Since the beginning of the 1980s, cryo-electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3-d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo-electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3-d reconstruction

A newly designed high‐pressure freezing machine for cryofixation was established and tested (Leica EMPACT), based on ideas originally proposed by Moor & Riehle in 1968. The new machine, essentially an improved version of our prototype, pressurizes the sample to 2000 bar in a small container (using methylcyclohexane as hydraulic fluid) and at the same time cools the outer surface of the container with a jet of liquid nitrogen. The advantage of this approach is that the machine uses little liquid nitrogen and can be built small and light. The machine is able to vitrify and freeze well a variety of specimens, for example, plant leaves, yeast cells, liver or nerve tissue (more samples are shown at: http://www.ana.unibe.ch/empact). Cooling efficiency is the same as in the traditional machines that use liquid nitrogen to pressurize and simultaneously cool the sample.

Summary For more than 20 years, high‐pressure freezing has been used to cryofix bulk biological specimens and reports are available in which the potential and limits of this method have been evaluated mostly based on morphological criteria. By evaluating the presence or absence of segregation patterns, it was postulated that biological samples of up to 600 μm in thickness could be vitrified by high‐pressure freezing. The cooling rates necessary to achieve this result under high‐pressure conditions were estimated to be of the order of several hundred degrees kelvin per second. Recent results suggest that the thickness of biological samples which can be vitrified may be much less than previously believed. It was the aim of this study to explore the potential and limits of high‐pressure freezing using theoretical and experimental methods. A new high‐pressure freezing apparatus (Leiċa EM HPF), which can generate higher cooling rates at the sample surface than previously possible, was used. Using bovine articular cartilage as a model tissue system, we were able to vitrify 150‐μm‐thick tissue samples. Vitrification was proven by subjecting frozen‐hydrated cryosections to electron diffraction analysis and was found to be dependent on the proteoglycan concentration and water content of the cartilage. Only the lower radical zone (with a high proteoglycan concentration and a low water content compared to the other zones) could be fully vitrified. Our theoretical calculations indicated that applied surface cooling rates in excess of 5000 K/s can be propagated into specimen centres only if samples are relatively thin (<200 μm). These calculations, taken together with our zone‐dependent attainment of vitrification in 150‐μm‐thick cartilage samples, suggest that the critical cooling rates necessary to achieve vitrification of biological samples under high‐pressure freezing conditions are significantly higher (1000–100 000 K/s) than previously proposed, but are reduced by about a factor of 100 when compared to cooling rates necessary to vitrify biological samples at ambient pressure.

Transmission electron microscopy has provided most of what is known about the ultrastructural organization of tissues, cells, and organelles. Due to tremendous advances in crystallography and magnetic resonance imaging, almost any protein can now be modeled at atomic resolution. To fully understand the workings of biological "nanomachines" it is necessary to obtain images of intact macromolecular assemblies in situ. Although the resolution power of electron microscopes is on the atomic scale, in biological samples artifacts introduced by aldehyde Wxation, dehydration and staining, but also section thickness reduces it to some nanometers. CryoWxation by high pressure freezing circumvents many of the artifacts since it allows vitrifying biological samples of about 200 m in thickness and immobilizes complex macromolecular assemblies in their native state in situ. To exploit the perfect structural preservation of frozen hydrated sections, sophisticated instruments are needed, e.g., high voltage electron microscopes equipped with precise goniometers that work at low temperature and digital cameras of high sensitivity and pixel number. With them, it is possible to generate high resolution tomograms, i.e., 3D views of subcellular structures. This review describes theory and applications of the high pressure cryoWxation methodology and compares its results with those of conventional procedures.Moreover, recent Wndings will be discussed showing that molecular models of proteins can be Wtted into depicted organellar ultrastructure of images of frozen hydrated sections. High pressure freezing of tissue is the base which may lead to precise models of macromolecular assemblies in situ, and thus to a better understanding of the function of complex cellular structures.

|Biomacromolecules 2011, 12, 3666-3673 Biomacromolecules ARTICLE when generalizing our findings to other cellulose nanowhisker types, because surface chemistry, surface charges, as well as the length, aspect ratio, and stiffness vary considerably and are likely to influence interactions with mammalian cells.

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Matrilin-2 is a member of the protein superfamily with von Willebrand factor type A-like modules. Mouse matrilin-2 cDNA fragments were expressed in 293-EBNA cells, and the protein was purified, characterized, and used to immunize rabbits. The affinity-purified antiserum detects matrilin-2 in dense and loose connective tissue structures, subepithelial connective tissue of the skin and digestive tract, specialized cartilages, and blood vessel walls. In situ hybridization of 35 S-labeled riboprobes localizes the matrilin-2 mRNA to fibroblasts of dermis, tendon, ligaments, perichondrium, and periosteum; connective tissue elements in the heart; smooth muscle cells; and epithelia and loose connective tissue cells of the alimentary canal and respiratory tract. RNA blot hybridization and immunoblotting revealed both matrilin-2 mRNA and protein in cultures of a variety of cell types, confirming the tissue distribution. Alternative splicing affects a module unique for matrilin-2 in all of the above RNA sources. SDS-polyacrylamide gel electrophoresis and electron microscopy reveals matrilin-2 from tissue extracts and cell line cultures as a mixture of mono-, di-, tri-, and tetramers. Matrilin-2 is substituted with N-linked oligosaccharides but not with glycosaminoglycans. Because of other, yet unidentified, cell-type dependent posttranslational modifications, the monomer is heterogeneous in size. Immunofluorescence showed that matrilin-2 functions by forming an extracellular, filamentous network.Extracellular matrix provides physical support to the cells, delineates pathways for cell migration during differentiation and tissue regeneration, and provides the necessary milieu for the normal cell metabolism and development. Collagen fibers and proteoglycan aggregates provide the structural basis for matrix architecture. Noncollagenous proteins modulate the organization of these elements, form collagen-associated or independent networks, and are parts of cell migratory pathways.The matrix molecules share homologous modules, protein domains of common evolutionary origin, but a great functional variability of the homologous modules in different proteins has been observed. The recently discovered matrilins (for a review, see Ref. 1) are typical modular proteins belonging to the superfamily with von Willebrand factor type A-like (vWFA) 1 modules. Members of the matrilin family are found in a wide variety of extracellular matrices. Matrilin-1, formerly called cartilage matrix protein, and matrilin-3 (2, 3) are abundant in cartilage, while matrilin-2 (4) and matrilin-4 (5) show a broader tissue distribution. Thus, all forms of connective tissue appear to contain at least one form of matrilin, indicating a general and important function for this protein family.Matrilin-2 was found to contain the same protein modules in the same order as matrilin-1 (4). The precursor protein in mouse is 956 amino acids long and consists of a putative signal peptide, two vWFA domains connected by 10 epidermal growth factor-like modules, a potential oli...

Background and PurposeAlthough the relevance of understanding spinal kinematics during functional activities in patients with complex spinal deformities is undisputed among researchers and clinicians, evidence using skin marker-based motion capture systems is still limited to a handful of studies, mostly conducted on healthy subjects and using non-validated marker configurations. The current study therefore aimed to explore the validity of a previously developed enhanced trunk marker set for the static measurement of spinal curvature angles in patients with main thoracic adolescent idiopathic scoliosis. In addition, the impact of inaccurate marker placement on curvature angle calculation was investigated.MethodsTen patients (Cobb angle: 44.4±17.7 degrees) were equipped with radio-opaque markers on selected spinous processes and underwent a standard biplanar radiographic examination. Subsequently, radio-opaque markers were replaced with retro-reflective markers and the patients were measured statically using a Vicon motion capture system. Thoracolumbar / lumbar and thoracic curvature angles in the sagittal and frontal planes were calculated based on the centers of area of the vertebral bodies and radio-opaque markers as well as the three-dimensional position of the retro-reflective markers. To investigate curvature angle estimation accuracy, linear regression analyses among the respective parameters were used. The impact of inaccurate marker placement was explored using linear regression analyses among the radio-opaque marker- and spinous process-derived curvature angles.Results and DiscussionThe results demonstrate that curvatures angles in the sagittal plane can be measured with reasonable accuracy, whereas in the frontal plane, angles were systematically underestimated, mainly due to the positional and structural deformities of the scoliotic vertebrae. Inaccuracy of marker placement had a greater impact on thoracolumbar / lumbar than thoracic curvature angles. It is suggested that spinal curvature measurements are included in marker-based clinical gait analysis protocols in order to enable a deeper understanding of the biomechanical behavior of the healthy and pathological spine in dynamic situations as well as to comprehensively evaluate treatment effects.