal year (October 1 - September 30). Project approval is based upon the scientific excellence and relevance of proposed collaborative research.

A standard Philips CM200FEG electron microscope, without the special Lorentz lens, has been optimized for Lorentz imaging. The necessary field-free sample region is obtained by switching off the objective lens in the free lens mode. The limited range of magnification is compensated for by a post-column Gatan image filter (GIF) which magnifies by a factor of _ 20. Fresnel imaging is performed by defocusing with the diffraction lens. The use of low angle diffraction, in combination with the apertures located at the selected area aperture plane, allow Foucault imaging. The TEM analog of differential phase contrast (DPC) imaging has been implemented. This method makes it possible to obtain quantitave induction maps of the in-plane magnetization. TEM DPC is based on a series of Foucault images, recorded with different incremental beam tilts, which are processed to yield images equivalent to the quadrant signals obtained by the STEM DPC technique.

Five applications of analytical light microscopy to problem-solving in the pharmaceutical industry are described and illustrated by case examples. Problems included are (1) the caking of pharmaceutical products, (2) chemical incompatibility, (3) nucleation and precipitation, (4) recognition of hydrates and polymorphs, and (5) identification and sourcing of contaminant particles.

Considerable effort goes into the sizing of solid pharmaceutical raw materials and products. In many instances, particle size is used to help control the rate and uniformity of dissolution, which in turn affects the kinetics of drug delivery. Caking is a macroscopic description of the change in a sample from a finely-divided powder to a single solid molded aggregate. Caking obviously has a severe impact on handling and dissolution characteristics.

Caking of a powder is almost always caused by moisture through the cyclical dissolution and precipitation on the surfaces of the particles. When exposed to sufficient vapor pressure, often through changes in humidity or temperature,

Several case studies are shown as examples of the results of analyses using various microscopical instruments to characterize the particles in building dusts. Characterizing the particles and identifying possible sources of particles in dusts are often important parts of solving indoor indoor air quality (IAQ) and environmental problems. Samples of the dust particles can be collected by filtering the air with cassette air filters or by gathering the dust that has accumulated on surfaces. Depending on the size and types of particles in the dust either polarized light microscopy (PLM), scanning electron microscopy with x-ray analysis (SEM/EDS), analytical transmission electron microscopy (ATEM) or Fourier transform infrared microspectroscopy (FTIR) may be used solely or in combination. PLM can characterize most of the common dust particle in residential and commercial buildings. SEM/EDS is helpful in identifying metal particles and when used in the backscatter electron mode, a large number of particles can be surveyed and those containing heavy metals such as lead and cadmium can be identified quickly.

Many different microscopic techniques are utilized in the development of new pharmaceuticals. For that reason, pharmaceutical companies have supported microscopy and microscopists even in the face of retrenchments in many other industries. Microscopy has the following three major uses in pharmaceutical development: 1) solid-state characterization of drug substance; 2) particle size analysis; 3) contaminant identification. Microscopy is also an important component of the study of the biological activity of drugs but that subject will not be discussed here.

The analysis of the solid-state properties of new drug candidates is probably the most important and challenging subject of study for pharmaceutical microscopists.1 The primary objective of these studies is identification of the most suitable solid-state form (i.e. polymorph, solvate, hydrate) of the drug substance for development. Polarized light microscopy (PLM), thermal microscopy (TM), infrared microspectroscopy (IR), and Raman microspectroscopy (RM) are all utilized in these studies. Both PLM and TM are used to discover new polymorphs and solvates of the chemical.

A BNL-Grace process has been developed to chemically convert in-situ, the chrysotile fibers of sprayed-on fireproofing products to an unregulated glassy material. The effectiveness of this process has been convincingly demonstrated using Analytical Electron Microscopy and X-Ray Diffraction techniques.

Asbestos minerals were used in fireproofing materials because of their excellent physical properties including fire resistance, high tensile strength, heat and electrical insulation, and resistance to acids and alkali. But in 1975 the Environmental Protection Agency began regulating materials containing > 1% asbestos.

The new in-situ BNL-Grace process, which uses a foamy solution sprayed directly onto asbestos-containing fireproofing chemically digests essentially all the asbestos fibers, transforming them into harmless materials. After treatment, the fireproofing is no longer a regulated material. The process produces essentially no waste.

Our problem was to demonstrate that all of the asbestos was converted and that the remaining materials were no longer regulated. Typical analysis methods use conventional optical and Polarized Light Microscopy (PLM) to measure and observe fibers.

During the past twelve months RJ Lee Group has manually collected and published over one hundred thousand composite SEM images consisting of three components: a low magnification image, a high magnification image, and an x-ray spectrum. In addition, about one and one-half million discrete features have been automatically located and compositionally/morphologically characterized, with about one-fourth of those features then having ancillary images collected automatically based upon characterization criteria. Finally between ten and twenty thousand images have been collected for the production of image montages. The output of this effort has been systematically entered into networked databases where it is accessed for post-processing, analysis, and report generation.