Modern field emission SEMs fitted with in-lens or near-lens (W.D. = l-3mm.) stages and above-lens SE detectors are capable of imaging 1-5 nm features at high voltage (15-30kV) and 5-10 nm features at low voltage (l-5kV). Ultrathin fine-grain refractory metal coatings were shown to optimize higher-solution (HR) secondary electron imaging of light element specimens especially biological samples. Ambient temperature dried specimens and frozen-hydrated cryo-specimens coated with continuous 1nm-thick metal films and staged in-lens have revealed biologically significant features of molecular dimension in tandem HRSEM and scanning transmission (STEM) modes. Although positive and negative stains have proved useful for general contrast enhancement of TEM specimens these stains alter many of our new genetically synthesized block protein copolymers. Therefore we have turned to non-evaporated metal coatings for contrast enhancement of these specimens in the TEM.

Macromolecular structure can be solved by x-ray crystallography to atomic resolution provided that the molecule can be crystallized, that the crystals diffract x-rays to high resolution, and that the phases of the diffracted x-rays can be determined. Though the resolution of single particle imaging by electron microscopy is lower than that of x-ray diffraction by crystals, electron microscopy can directly image a large molecular weight range of macromolecules in a non-crystalline environment, and provide the basis for the three-dimensional reconstruction of these structures. To investigate combining structural information from x-ray crystallography and electron microscopy for unknown structures, we have imaged a small protein of known structure (1), the 35 kDa human complement protein fragment C3d, in a scanning transmission electron microscope (STEM). The intention is to eventually combine the knowledge of electron densities and molecular boundaries from electron microscopy to assist in phase determination in x-ray crystallography.

Plant organelles, including the dominant chloroplasts, migrate intracellularly on cytoplasmic strands (Fig. 1A-D). The chloroplasts in the leaf cells orient and redistribute in response to light to ensure maximum photosynthetic productivity. Their orderly distribution is also essential for proper transmission of organelle genome during cell proliferation. The movement and positioning of chloroplasts have been suggested to be mediated by the actin and tubulin-based cytoskeleton in green algae and higher plants. However, the actin structures controlling these processes have not been clearly delineated because of the difficulty in preserving and detecting the fine actin filaments in plant cells using conventional fixation methods and currently available antibodies.

We investigated the role of the actin cytoskeleton in the regulation of chloroplast movement and positioning by studying: 1) the structural relationships of microfilaments and chloroplasts in leaf cells of Arabidopsis; and 2) effects of an anti-actin drug, Latrunculin B (LAT-B), on intracellular distribution of chloroplasts.

All microscopy is limited by the quality of the specimen under study. Three-dimensional (3-D) visualization of antigen localization using the electron microscope (EM) is particularly challenging due to the need to maintain the activity of some epitopes while preserving cellular ultrastructure. We have used rapid freezing to immobilize all cellular constituents almost instantaneously. Freeze-substitution of the frozen samples was used to stabilize the specimen and to accomplish low-temperature dehydration, minimizing perturbation of cellular structure. We have found that high pressure freezing, double jet freezing and plunge freezing are all useful for achieving high quality structural preservation for some cell types or for particular applications. For immunolocalization, we have had most success freeze-substituting into acetone containing 0.2% glutaraldehyde and 0.1 % uranyl acetate. We have utilized low-temperature acrylic embedding resins, Lowicryl HM20 and LRGold, to further maintain structure and decrease protein insolubility. Both of these resins have proven suitable for cutting serial thin sections.

The preservation of S. cerevisiae ultrastructure is not a trivial task, because the cell wall serves as a diffusion barrier during all steps of preparation. In addition, a high density of proteins in the cytoplasm and in the nucleus renders the visualization of cellular details difficult.

High pressure freezing in combination with freeze substitution has proven to be an advantageous technique for the preservation of the yeast mitotic spindle (1). However, the composition of the freeze substitution “cocktail” is crucial, when immunolabeling of antigens in the sample is intended. A high concentration of crosslinking agents might improve ultrastructural preservation of the sample, but will possibly destroy the antigenicity of the target proteins. In contrast, under conditions of high labeling efficiency, the ultrastructural detail may be lost. We were interested in a method, which gives a good ultrastructural preservation of the mitotic spindle and maintains the antigenicity of proteins associated with the spindle.

Immunocytochemistry describes methods for visualization of specific intracellular molecules within cells. These methods use high affinity markers to specifically bind to target molecules in tissues or cells. For electron microscopy, the sites where these markers are bound are visualized using electron opaque probes. Successful immunocytochemistry depends upon immobilization of target molecules, retention of subcellular morphology, and upon total accessibility of affinity marker to the target molecules.

Accessibility to extracellular molecules is not usually a problem, but for intracellular molecules, labeling protocols must be able to preserve subcellular morphology and allow the affinity markers to gain access to their target molecules. Currently, the best way to gain access to the inside of cells for electron microscopy (TEM) is to cut thin sections through them. In this way, morphology is retained and affinity markers can be applied to the cut face of the cells.

Cryopreservation has many advantages over routine chemical fixation. Two of these are the almost spontaneous arrest of biological activity and the retention of material that normally would be lost with chemical fixation and dehydration. In the current study 2 systems were evaluated. Hyaluronic acid (HA) was covalently bound to 1.4nm nanogold® and applied to astrocytoma cell cultures and the uptake monitored over time using both cryopreparative methods and conventional chemical fixation. The second system evaluated was the detection of liposomes containing CFTR covalently bound to 1.4nm gold in the airways of mice from a phenotype of an inbred congenic strain of CFTR-knockout mice that spontaneously develop lung disease.

In the HA uptake experiment, astrocytoma cell cultures were treated with HA gold for either 5, 10 or 20 minutes. At the appropriate time point, cells were either plunge frozen in liquid ethane or fixed with 2.5% glutaraldehyde in phosphate buffer.

Water is the most abundant and most important molecule in the hydrosphere, outer lithosphere and the biosphere of our planet. It is also the most abundant and energetically the least expensive building block of living material, forms an integral parts of natural inorganic matrices such as soil and is a constituent of many synthetic organic materials such as paints and polymers. Paradoxically, water does not exist naturally, in the pure state. Water, when converted to the solid state, can provide the perfect matrix in which we may observe the structure and study the in situ chemistry of hydrated samples. We will consider the nature of this solid matrix, and its constituent components in a range of sample, and show how it may be formed, manipulated, examined and analysed. In the short amount of time and space available, one can do little more than highlight the main features of the subject.

Poor specimen preparation techniques affect all subsequent EM imaging operations, whether simple descriptions or sophisticated computer reconstruction and analysis. Therefore, EM researchers continue to put effort into developing methods that will preserve the fine structure of the cell as close to the native state as possible. Much attention has been paid to the primary or initial fixation because this is probably the singlemost important step in EM processing. Evidence from a variety of sources. shows that cryofixation is more likely to give good preservation than conventional chemical fixation for most cell types. Among the choices for cryofixation, high pressure freezing is probably the best for most samples, even those that are small enough to be frozen by other means.

Dehydration is another key point in processing where high resolution structure can be lost or distorted, though it has received relatively little attention compared to primary fixation.

Cryo-electron tomography offers a means of obtaining the 3-D ultrastructure of specimens that have not been chemically fixed or stained. The technique is still under development, but it has already been applied to several biological specimens including prokaryotic cells, centrioles, sperm axonemes, mitochondria, spindle-pole bodies, lipid vesicles, isolated skeletal-muscle triad junctions, chromatin fragments, actin bundles, and macromolecules.

Electron tomography itself is a nearly mature technology. It is most often used with conventional plastic sections 100- 1000nm thick. The specimen is imaged in the TEM, using a tilt stage to record a series of projection images over a large angular range. Using image-processing techniques, the tilt images are back-projected to form a reconstructed volume of the specimen. The reconstruction is a 3-D array of pixels or volume elements (voxels), and can be sliced in any direction to form a series of 2-D images.

The value of a Cryo SEM system, attached to a conventional SEM, has already been demonstrated in the biological, pharmaceutical, oil, paint, plastics and food industry. Specimens are first rapidly frozen to below -160°C, transferred under vacuum to a cryo preparation chamber, fractured and examined at low KV (uncoated ) in the SEM. The sample is then usually etched, sputter coated and finally examined at 5-15KV. On conventional microscopes, successful cryo images can be obtained at 30,000 - 50,000X magnification at high KV.

With the invention of field emission gun technology, much higher resolution is now achievable at a lower KV. This allows higher resolution information to be visualised from the surface of the sample. To allow this successful resolution, with the low atomic number samples mentioned above, we have to put down a fine grain, artefact free coating of high atomic number material to prevent charging and help reduce beam penetration.

Introduction

Cryo Ultramicrotomy gains increasing importance for immunocyto- and immunohisto- chemistry, element analysis, morphological studies and other investigations.

Major advances in immunocyto-chemistry and the sectioning of frozen hydrated specimens have been realized with the appropriate instrumentation and tools (1,2).

In immuno-electron microscopy it has been found that a considerable reduction of structural damage in tissue and cells can be obtained with a modified sectioning and pick-up method using sucrose/methyl cellulose solution (3, 4).

In recent years various scientists have tried to understand cryo sectioning and the problems related to the cry-osectioning process (5, 6, 7, 8).

A promising attempt to eliminate one of the major artefacts in ultramicrotomy, that of compression", was made with the development of the oscillating diamond knife (9).

In polymer research cryo ultramicrotomy is a well established technique.

However, only little information is available about the the cryo sectioning process for the following reasons: Much cryo sectioning is performed in polymer companies.

Latexes are dispersions of homopolymers and copolymers, usually in water. Uses of these latexes are many such as protective coatings and adhesives. In order to form a continuous film the polymer must have film forming properties such as a low glass transition temperature (Tg). Latexes are being designed such as one polymer in the core of the particle and a shell of another polymer or perhaps a series of shell layers. Microscopy has become a powerful tool in the examination of the morphology of the latex particles. Because of the use of low Tg polymers, sample preparation and examination by electron microscopy at temperatures above the Tg of the polymer causes the particles to become distorted and no longer representative of their true morphology. Low temperature methods therefore have become crucial in the field of latex microscopy.

Labeling specific tissues, cells, cell components, or individual molecular species is often critical to correlative microscopic studies. In the case of larger structures such as tissues or cells it is often possible to use fluoresecent labels or cytologic/cytochemical based stains which are observable via photon based imaging systems. Specific locations on cells and tissues identified by the labeling or staining procedure can be delineated and then can be viewed via electron or force based instrumentation. This is provided preparative methodologies can be designed which: 1. are compatible with observation of the particular sample by the various desired imaging modes and 2. do not compromise structure viewed at the higher levels of resolution provided by electron and force based imaging technologies. This approach is particularly valuable where simultaneous multiple labeling is required since color differences permit discrimination of differing fluorescent and/or chromatic stains.

With the advent of new ways to image biological material, to tag biological molecules, and to prepare samples, much progress has been made in understanding cellular and subcellular function without resorting to classical electron microscopy. Newer ways to image include the use of microscopies such as: differential interference, video, confocal, near field scanning optical, and magnetic resonance imaging. The use of tags such as cell-permeant organelle-specific markers and green fluorescent protein has further increased our ability to vision subcellular events in living cells, providing us with functional correlations of gene activity and cellular function in a number of biological systems. Techniques such as high pressure freezing coupled with freeze substitution have expanded the range of tissues and organisms that can be optimally preserved and are changing our understanding of cellular fine structure. However, even when electron microscopy is mandated, 1-2 angstrom resolution is rarely indicated except for investigations of molecular structure.

Since it was introduced by Faulk and Taylor in 1971, colloidal gold (cAu) has become the predominant marker used for EM cell labeling. Double labeling, using cAu of two distinct sizes, was pioneered a decade later. However, as interest continues to grow of how the myriad proteins, enzymes, receptors, and other macromolecules function with respect to one another in the cellular environment, this method has proven largely inadequate for high resolution, multiple labeling because each cAu label must be of sufficiently distinct size to prevent size overlap and confusion between different labels. As the particle size increases, so does steric hindrance between different labels competing for binding sites, while the spatial resolution of the labeling and overall labeling efficiency decrease. In order to address these concerns and surmount the limitations of current labeling methodologies, we have developed colloidal particles of uniform size from various metals including Ag, Pt, Pd, Rh, Ru, Ni, and Cu, in addition to Au.

Energy filtered transmission electron microscopy has the potential to provide high resolution, spatially resolved, atomic and chemical information. However, aberrations generated by the electron spectrometer blur the energy resolution and limit the atomic or molecular distributions that can be studied. Energy absorptions corresponding to the visible light range fall below an energy loss of 5 eV. The selection of electrons that have lost an amount of energy corresponding to chromophore absorption by the sample thus requires a spectrometer with a high energy resolution over the full image plane. A corrected prism-mirror-prism filter that has a resolution of 1.1 eV, sufficient to select these low energy loss electrons, was developed and installed by us in a Zeiss EM902. Its imaging capability was verified for a number of different chromophores. The chromophore currently under study is that of the green fluorescent protein (GFP).

Gold clusters are gold compounds with a core of gold atoms and organic groups covalently bound to the surface gold atoms. An example is undecagold, Au11(P(C6H5)3)7, whose structure was solved by x-ray crystallography using 3-dimensional crystals. These differ from colloidal gold, which are suspensions of metal particles, usually formed by metal ion reduction; although the particles may be approximately the same size, they vary due to the statistical process of formation. Gold clusters are compounds with a definite formula, and should all be perfectly identical. However, it is known that there is a family of stable gold cluster compounds, such as Au6, Au11, Au13, AU55, Au67, etc. In a given preparation of gold clusters, there is usually some mixture of these, thus leading to some size variation. Methods such as gel filtration column chromatography and ultrafiltration can be used to separate most of these species, so that relatively pure preparations may be achieved.

Immunocytochemistry generally refers to methods directed toward obtaining information on the in situ distribution of antigens in cells and tissues. Immunocytochemical methods can be applied at either the light or electron microscope levels, or both in concert. The detection of antibody recognition of an antigen (i.e., localization of an antigen) relies upon a reporter system. At the light microscope level, enzymes (e.g., horseradish peroxidase) or fluorochromes are the most widely used reporters in immunocytochemistry. At the electron microscope level, particulate probes (e.g., colloidal gold) are the most widely used reporters. However, enzymes and even fluorochromes can be used at the EM level.

In this abstract, we discuss our use of gold cluster immunoprobes as the reporter system in both light and electron microscope level immuncytochemistry. These gold cluster immunoprobes, are commercially known as NanogoldTM (NG). These probes are very small with a diameter of 1.4-nm.

Immunocytochemistry provides unique information concerning the spatial and temporal distribution of biomolecules in cells and tissues. While techniques associated with immunocytochemical labeling are well developed, there are always improvements that can be achieved. In this abstract, a modification to immunocytochemical methodology that increases the labeling efficiency for certain antigens is described. This modification includes incubation of the sample with sodium dodecyl sulfate (SDS) prior to application of the primary antibody.

Tubulin in microtubules of human neutrophils has been extremely difficult to analyze with antibodies to α-tubulin or β-tubulin using immunocytochemical methods. Using an immunofluorescence assay, we have analyzed several preparative procedures in order to improve the labeling efficiency of these microtubules using anti-tubulin antibodies. We find that treatment of chemically fixed neutrophils with the chaotropic agent SDS improves labeling efficiency for neutrophil microtubules in the immunofluorescence assay.