Sophisticated tools are needed to examine the results of cyro-microscopy. As the size and resolution of three dimensional macromolecular structures steadily improve, and the speed at with which they can be generated increases, researchers are finding they are inundated with larger datasets and at the same time are compelled to expediently evaluate these structures in unforeseen ways. Integration of EM data with other types of information is becoming necessary and routine; for example X-ray data, 3D EM reconstructions, and theoretical models, must be evaluated in concert to discount or propose hypothesis. To create such tools, the developer must take into account not only the empirical and theoretical possibilities, but also they must master the human factors and computational limits. During the last five years, the National Center for Macromolecular Imaging (NCMI) has progressed from a remedial 3D visualization capability to a collection of visualization tools allowing researchers to focus on the discovery phase of biological research.

PRD1 is a ds-DNA bacteriophage from the Tectiviridae family with an unusual structural feature: the viral genome is enclosed by a protein-rich membrane, which is in turn enclosed by an external icosahedral protein shell (capsid). Three-dimensional reconstructions from cryo-electron microscopy (cryo-EM) images have revealed the structure of the PRD1 capsid at moderate resolution (28 Å), while X-ray crystallographic studies have recently provided a high resolution (1.85 Å) picture of the major coat protein, P3. We have now combined these results from different imaging methods to obtain a more detailed understanding of the virion organization. The combination has been made in a cyclic process: a preliminary fitting of the atomic structure of P3 to each one of its independent positions in the cryo-EM maps of the capsids provided initial models that could be used to improve the reconstructions; the refined maps then served as a base frame for an optimized fit. This process allows us to study the viral particle structure at “quasi-atomic” resolution.

Human adenovirus (Ad) provides unique model system for studying the multistage events in viral entry. We are applying cryo-electron microscopy and single particle reconstruction methods to study the cell entry pathway of Ad as well as to examine the structure of reengineered Ad vectors. Ad cell entry is mediated by interactions with two different cellular receptors: the fiber receptor, CAR for most Ad serotypes or a 50 kDa receptor for a subset of serotypes including Ad37, for attachment; and the αvβ3 and αvβ5 integrins for internalization. We have previously reconstructed both Ad2 and Adl2 virus particles complexed with a soluble form of αvβ5 integrin. We learned during our comparative cryo-EM study of the Ad2/αvβ5 and Adl2/αvβ5 complexes that the length of the variable RGD loop in the penton base affects the relative height of the integrin density over the penton base.

For several years we have been developing a software application, called Leginon, for the automated control and acquisition of images from a transmission electron microscope. The system has been developed around a Philips CM200 and a Gatan MSC CCD camera. One of the primary motivations in developing this software is to provide for a system that can acquire many hundreds of images under low dose conditions from a specimen embedded in vitreous ice. In order to set up and manage this system we have developed a number of simple interactive graphical tools that enable the user to design, oversee and manage protocols for controlling the microscope and collecting the images. Many of these simple tools have also proved generally useful as stand alone applications.

For several years we have been developing a system for the automated control and acquisition of images from a transmission electron microscope [1,2,3]. The system has been developed around a Philips CM200 equipped with a Gatan cryostage and a Gatan MSC CCD camera. One of the primary motivations in developing this software is to provide for a system that can acquire many hundreds of images over several days of completely unattended operation.

The current implementation of our automated system allows data to be automatically collected from the TEM for more than 36 hours. There is no intervention required from the operator except for one critical exception, refilling the Gatan cryostage. The cryostage, which maintains the temperature of the vitreous ice specimens, must be refilled with liquid nitrogen (LN2) approximately every two hours.

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.