Scanning Probe Microscopy Bone Marrow Determination of Steogenic Differentiation of Mesenchymal Stem Cells.

To address the question of determining the osteogenic differentiation of mesenchymal stem cells, the bone marrow studies were performed using probe microscopy. All adherent bone marrow was used to isolate the bone marrow mesenchymal stem cells and expanded and purified in vitro. Its morphology under an inverted microscope was observed. We used Zuogui Pills to differentiate the separation methods. Alcian blue staining, modified calcium cobalt alkaline phosphatase staining, and neuron-specific enolase immunohistochemical staining were performed. The experimental results are shown below. The morphology of the isolated and purified cells was analyzed with an inverted microscope, and the isolated and purified cells were analyzed with Zuogui Pill. Alcian blue staining, modified calcium cobalt alkaline phosphatase staining, and neuron-specific enolase immunohistochemical staining confirmed that the cells differentiated into cartilage and osteoblasts, and the cell structure and morphology were similar to those of the bone marrow mesenchymal stem cells. The results showed that the adherent mode of cells obtained from the whole bone marrow was the rat bone marrow mesenchymal stem cells, and the Zuogui Pills could induce multidirectional differences in the bone marrow mesenchymal stem cells.

High resolution imaging of viruses: Scanning probe microscopy and related techniques.

Scanning probe microscopy is a group of measurements that provides 3D visualization of viruses in different environmental conditions including liquids and air. Besides 3D topography it is possible to measure the properties like mechanical rigidity and stability, adhesion, tendency to crystallization, surface charge, etc. Choosing the right substrate and scanning parameters makes it much easier to obtain reliable data. Rational interpretation of experimental results should take into account possible artifacts, proper filtering and data presentation using specially designed software packages. Animal and human virus characterization is in the focus of many intensive studies because of their potential harm to higher organisms. The article focuses on high-resolution visualization of plant viruses. Tobacco mosaic virus, potato viruses X and B and others are not dangerous for the human being and are widely used in different applications such as vaccine preparation, construction of building units in nanotechnology and material science applications, nanoparticle production and delivery, and even metrology. The methods of virus's deposition, visualization, and consequent image processing and interpretation are described in details. Specific examples of viruses imaging are illustrated using the FemtoScan Online software, which has typical and all the necessary built-in functions for constructing three-dimensional images, their processing and analysis. Despite visible progress in visualizing the viruses using probe microscopy, many unresolved problems still remain. At present time the probe microscopy data on viruses is not systemized. There is no descriptive atlas of the images and morphology as revealed by this type of high resolution microscopy. It is worth emphasizing that new virus investigation methods will appear due to the progress of science.

Time-Resolved Scanning Ion Conductance Microscopy for Three-Dimensional Tracking of Nanoscale Cell Surface Dynamics.

Nanocharacterization plays a vital role in understanding the complex nanoscale organization of cells and organelles. Understanding cellular function requires high-resolution information about how the cellular structures evolve over time. A number of techniques exist to resolve static nanoscale structure of cells in great detail (super-resolution optical microscopy, EM, AFM). However, time-resolved imaging techniques tend to either have a lower resolution, are limited to small areas, or cause damage to the cells, thereby preventing long-term time-lapse studies. Scanning probe microscopy methods such as atomic force microscopy (AFM) combine high-resolution imaging with the ability to image living cells in physiological conditions. The mechanical contact between the tip and the sample, however, deforms the cell surface, disturbs the native state, and prohibits long-term time-lapse imaging. Here, we develop a scanning ion conductance microscope (SICM) for high-speed and long-term nanoscale imaging of eukaryotic cells. By utilizing advances in nanopositioning, nanopore fabrication, microelectronics, and controls engineering, we developed a microscopy method that can resolve spatiotemporally diverse three-dimensional (3D) processes on the cell membrane at sub-5-nm axial resolution. We tracked dynamic changes in live cell morphology with nanometer details and temporal ranges of subsecond to days, imaging diverse processes ranging from endocytosis, micropinocytosis, and mitosis to bacterial infection and cell differentiation in cancer cells. This technique enables a detailed look at membrane events and may offer insights into cell-cell interactions for infection, immunology, and cancer research.

Reactive oxygen FIB spin milling enables correlative workflow for 3D super-resolution light microscopy and serial FIB/SEM of cultured cells.

Correlative light and electron microscopy (CLEM) is a powerful tool for defining the ultrastructural context of molecularly-labeled biological specimens, particularly when superresolution fluorescence microscopy (SRM) is used for CLEM. Current CLEM, however, is limited by the stark differences in sample preparation requirements between the two modalities. For CLEM using SRM, the small region of interest (ROI) of either or both modalities also leads to low success rate and imaging throughput. To overcome these limitations, here we present a CLEM workflow based on a novel focused ion beam/scanning electron microscope (FIB/SEM) compatible with common SRM for imaging biological specimen with ultrahigh 3D resolution and improved imaging throughput. By using a reactive oxygen source in a plasma FIB (PFIB) and a rotating sample stage, the novel FIB/SEM was able to achieve several hundreds of micrometer large area 3D analysis of resin embedded cells through a process named oxygen serial spin mill (OSSM). Compared with current FIB mechanisms, OSSM offers gentle erosion, highly consistent slice thickness, reduced charging during SEM imaging, and improved SEM contrast without increasing the dose of post-staining and fixation. These characteristics of OSSM-SEM allowed us to pair it with interferometric photoactivated localization microscopy (iPALM), a recent SRM technique that affords 10-20 nm isotropic spatial resolution on hydrated samples, for 3D CLEM imaging. We demonstrate a CLEM workflow generalizable to using other SRM strategies using mitochondria in human osteosarcoma (U2OS) cells as a model system, where immunostained TOM20, a marker for the mitochondrial outer membrane, was used for iPALM. Owing to the large scan area of OSSM-SEM, it is now possible to select as many FOVs as needed for iPALM and conveniently re-locate them in EM, this improving the imaging throughput. The significantly reduced dose of post-fixation also helped to better preserve the sample ultrastructures as evidenced by the excellent 3D registration between OSSM-SEM and iPALM images and by the accurate localization of TOM20 (by iPALM) to the peripheries of mitochondria (by OSSM-SEM). These advantages make OSSM-SEM an ideal modality for CLEM applications. As OSSM-SEM is still in development, we also discuss some of the remaining issues and the implications to biological imaging with SEM alone or with CLEM.

A de-waxing methodology for scanning probe microscopy.

A de-waxing protocol that successfully removes paraffin from tissue microarray (TMA) cores of fixed tissue obtained from oral cancer is described. The success of the protocol is demonstrated by the comparison of Fourier transform infrared (FTIR) results obtained on paraffin-embedded and de-waxed tissue and the absence of any significant correlations between infrared scanning near-field optical microscopy (SNOM) images of de-waxed tissue obtained at the three main paraffin IR peaks. The success of the protocol in removing paraffin from tissue is also demonstrated by images obtained with scanning electron microscopy (SEM) and by energy dispersive spectra (EDS) of a de-waxed CaF2 disc which shows no significant contribution from carbon. The FTIR spectra of the de-waxed TMA core overlaps that obtained from OE19 oesophageal cancer cells which had never been exposed to paraffin.

Sepsis Reduces Bone Strength Before Morphologic Changes Are Identifiable.

OBJECTIVES: Survivors of critical illness have an increased prevalence of bone fractures. However, early changes in bone strength, and their relationship to structural changes, have not been described. We aimed to characterize early changes in bone functional properties in critical illness and their relationship to changes in bone structure, using a sepsis rodent model. DESIGN: Experimental study. SETTING: Animal research laboratory. SUBJECTS: Adult Sprague-Dawley rats. INTERVENTIONS: Forty Sprague-Dawley rats were randomized to cecal ligation and puncture or sham surgery. Twenty rodents (10 cecal ligation and puncture, 10 sham) were killed at 24 hours, and 20 more at 96 hours. MEASUREMENTS AND MAIN RESULTS: Femoral bones were harvested for strength testing, microCT imaging, histologic analysis, and multifrequency scanning probe microscopy. Fracture loads at the femoral neck were significantly reduced for cecal ligation and puncture-exposed rodents at 24 hours (83.39 ± 10.1 vs 103.1 ± 17.6 N; p = 0.014) and 96 hours (81.60 ± 14.2 vs 95.66 ± 14.3 N; p = 0.047). Using multifrequency scanning probe microscopy, collagen elastic modulus was lower in cecal ligation and puncture-exposed rats at 24 hours (1.37 ± 0.2 vs 6.13 ± 0.3 GPa; p = 0.001) and 96 hours (5.57 ± 0.5 vs 6.13 ± 0.3 GPa; p = 0.006). Bone mineral elastic modulus was similar at 24 hours but reduced in cecal ligation and puncture-exposed rodents at 96 hours (75.34 ± 13.2 vs 134.4 ± 8.2 GPa; p < 0.001). There were no bone architectural or bone mineral density differences by microCT. Similarly, histologic analysis demonstrated no difference in collagen and elastin staining, and C-X-C chemokine receptor type 4, nuclear factor kappa beta, and tartrate-resistant acid phosphatase immunostaining. CONCLUSIONS: In a rodent sepsis model, trabecular bone strength is functionally reduced within 24 hours and is associated with a reduction in collagen and mineral elastic modulus. This is likely to be the result of altered biomechanical properties, rather than increased bone mineral turnover. These data offer both mechanistic insights and may potentially guide development of therapeutic interventions.

Migration mechanism of mesenchymal stem cells studied by QD/NSOM.

The migration of mesenchymal stem cells (MSCs) plays a key role in tumor-targeted delivery vehicles and tumor-related stroma formation. However, there so far has been no report on the distribution of cell surface molecules during the VEGF-induced migration of MSCs. Here, we have utilized near-field scanning optical microscopy (NSOM) combined with fluorescent quantum dot (QD)-based nano-technology to capture the functional relationship between CD44 and CD29 adhesion molecules on MSCs and the effect of their spatial rearrangements. Before VEGF-induced migration of MSCs, both CD44 and CD29 formed 200-220 nm nano-domains respectively, with little co-localization between the two types of domains. Surprisingly, the size of the CD44 nano-domain rapidly increased in size to 295 nm and apparently larger aggregates were formed following MSC treatment with VEGF for 10 min, while the area of co-localization increased to 0.327 µm2. Compared with CD44, CD29 was activated obviously later, for the fact that CD29 aggregation didn't appear until 30 min after VEGF treatment. Consistently, its co-localization area increased to 0.917 µm2. The CD44 and CD29 nano-domains further aggregated into larger nano-domains or even formed micro-domains on the membrane of activated MSCs. The aggregation and co-localization of these molecules promoted FAK formation and cytoskeleton rearrangement. All of the above changes induced by VEGF contributed to MSC migration. Taken together, our data of NSOM-based dual color fluorescent imaging demonstrated for the first time that CD44, together with CD29, involved in VEGF-induced migration of MSCs through the interaction between CD44 and its co-receptor of VEGFR-2.

Leakage radiation microscope for observation of non-transparent samples.

We describe a leakage radiation microscope technique that can be used to extend the leakage radiation microscopy to optically non-transparent samples. In particular, two experiments are presented, first to demonstrate that acquired images with our configuration correspond to the leakage radiation phenomenon and second, to show possible applications by directly imaging a plasmonic structure that previously could only be imaged with a near-field scanning optical microscope. It is shown that the measured surface plasmon wavelength and propagation length agree with theoretically-calculated values. This configuration opens the possibility to study important effects where samples are optically non-transparent, as in plasmonic cavities and single hole plasmonic excitation, without the use of time-consuming near-field scanning optical microscopy.

Assessment of multidrug resistance on cell coculture patterns using scanning electrochemical microscopy.

The emergence of resistance to multiple unrelated chemotherapeutic drugs impedes the treatment of several cancers. Although the involvement of ATP-binding cassette transporters has long been known, there is no in situ method capable of tracking this transporter-related resistance at the single-cell level without interfering with the cell's environment or metabolism. Here, we demonstrate that scanning electrochemical microscopy (SECM) can quantitatively and noninvasively track multidrug resistance-related protein 1-dependent multidrug resistance in patterned adenocarcinoma cervical cancer cells. Nonresistant human cancer cells and their multidrug resistant variants are arranged in a side-by-side format using a stencil-based patterning scheme, allowing for precise positioning of target cells underneath the SECM sensor. SECM measurements of the patterned cells, performed with ferrocenemethanol and [Ru(NH3)6](3+) serving as electrochemical indicators, are used to establish a kinetic "map" of constant-height SECM scans, free of topography contributions. The concept underlying the work described herein may help evaluate the effectiveness of treatment administration strategies targeting reduced drug efflux.

Surface properties and interaction forces of biopolymer-doped conductive polypyrrole surfaces by atomic force microscopy.

Surface properties and electrical charges are critical factors elucidating cell interactions on biomaterial surfaces. The surface potential distribution and the nanoscopic and microscopic surface elasticity of organic polypyrrole-hyaluronic acid (PPy-HA) were studied by atomic force microscopy (AFM) in a fluid environment in order to explain the observed enhancement in the attachment of human adipose stem cells on positively charged PPy-HA films. The electrostatic force between the AFM tip and a charged PPy-HA surface, the tip-sample adhesion force, and elastic moduli were estimated from the AFM force curves, and the data were fitted to electrostatic double-layer and elastic contact models. The surface potential of the charged and dried PPy-HA films was assessed with Kelvin probe force microscopy (KPFM), and the KPFM data were correlated to the fluid AFM data. The surface charge distribution and elasticity were both found to correlate well with the nodular morphology of PPy-HA and to be sensitive to the electrochemical charging conditions. Furthermore, a significant change in the adhesion was detected when the surface was electrochemically charged positive. The results highlight the potential of positively charged PPy-HA as a coating material to enhance the stem cell response in tissue-engineering scaffolds.

Muscle contraction mechanism based on single molecule measurements.

Single molecule measurements have shown that a muscle myosin step is driven by biased Brownian movement. Furthermore, they have also demonstrated that in response to strain in the backward direction a detached myosin head preferentially attaches to the forward direction due to an accelerated transition from a weak binding to strong binding state. Because they are consistent with the original Huxley model for muscle contraction, we have built a model that describes macroscopic muscle characteristics based on these single molecule results.

A hybrid scanning mode for fast scanning ion conductance microscopy (SICM) imaging.

We have developed a new method of controlling the pipette for scanning ion conductance microscopy to obtain high-resolution images faster. The method keeps the pipette close to the surface during a single line scan but does not follow the exact surface topography, which is calculated by using the ion current. Using an FPGA platform we demonstrate this new method on model test samples and then on live cells. This method will be particularly useful to follow changes occurring on relatively flat regions of the cell surface at high spatial and temporal resolutions.

Polyglutamine aggregate structure in vitro and in vivo; new avenues for coherent anti-Stokes Raman scattering microscopy.

Coherent anti-Stokes Raman scattering (CARS) microscopy is applied for the first time for the evaluation of the protein secondary structure of polyglutamine (polyQ) aggregates in vivo. Our approach demonstrates the potential for translating information about protein structure that has been obtained in vitro by X-ray diffraction into a microscopy technique that allows the same protein structure to be detected in vivo. For these studies, fibres of polyQ containing peptides (D(2)Q(15)K(2)) were assembled in vitro and examined by electron microscopy and X-ray diffraction methods; the fibril structure was shown to be cross ß-sheet. The same polyQ fibres were evaluated by Raman spectroscopy and this further confirmed the ß-sheet structure, but indicated that the structure is highly rigid, as indicated by the strong Amide I signal at 1659 cm(-1). CARS spectra were simulated using the Raman spectrum taking into account potential non-resonant contributions, providing evidence that the Amide I signal remains strong, but slightly shifted to lower wavenumbers. Combined CARS (1657 cm(-1)) and multi-photon fluorescence microscopy of chimeric fusions of yellow fluorescent protein (YFP) with polyQ (Q40) expressed in the body wall muscle cells of Caenorhabditis elegans nematodes (1 day old adult hermaphrodites) revealed diffuse and foci patterns of Q40-YFP that were both fluorescent and exhibited stronger CARS (1657 cm(-1)) signals than in surrounding tissues at the resonance for the cross ß-sheet polyQ in vitro.

Inhibition of the MRP1-mediated transport of the menadione-glutathione conjugate (thiodione) in HeLa cells as studied by SECM.

Oxidative stress induced in live HeLa cells by menadione (2-methyl-1,4-napthaquinone) was studied in real time by scanning electrochemical microscopy (SECM). The hydrophobic molecule menadione diffuses through a living cell membrane where it is toxic to the cell. However, in the cell it is conjugated with glutathione to form thiodione. Thiodione is then recognized and transported across the cell membrane via the ATP-driven MRP1 pump. In the extracellular environment, thiodione was detected by the SECM tip at levels of 140, 70, and 35 µM upon exposure of the cells to menadione concentrations of 500, 250, and 125 µM, respectively. With the aid of finite element modeling, the kinetics of thiodione transport was determined to be 1.6 10(-7) m/s, about 10 times faster than menadione uptake. Selective inhibition of these MRP1 pumps inside live HeLa cells by MK571 produced a lower thiodione concentration of 50 µM in presence of 500 µM menadione and 50 µM MK571. A similar reduced (50% drop) thiodione efflux was observed in the presence of monoclonal antibody QCRL-4, a selective blocking agent of the MRP1 pumps. The reduced thiodione flux confirmed that thiodione was transported by MRP1, and that glutathione is an essential substrate for MRP1-mediated transport. This finding demonstrates the usefulness of SECM in quantitative studies of MRP1 inhibitors and suggests that monoclonal antibodies can be a useful tool in inhibiting the transport of these MDR pumps, and thereby aiding in overcoming multidrug resistance.

Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy.

We describe voltage-switching mode scanning electrochemical microscopy (VSM-SECM), in which a single SECM tip electrode was used to acquire high-quality topographical and electrochemical images of living cells simultaneously. This was achieved by switching the applied voltage so as to change the faradaic current from a hindered diffusion feedback signal (for distance control and topographical imaging) to the electrochemical flux measurement of interest. This imaging method is robust, and a single nanoscale SECM electrode, which is simple to produce, is used for both topography and activity measurements. In order to minimize the delay at voltage switching, we used pyrolytic carbon nanoelectrodes with 6.5-100 nm radii that rapidly reached a steady-state current, typically in less than 20 ms for the largest electrodes and faster for smaller electrodes. In addition, these carbon nanoelectrodes are suitable for convoluted cell topography imaging because the RG value (ratio of overall probe diameter to active electrode diameter) is typically in the range of 1.5-3.0. We first evaluated the resolution of constant-current mode topography imaging using carbon nanoelectrodes. Next, we performed VSM-SECM measurements to visualize membrane proteins on A431 cells and to detect neurotransmitters from a PC12 cells. We also combined VSM-SECM with surface confocal microscopy to allow simultaneous fluorescence and topographical imaging. VSM-SECM opens up new opportunities in nanoscale chemical mapping at interfaces, and should find wide application in the physical and biological sciences.

Scanning probe-enabled nanocombinatorics define the relationship between fibronectin feature size and stem cell fate.

We report the development of a powerful analytical method that utilizes a tilted elastomeric pyramidal pen array in the context of a scanning probe lithography experiment to rapidly prepare libraries having as many as 25 million features over large areas with a range of feature sizes from the nano- to microscale. This technique can be used to probe important chemical and biological processes, opening up the field of nanocombinatorics. In a proof-of-concept investigation of mesenchymal stem cell (MSC) differentiation, combinatorial patterns first enabled a rapid and systematic screening of MSC adhesion, as a function of feature size, while uniform patterns were used to study differentiation with statistically significant sample sizes. Without media containing osteogenic-inducing chemical cues, cells cultured on nanopatterned fibronectin substrates direct MSC differentiation towards osteogenic fates when compared to nonpatterned fibronectin substrates. This powerful and versatile approach enables studies of many systems spanning biology, chemistry, and engineering areas.

Intrinsic material properties of trabecular bone by nanoindentation testing of biopsies taken from healthy women before and after menopause.

Postmenopausal osteoporosis in women is characterized by an increase in bone fragility and risk of fracture. In addition to transmenopausal decline in three-dimensional trabecular bone architecture, changes in intrinsic material properties (local stiffness, damping, and hardness) may contribute to increased bone fragility. In this study, nanoindentation was used to quantify transmenopausal changes in the intrinsic properties of trabecular bone. Paired transilial biopsy specimens were used from a previously reported study in which bone biopsies were obtained from women prior to menopause (premenopausal, age 49.0 ± 1.9) and at 12 months past the last menstrual period (postmenopausal, age 54.6 ± 2.2). Elastic and viscoelastic material properties of the trabecular bone were measured using quasi-static and dynamic nanoindentation techniques, respectively. Paired Student's t tests (n = 15) were performed to assess the significance of the measured intrinsic properties. Trabecular bone microarchitecture is compromised in postmenopausal women, and although this loss is associated with a trend toward reduction in some intrinsic properties (storage modulus), we found no statistically significant changes in bone intrinsic properties between healthy pre- and postmenopausal biopsies in the quasi-static results and frequency-averaged dynamic results.

Peptide-antibody complex as handle for single-molecule cut & paste.

Molecule-by-molecule arrangement of proteins, for example, in enzymatic networks of predefined composition and proximity, is a major goal that may be accomplished by the single-molecule cut-and-paste technique (SMC&P). For this purpose, co-expressed anchors and handles as protein tags should be employed. As a first step in this direction, the authors develop an SMC&P design which exploits an antibody-peptide complex as a molecular handle.

High-frequency electromagnetic dynamics properties of THP1 cells using scanning microwave microscopy.

Microwave measurements combined with scanning probe microscopy is a novel tool to explore high-localized mechanical and electrical properties of biological species. Complex permittivities and permeabilities are detected through slight variations of an incident microwave signal. Here we report the high-frequency dependence of the electromagnetic dynamic characteristics in human monocytic leukemia cells (THP1) through local measurements by scanning microwave microscopy (SMM). The amplitude and phase images were shown to depend on the applied resonance frequency. While the amplitude yields information about the resistivity determined by the water and the ionic strength, the phase information reflects the dielectric losses arising from the fluid density.

Estudian las características histopatológicas y moleculares de las células de Langerhans en la mucosa bucal / Langerhans cells in oral mucosa: hystopatological and molecular characterization

Actualmente, las células dendríticas en tejidos periféricos, piel y mucosas son centro de numerosas publicaciones. De acuerdo con su localización se clasifican en subtipos de diferente denominación. Su posición apropiada les permite ejercer un papel crucial en la detección y la captación de antígenos y su presentación a los linfocitos T en centros linfoides, iniciando la inmunidad innata y la inmunidad adaptativa. Debe destacarse además su actuación en la tolerancia inmunitaria y su participación en diversas enfermedades autoinmunitarias como la enfermedad periodontal e incluso el cáncer de células escamosas. Presentamos una actualización resumida de los últimos estudios realizados y destacamos el conocimiento de los marcadores inmunológicos y las características fundamentales de los subtipos, células de Langerhans y células plasmocitoides, especialmente en la mucosa oral. La disminución de los distintos fenotipos de células dendríticas en lesiones cancerizables y en la vejez, como su ausencia en neoplasias malignas son aspectos destacables. Comunicamos a la vez nuestro primer acercamiento a la microscopia electrónica de barrido para observarlas en tejido mucoso sano y de tres lesiones: mucosa hiperplásica, gingivitis descamativa y úlceras aftosas recidivantes con vasculitis.