Mechanical control of mitotic progression in single animal cells.

Despite the importance of mitotic cell rounding in tissue development and cell proliferation, there remains a paucity of approaches to investigate the mechanical robustness of cell rounding. Here we introduce ion beam-sculpted microcantilevers that enable precise force-feedback-controlled confinement of single cells while characterizing their progression through mitosis. We identify three force regimes according to the cell response: small forces (∼5 nN) that accelerate mitotic progression, intermediate forces where cells resist confinement (50-100 nN), and yield forces (>100 nN) where a significant decline in cell height impinges on microtubule spindle function, thereby inhibiting mitotic progression. Yield forces are coincident with a nonlinear drop in cell height potentiated by persistent blebbing and loss of cortical F-actin homogeneity. Our results suggest that a buildup of actomyosin-dependent cortical tension and intracellular pressure precedes mechanical failure, or herniation, of the cell cortex at the yield force. Thus, we reveal how the mechanical properties of mitotic cells and their response to external forces are linked to mitotic progression under conditions of mechanical confinement.

Probing the mystery of Liesegang band formation: revealing the origin of self-organized dual-frequency micro and nanoparticle arrays.

Periodic precipitation processes in gels can result in impressive micro- and nanostructured patterns known as periodic precipitation (or Liesegang bands). Under certain conditions, the silver nitrate-chromium(vi) system exhibits the coexistence of two kinds of Liesegang bands with different frequencies. We now present that the two kinds of bands form independently on different time scales and the pH-dependent chromate(vi)-dichromate(vi) equilibrium controls the formation of the precipitates. We determined the spatial distribution and constitution of the particles in the bands using focused ion beam-scanning electron microscopy (FIB-SEM) and scanning transmission X-ray spectromicroscopy (STXM) measurements. This provided the necessary empirical input data to formulate a model for the pattern formation; a model that quantitatively reproduces the experimental observations. Understanding the pattern-forming process at the molecular level enables us to tailor the size and the shape of the bands, which, in turn, can lead to new functional architectures for a range of applications.

Supramolecular Organization of Collagen Fibrils in Healthy and Osteoarthritic Human Knee and Hip Joint Cartilage.

Cartilage matrix is a composite of discrete, but interacting suprastructures, i.e. cartilage fibers with microfibrillar or network-like aggregates and penetrating extrafibrillar proteoglycan matrix. The biomechanical function of the proteoglycan matrix and the collagen fibers are to absorb compressive and tensional loads, respectively. Here, we are focusing on the suprastructural organization of collagen fibrils and the degradation process of their hierarchical organized fiber architecture studied at high resolution at the authentic location within cartilage. We present electron micrographs of the collagenous cores of such fibers obtained by an improved protocol for scanning electron microscopy (SEM). Articular cartilages are permeated by small prototypic fibrils with a homogeneous diameter of 18 ± 5 nm that can align in their D-periodic pattern and merge into larger fibers by lateral association. Interestingly, these fibers have tissue-specific organizations in cartilage. They are twisted ropes in superficial regions of knee joints or assemble into parallel aligned cable-like structures in deeper regions of knee joint- or throughout hip joints articular cartilage. These novel observations contribute to an improved understanding of collagen fiber biogenesis, function, and homeostasis in hyaline cartilage.

Micromechanical cantilever array sensors for selective fungal immobilization and fast growth detection.

We demonstrate the use of micromechanical cantilever arrays for selective immobilization and fast quantitative detection of vital fungal spores. Micro-fabricated uncoated as well as gold-coated silicon cantilevers were functionalized with concanavalin A, fibronectin or immunoglobulin G. In our experiments two major morphological fungal forms were used--the mycelial form Aspergillus niger and the unicellular yeast form Saccharomyces cerevisiae, as models to explore a new method for growth detection of eukaryotic organisms using cantilever arrays. We exploited the specific biomolecular interactions of surface grafted proteins with the molecular structures on the fungal cell surface. It was found that these proteins have different affinities and efficiencies to bind the spores. Maximum spore immobilization, germination and mycelium growth was observed on the immunoglobulin G functionalized cantilever surfaces. We show that spore immobilization and germination of the mycelial fungus A. niger and yeast S. cerevisiae led to shifts in resonance frequency within a few hours as measured by dynamically operated cantilever arrays, whereas conventional techniques would require several days. The biosensor could detect the target fungi in a range of 10(3) - 10(6) CFUml(-1). The measured shift is proportional to the mass of single fungal spores and can be used to evaluate spore contamination levels. Applications lie in the field of medical and agricultural diagnostics, food- and water-quality monitoring.

Morphological changes of tungsten surfaces by low-flux helium plasma treatment and helium incorporation via magnetron sputtering.

The effect of helium on the tungsten microstructure was investigated first by exposure to a radio frequency driven helium plasma with fluxes of the order of 1 × 10(19) m(-2) s(-1) and second by helium incorporation via magnetron sputtering. Roughening of the surface and the creation of pinholes were observed when exposing poly- and nanocrystalline tungsten samples to low-flux plasma. A coating process using an excess of helium besides argon in the process gas mixture leads to a porous thin film and a granular surface structure whereas gas mixture ratios of up to 50% He/Ar (in terms of their partial pressures) lead to a dense structure. The presence of helium in the deposited film was confirmed with glow-discharge optical emission spectroscopy and thermal desorption measurements. Latter revealed that the highest fraction of the embedded helium atoms desorb at approximately 1500 K. Identical plasma treatments at various temperatures showed strongest modifications of the surface at 1500 K, which is attributed to the massive activation of helium singly bond to a single vacancy inside the film. Thus, an efficient way of preparing nanostructured tungsten surfaces and porous tungsten films at low fluxes was found.

Early detection of aging cartilage and osteoarthritis in mice and patient samples using atomic force microscopy.

The pathological changes in osteoarthritis--a degenerative joint disease prevalent among older people--start at the molecular scale and spread to the higher levels of the architecture of articular cartilage to cause progressive and irreversible structural and functional damage. At present, there are no treatments to cure or attenuate the degradation of cartilage. Early detection and the ability to monitor the progression of osteoarthritis are therefore important for developing effective therapies. Here, we show that indentation-type atomic force microscopy can monitor age-related morphological and biomechanical changes in the hips of normal and osteoarthritic mice. Early damage in the cartilage of osteoarthritic patients undergoing hip or knee replacements could similarly be detected using this method. Changes due to aging and osteoarthritis are clearly depicted at the nanometre scale well before morphological changes can be observed using current diagnostic methods. Indentation-type atomic force microscopy may potentially be developed into a minimally invasive arthroscopic tool to diagnose the early onset of osteoarthritis in situ.

Influence of size, shape, and flexibility on bacterial passage through micropore membrane filters.

Sterilization of fluids by means of microfiltration is commonly applied in research laboratories as well as in pharmaceutical and industrial processes. Sterile micropore filters are subject to microbiological validation, where Brevundimonas diminuta is used as a standard test organism. However, several recent reports on the ubiquitous presence of filterable bacteria in aquatic environments have cast doubt on the accuracy and validity of the standard filter-testing method. Six different bacterial species of various sizes and shapes (Hylemonella gracilis, Escherichia coli, Sphingopyxis alaskensis, Vibrio cholerae, Legionella pneumophila, and B. diminuta) were tested for their filterability through sterile micropore filters. In all cases, the slender spirillum-shaped Hylemonella gracilis cells showed a superior ability to pass through sterile membrane filters. Our results provide solid evidence that the overall shape (including flexibility), instead of biovolume, is the determining factor for the filterability of bacteria, whereas cultivation conditions also play a crucial role. Furthermore, the filtration volume has a more important effect on the passage percentage in comparison with other technical variables tested (including flux and filter material). Based on our findings, we recommend a re-evaluation of the grading system for sterile filters, and suggest that the species Hylemonella should be considered as an alternative filter-testing organism for the quality assessment of micropore filters.

An antibody-sensitized microfabricated cantilever for the growth detection of Aspergillus niger spores.

We demonstrate a new sensitive biosensor for detection of vital fungal spores of Aspergillus niger. The biosensor is based on silicon microfabricated cantilever arrays operated in dynamic mode. The change in resonance frequency of the sensor is a function of mass binding to the cantilever surface. For specific A. niger spore immobilization on the cantilever, each cantilever was individually coated with anti-Aspergillus niger polyclonal antibodies. We demonstrate the detection of single A. niger spores and their subsequent growth on the functionalized cantilever surface by online measurements of resonance frequency shifts. The new biosensor operating in humid air allows quantitative and qualitative detection of A. niger spores as well as detection of vital, functional spores in situ within approximately 4 h. The detection limit of the sensor is 103 CFU mL-1. Mass sensitivity of the cantilever sensor is approximately 53 pg Hz-1.