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Combining high-resolution micro-computed tomography with material composition to define the quality of bone tissue.

Atraumatic fractures of the skeleton in osteoporotic patients are directly related to a deterioration of bone strength. However, the failure of the bone tissue to withstand functional load bearing cannot be explained as a simple decrease in bone mineral density (quantity); strength is also significantly dependent upon bone quality. While a formal definition of bone quality is somewhat elusive, at the very least, it incorporates architectural, physical, and biologic factors that are critical to bone strength. Such factors include bone morphology (ie, trabecular connectivity, cross-sectional geometry, longitudinal curvature); the tissue's material properties (eg, stiffness, strength); its chemical composition and architecture (eg, ratio of calcium to other components of the organic and/or inorganic phase, collagen orientation, porosity, permeability); and the viability of the tissue (eg, responsivity of the bone cell population). Combining high-resolution structural indices of bone, as determined by micro-computed tomography; material properties determined by nanoindentation; and the chemical make-up of bone, as determined by infrared spectroscopy, helps to provide critical information toward a more comprehensive assessment of the interdependence of bone quality, quantity, and fracture risk.

Animals↗

Study of the surface morphology of a cholesteryl tethering system for lipidic bilayers.

The immobilization of functional molecules embedded in lipidic membranes onto inorganic substrates is of great interest for numerous applications in the fields of biosensors and biomaterials. We report on the preparation and the morphological characterization of a tethering system for lipidic bilayers, which is based on cholesteryl derivatives deposited on hydrophilic surfaces by self-assembling and microcontact printing techniques. The investigation of the structural properties of the realized films by atomic, lateral, and surface potential microscopy allowed us to assess the high quality of the realized cholesteryl layers.

Cholesterol↗

Effects of anthracycline derivatives on human leukemia K562 cell growth and differentiation.

New derivatives of daunorubicin (DRB), doxorubicin (DOX), and epidoxorubicin (EDOX) with an amidine group bonded to C-3' of daunosamine moiety with either morpholine or hexamethyleneimine ring attached to the amidine group are studied in this paper. We have shown that all of these newly synthesized anthracycline derivatives inhibit human leukemia K562 cell line proliferation but only some of them induce erythroid differentiation when used at subtoxic concentrations. Morpholine derivative of DOX has the greatest potential to inhibit proliferation and to induce differentiation in vitro. The correlation between these two cellular processes was also significant for other tested compounds. In cell cycle analysis, we have demonstrated that those anthracycline derivatives that exert the greatest cytostatic potential caused G(2)/M arrest, which in turn, might contribute to the development of a differentiating phenotype. The concentrations of the compounds used in the study are pharmacologically relevant. These new potent inducers of differentiation might be exploited as anticancer drugs for treatment of leukemia by differentiation therapy.

Anthracyclines↗

Multi-channel 3-D cell culture device integrated on a silicon chip for anticancer drug sensitivity test.

A novel three-dimensional cell culture system was constructed with an array of cell panels (4 x 5) in a silicon chip, together with multi-channel drug containers. Human breast cancer (MCF-7) cells were embedded in a collagen-gel matrix and entrapped in a pyramidal-shaped silicon hole. Each cell panel can be isolated by a channel composed of a microfluid part and a reservoir. A cell panel was exposed to 200 mm KCN for 2 days to demonstrate that each cell panel could be independently evaluated under various stimulation conditions. Based on the cellular respiration activity, the proliferation behavior was continuously monitored on the silicon-based cell array for 5 days using scanning electrochemical microscopy (SECM). The cells entrapped in the device (3-D culture) proliferated normally, and the proliferation rate was lower than that of cells grown in a monolayer cell culture (2-D culture). The effects of three anticancer drugs measured simultaneously on the cell chip were in good agreement with those obtained by a conventional colorimetric assay. Our results suggest that the silicon-based device for 3D culture is appropriate for a chemosensitivity assay involving multi-chemical stimulation.

Antineoplastic Agents↗

Magnetic force microscopy analysis of apoptosis of HL-60 cells induced by complex of antisense oligonucleotides and magnetic nanoparticles.

Magnetic force microscopy (MFM) has been employed to observe antisense oligonucleotides (ASOs)-coupled silica-coated magnetic iron oxide nanoparticles (SMNPs) internalized into human leukemia (HL-60) cells. The experiment demonstrated that the ASOs-coupled SMNPs delivery into the cells really occurred. The nanoparticles were internalized into the cells and the apoptotic topography can be directly visualized simultaneously with MFM technology. These present observations offer direct morphology evidence on studying the apoptosis of tumor cells and provide useful information for better design of new diagnostic and therapeutic tools in tumor treatment.

Apoptosis↗

Elasticity and adhesion of resting and lipopolysaccharide-stimulated macrophages.

Colloidal Force Microscopy was employed to study the viscoelastic and adhesive properties of macrophages upon stimulation with lipopolysaccharide (LPS). Force vs. distance measurements were performed. The adhesion of LPS-stimulated cells (separation force=37+/-3 nN) was almost twice as high as that of resting macrophages (16+/-1 nN). Upon retraction pulling of membrane tethers was observed. Tether lengths and forces at which rupture take place did not depend on stimulation. The reduced Young's modulus K, a measure of cytoskeleton elasticity, was three times lower than that of the control. The data show that LPS has profound effects on cytomechanical and adhesion properties of macrophages.

Cell Adhesion↗

Optically detected nuclear magnetic resonance at the sub-micron scale.

Nuclear magnetic resonance is arguably one of the most powerful techniques available today to characterize diverse systems. However, the low sensitivity of the standard detection method constrains the applicability of this technique to samples having effective dimensions not less than a few microns. Here, we propose a novel scheme and device for the indirect detection of the nuclear spin signal at a submicroscopic scale. This approach--for which the name Dipolar Field Microscopy is suggested--is based on the manipulation of the long-range nuclear dipolar interaction created between the sample and a semiconductor tip located close to its surface. After a preparation interval, the local magnetization of the sample is used to modulate the nuclear magnetization in the semiconductor tip, which, in turn is determined by an optical inspection. Based on results previously reported, it is shown that, in principle, images and/or localized high-resolution spectra of the sample can be retrieved with spatial resolution proportional to the size of the tip.

Image Enhancement↗

Observation of biological samples using a scanning microwave microscope.

We present the application of a scanning microwave microscope technique to biological samples. Since dielectric properties of most biological samples originate mainly from the water they contain, we were able to obtain microscope images of biological samples by our scanning microwave microscope technique. As a model system, we have measured the electrical properties of water in the microwave region. The high dielectric constant and the large loss tangent of water were verified. Furthermore, we have measured the properties of water with differing amounts of sodium chloride concentration ranging from de-ionized water to the saturated solution. We have observed a significant change in the resonant frequency and Q value of the resonator as a function of sodium chloride concentration. The concentration dependence of the signals shows that our scanning microwave microscope technique can be useful for investigating the local electric behavior of biological samples with a simple model of ionic conduction.

Microscopy, Scanning Probe↗

Effective mass and flow patterns of fluids surrounding microcantilevers.

An analytical approach to determine the streamlines of fluid flow adjacent to the surfaces of vibrating cantilevers is presented. Fluid flow over the top and bottom surfaces of a microcantilever is established by solving two-dimensional Navier-Stokes equations for viscous flow. The x and y velocity components are used to establish streamlines for absolute fluid motion. These streamlines show a central stagnation core perpendicular and central to the cantilever surface extending along the full length of cantilevers, which most likely accounts for the added mass effect (induced mass) of fluid media around vibrating microcantilevers.

Air↗

Effect of nanometer surface morphology on surface stress and adsorption kinetics of alkanethiol self-assembled monolayers.

Microcantilevers undergo quasi-static bending due to adsorption-induced stress when adsorption is confined to a single surface. Understanding the origin of surface stress is crucial for optimizing sensor performance. We have investigated the effect of surface morphology of gold-coated cantilevers on the adsorption characteristics of self-assembled monolayers of alkanethiols. Self-assembly of alkanethiols from liquid phase closely follows Langmuir-type kinetics up to a single monolayer assembly. Our results indicate that alkanethiol adsorption-induced surface stress is largely unaffected by surface roughness of the cantilever. Unlike prior reports that suggest surface roughness enhances adsorption-induced stress, we observe that nanometer-size roughness slightly decreases surface stress and adsorption kinetics.

Alkanes↗

Microscale Marangoni actuation: all-optical and all-electrical methods.

We present experimental results from an all-optical microfluidic platform that may be complimented by a thin film all-electrical network. Using these configurations we have studied the microfluidic convective flow systems of silicone oil, glycerol, and 1,3,5-trinitrotoluene on open surfaces through the production of surface tension gradients derived from thermal gradients. We show that sufficient localized thermal variation can be created utilizing surface plasmons and/or engaging individually addressable resistive thermal elements. Both studies manipulate fluids via Marangoni forces, each having their unique exploitable advantages. Surface plasmon excitation in metal foils are the driving engine of many physical-, chemical-, and bio-sensing applications. Incorporating, for the first time, the plasmon concept in microfluidics, our results thus demonstrate great potential for simultaneous fluid actuation and sensing.

Glycerol↗

Nanobubbles and the hydrophobic attraction.

The evidence for nanobubbles as the origin of the long-ranged attractions measured between hydrophobic surfaces immersed in water is reviewed by focusing upon several unique features of the force curves. Also covered is the morphology of nanobubbles as revealed by direct imaging with tapping mode atomic force microscopy. A discussion of the origin, thermodynamic stability and practical implications of nanobubbles is given.

Biophysical Phenomena↗

Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching.

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of approximately 13 s.

Bacterial Proteins↗

Abeta42-peptide assembly on lipid bilayers.

One of the major pathological features of Alzheimer's disease (AD) is the presence of extracellular amyloid plaques that are composed predominantly of the amyloid-beta peptide (Abeta). Diffuse plaques associated with AD are composed predominantly of Abeta42, whereas senile plaques contain both Abeta40 and Abeta42. Recently, it has been suggested that diffuse plaque formation is initiated as a plasma membrane-bound Abeta species and that Abeta42 is the critical component. In order to investigate this hypothesis, we have examined Abeta42-membrane interactions using in situ atomic force microscopy and fluorescence spectroscopy. Our studies demonstrate the association of Abeta42 with planar bilayers composed of total brain lipids, which results initially in peptide aggregation and then fibre formation. Modulation of the cholesterol content is correlated with the extent of Abeta42-assembly on the bilayer surface. Although Abeta42 was not visualized directly on cholesterol-depleted bilayers, fluorescence anisotropy and fluorimetry demonstrate Abeta42-induced membrane changes. Our results demonstrate that the composition of the lipid bilayer governs the outcome of Abeta interactions.

Amyloid beta-Peptides↗

Arrays of transfected mammalian cells for high content screening microscopy.

In this chapter we describe protocols for reverse transfection to generate mammalian cell arrays for systematic gene knock-downs by RNAi or knock-ins by ectopic cDNA expression. The method is suitable for high content screening microscopy at a high spatial and temporal resolution allowing even time-lapse analysis of hundreds of samples in parallel.

Animals↗

PEO-grafting on PU/PS IPNs for enhanced blood compatibility--effect of pendant length and grafting density.

Polyurethane (PU) homopolymers and PU/polystyrene (PS) interpenetrating polymer networks (IPNs) were successfully synthesized changing the length of the pendant poly(ethylene oxide) (PEO) chains and the grafting density of PEO chains. All the PU/PS IPNs had the microphase-separated structures in which the PS-rich phase domains were dispersed in the matrix of the PU-rich phase. The domain size decreased a little, as the degree of grafting with PEO chains was increased. The water swelling ratio increased, and the interfacial energy decreased, as the length of the pendant PEO chains, and the grafting density of PEO chains of the PEO-grafted PU/PS IPNs were increased, since the mobile hydrophilic pendant PEO chains effectively induced and absorbed the water, when they were contacted with water. The hydrophilic and highly concentrated pendant PEO chains could easily prohibit the adhesion of the fibrinogens and the platelets on the surface, and the blood compatibility of IPNs was enhanced by increasing of grafting with PEO chains. The adsorption of the fibrinogens and the platelets was suppressed, as the length of pendant PEO chains, and the grafting density were increased.

Adsorption↗

Stochastic properties of actomyosin motor.

The epoch-making techniques for manipulating a single myosin molecule have recently been developed, and the unitary mechanical reactions of a single actomyosin, muscle motor molecule, are directly measured. The data show that the unitary mechanical step during sliding along an actin filament of approximately 5.5 nm, but groups of two to five rapid steps in succession produce displacements of approximately 11-30 nm. The instances of multiple stepping are produced by single myosin heads during one biochemical cycle of ATP hydrolysis. Thus, the coupling between ATP hydrolysis cycle and mechanical step is variable, i.e. loose-coupling. Such a unique operation of actomyosin molecules is different from that of man-made machines, and most likely explains the flexible and effective mechanisms of molecular machines in the biosystems.

Actins↗