Cellular Force Microscopy to Measure Mechanical Forces in Plant Cells.

Cellular force microscopy (CFM) is a noninvasive microindentation method used to measure plant cell stiffness in vivo. CFM is a scanning probe microscopy technique similar in operation to atomic force microscopy (AFM); however, the scale of movement and range of forces are much larger, making it suitable for stiffness measurements on turgid plant cells in whole organs. CFM experiments can be performed on living samples over extended time periods, facilitating the exploration of the dynamics of processes involving mechanics. Different sensor technologies can be used, along with a variety of probe shapes and sizes that can be tailored to specific applications. Measurements can be made for specific indentation depths, forces and timing, allowing for very precise mechanical stimulation of cells with known forces. High forces with sharp tips can also be used for mechanical ablation of cells with force feedback.

Characterizing inorganic crystals grown on organic self-assembled bilayers with scanning probe and electron microscopies.

Combined microscopy techniques are used to establish the usability of phosphonic acid layers as promoters of hydroxyapatite (HAp) growth. Using spread coating, octadecylphosphonic acid (OPA) self-assembled bilayers are delivered to the thin natural oxide layer of a titanium film surface with no prior treatment. These bilayers aggregate two major advantages of phosphonic moieties to titanium surfaces: nucleation of hydroxyapatite crystals from ionic solution and affinity for both titanium oxide surface and HAp crystals. The functionalized substrates and bare titanium (control) samples are immersed in an aqueous solution containing calcium and phosphorus ions. Over a 4-week immersion time, OPA-functionalized substrates present numerous large agglomerates of inorganic crystals, in contrast to control samples, with no significant amount of deposits. Initial sample characterization was performed with atomic force microscopy (AFM). Compositional and structural characterization of these agglomerates (using TEM, EDS, and electron diffraction), revealed that they are indeed HAp, the main component of the inorganic bone matrix.

Migrating oligodendrocyte progenitor cells swell prior to soma dislocation.

The migration of oligodendrocyte progenitor cells (OPCs) to the white matter is an indispensable requirement for an intact brain function. The mechanism of cell migration in general is not yet completely understood. Nevertheless, evidence is accumulating that besides the coordinated rearrangement of the cytoskeleton, a finetuned interplay of ion and water fluxes across the cell membrane is essential for cell migration. One part of a general hypothesis is that a local volume increase towards the direction of movement triggers a mechano-activated calcium influx that regulates various procedures at the rear end of a migrating cell. Here, we investigated cell volume changes of migrating OPCs using scanning ion conductance microscopy. We found that during accelerated migration OPCs undergo an increase in the frontal cell body volume. These findings are supplemented with time lapse calcium imaging data that hint an increase in calcium content the frontal part of the cell soma.

Site specific properties of carious dentin matrices biomodified with collagen cross-linkers.

PURPOSE: To assess in non-cavitated carious teeth the mechanical properties of dentin matrix by measuring its reduced modulus of elasticity and the effect of dentin biomodification strategies on three dentin matrix zones: caries-affected, apparently normal dentin below caries-affected zone and sound dentin far from carious site. METHODS: Nano-indentations were performed on dentin matrices of carious molars before and after surface modification using known cross-linking agents (glutaraldehyde, proanthocyanidins from grape seed extract and carbodiimide). RESULTS: Statistically significant differences were observed between dentin zones of demineralized dentin prior to surface biomodification (P < 0.05). Following surface modification, there were no statistically significant differences between dentin zones (P < 0.05). An average increase of 30-fold, 2-fold and 2.2-fold of the reduced modulus of elasticity was observed following treatments of the three dentin zones with proanthocyanidin, carbodiimide and glutaraldehyde, respectively.

Molecular characterization of organic electronic films.

Organic electronics have emerged as a viable competitor to amorphous silicon for the active layer in low-cost electronics. The critical performance of organic electronic materials is closely related to their morphology and molecular packing. Unlike their inorganic counterparts, polymers combine complex repeat unit structure and crystalline disorder. This combination prevents any single technique from being able to uniquely solve the packing arrangement of the molecules. Here, a general methodology for combining multiple, complementary techniques that provide accurate unit cell dimensions and molecular orientation is described. The combination of measurements results in a nearly complete picture of the organic film morphology.

Scanning electrochemical microscopy assay of DNA based on hairpin probe and enzymatic amplification biosensor.

A novel scheme for scanning electrochemical microscopy (SECM) assay of DNA based on hairpin probe and enzymatic amplification biosensor was described. In this method, streptavidin-horseradish peroxidase (HRP) was captured by double-stranded DNA (ds-DNA) modified gold substrate via biotin-streptavidin interaction after hybridization of target DNA to the immobilized hairpin probe functioned with a biotin at its 3' end. In the presence of H2O2, hydroquinone (H2Q) was oxidized to benzoquinone (BQ) at the modified substrate surface through the HRP catalytic reaction, and the generated BQ corresponding to the amount of target DNA was reduced in solution by a SECM tip. The resulting reduction current allowed concentration detection of target DNA and SECM imaging of hybridization between the target DNA and the immobilized hairpin probe. The detection limit of this method was as low as 17 pM for complementary target DNA and it had good selectivity to discriminate between the complementary sequence and one containing base mismatches.

Development and in-vitro evaluation of a colon-specific controlled release drug delivery system.

The major challenges in targeting drug to various parts of the gastrointestinal tract include control of drug release with respect to its environment and transit time. These two variables should be taken into consideration in designing a rational colonic drug delivery system. To this end, a swelling matrix core containing pectin, hydroxypropyl methylcellulose (HPMC), microcrystalline cellulose and 5-aminosalicylic acid was developed. This was subjected to a dual coating operation: an inner pH-sensitive enteric and an outer semi-permeable membrane coat with a pore former. In-vitro dissolution studies were carried out in USP apparatus-I using sequential pH media. The first 2 h of dissolution studies were done in HCl buffer at pH 1.5, the next 2 h in pH 5.5 and, finally, in phosphate buffer at pH 6.8 with and without pectinolytic enzyme present. Less than 2% drug was released in the first 6 h and about 90% released in the following 12 h in a controlled manner. The stability studies of the coated systems were performed for 90 days under various conditions and it was found that drug release was not adversely affected. Results indicate that this delivery system has potential for site-specific delivery of drugs to the colon irrespective of transit time and rapid changes in the proximal pH of the gastrointestinal tract.

SNOM images of X-ray radiographs at nano-scale stored in a thin layer of lithium fluoride.

In this work, we report a method to observe soft X-ray radiographs at nanoscale of various kind of samples, biological and metallic, stored in a thin layer of lithium fluoride, employing scanning near-field optical microscopy with an optical resolution that reaches 50 nm. Lithium fluoride material works as a novel image detector for X-ray nano-radiographs, due to the fact that extreme ultraviolet radiation and soft X-rays efficiently produce stable point defects emitting optically stimulated visible luminescence in a thin surface layer. The bi-dimensional distribution of the so-created defects depends on the local nanostructure of the investigated sample.

Asymmetric distribution of metals in the Xenopus laevis oocyte: a synchrotron X-ray fluorescence microprobe study.

The asymmetric distribution of many components of the Xenopus oocyte, including RNA, proteins, and pigment, provides a framework for cellular specialization during development. During maturation, Xenopus oocytes also acquire metals needed for development, but apart from zinc, little is known about their distribution. Synchrotron X-ray fluorescence microprobe was used to map iron, copper, and zinc and the metalloid selenium in a whole oocyte. Iron, zinc, and copper were asymmetrically distributed in the cytoplasm, while selenium and copper were more abundant in the nucleus. A zone of high copper and zinc was seen in the animal pole cytoplasm. Iron was also concentrated in the animal pole but did not colocalize with zinc, copper, or pigment accumulations. This asymmetry of metal deposition may be important for normal development. Synchrotron X-ray fluorescence microprobe will be a useful tool to examine how metals accumulate and redistribute during fertilization and embryonic development.

Local charge transport in two-dimensional PbSe nanocrystal arrays studied by electrostatic force microscopy.

Two-dimensional PbSe nanocrystal arrays on silicon nitride membranes were investigated using electrostatic force microscopy (EFM) and transmission electron microscopy (TEM). Changes in lattice and transport properties upon annealing in a vacuum were revealed. Local charge transport behavior was directly imaged by EFM and correlated to nanopatterns observed with TEM. Charge transport through nanochannels in complex two-dimensional nanocrystal networks was identified. Our results demonstrate the importance of measurements of local transport details complementary to the conventional current-voltage (I-V) measurements.

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.