Collection of highly germinative pseudochain conidia of Oidium neolycopersici from conidiophores by electrostatic attraction

A population of simultaneously germinating conidia is an ideal inoculum of the powdery mildew pathogen, Oidium neolycopersici. In conditions of no or low wind velocity, O. neolycopersici successively stacks mature conidia on conidiophores in a chain formation (pseudochain), without releasing the precedent mature conidia. These pseudochain conidia represent a perfect inoculum, in which all conidia used for inoculation germinate simultaneously. However, we found that conidia must be collected before they fall to the leaf surface, because the germination rate was lower among conidia deposited on the leaf surface. We used an electrostatic spore collector to collect the pseudochain conidia, and their high germination rate was not affected by this treatment. The spore collector consisted of an electrified insulator probe, which created an electrostatic field around its pointed tip, and attracted conidia within its electric field. The attractive force created by the probe tip was directly proportional to voltage, and was inversely proportional to the distance between the tip and a target colony on a leaf. Pseudochain conidia were successfully collected by bringing the electrified probe tip close to target colonies on leaves. In this way, conidia were collected from colonies at 3-d intervals. This effectively collected all conidia from conidiophores before they dropped to the leaf surface. A high germination rate was observed among conidia attracted to the probe tip (95.5 ± 0.6 %). Conidia were easily suspended in water with added surfactant, and retained their germination ability. These conidia were infective and produced conidia in pseudochains on conidiophores after inoculation. The electrostatic spore collection method can be used to collect conidia as they form on conidiophores, thus obtaining an inoculum population in which all of the conidia germinate simultaneously.     

The Supraclavicular Fossa Ultrasound View for Central Venous Catheter Placement and Catheter Change Over Guidewire

The supraclavicular fossa ultrasound view can be useful for central venous catheter (CVC) placement. Venipuncture of the internal jugular veins (IJV) or subclavian veins is performed with a micro-convex ultrasound probe, using a neonatal abdominal preset with a probe frequency of 10 Mhz at a depth of 10-12 cm. Following insertion of the guidewire into the vein, the probe is shifted to the right supraclavicular fossa to obtain a view of the superior vena cava (SVC), right pulmonary artery and ascending aorta. Under real-time ultrasound view, the guidewire and its J-tip is visualized and pushed forward to the lower SVC. Insertion depth is read from guidewire marks using central venous catheter. CVC is then inserted following skin and venous dilation. The supraclavicular fossa view is most suitable for right IJV CVC insertion. If other insertion sites are chosen the right supraclavicular fossa should be within the sterile field. Scanning of the IJVs, brachiocephalic veins and SVC can reveal significant thrombosis before venipuncture. Misplaced CVCs can be corrected with a change over guidewire technique under real-time ultrasound guidance. In conjunction with a diagnostic lung ultrasound scan, this technique has a potential to replace chest radiograph for confirmation of CVC tip position and exclusion of pneumothorax. Moreover, this view is of advantage in patients with a non-p-wave cardiac rhythm were an intra-cardiac electrocardiography (ECG) is not feasible for CVC tip position confirmation. Limitations of the method are lack of availability of a micro-convex probe and the need for training.     

A Two Compartment Model Method for Continuous Determination of Hydraulic Parameters of Higher Plant Cells by Pressure Probe Technique

Zhu, G-L. and Lou, C-H. 1988. A two compartment model methodfor continuous determination of hydraulic parameters of higherplant cells by pressure probe technique.—J. exp. BoL 39:961–971. The new model treats the whole measuring system as two compartments:the vacuole and the cavity of the probe, which are connectedby a microcapillary. Instead of a short injection, a continuousinjection of distilled water is used in this model. The mainprinciple of the calculation method is to estimate cell turgorpressure and hydraulic conductivity continuously from the resistanceof the microcapillary and the probe pressure as well as thecurrent flow through the microcapillary. The model avoids theeffect of membrane potential changes on the measurement of hydraulicparameters in pressure steps. In this model, microcapillarieswith a tip diameter smaller than flow rate-limiting dimensionscan be employed. The present method provides a way to monitorcontinuously cell hydraulic conductivity during the pressurerelaxation process with a time resolution in seconds. The calculationis performed automatically using a microcomputer. Key words: Two compartment model, cell pressure probe, computer     

Measurement of light gradients and spectral regime in plant tissue with a fiber optic probe

A method is described in which light gradients and spectral regime can be measured within plant tissue using fiber optics. A fiber optic probe was made by modifying a single optical fiber (200 μm diameter) so that it had a light harvesting end that was a truncated tip 20–70 μm in diameter. The probe was a directional sensor with a half-band acceptance angle of 17–20°. Light measurements were made as the fiber optic probe was driven through plant tissue by a motorized micromanipulator, and the light that entered the fiber tip was piped to a spectroradiometer. By irradiating green leaf tissue of the succulent Crassula falcata L. with collimated light and inserting the probe from different directions, it was possible to measure light quality and quantity at different depths. Collimated light was scattered completely by the initial 1.0 mm of leaf tissue, which also greatly attenuated all light except the green and far-red. Light scatter contributed significantly to light quantity and had a pronounced spectral structure. Immediately beneath the irradiated surface the amount of light at 550 nm was 1.2 times that of the incident light. The light gradient declined rapidly to 0.5 times incident light at 1.4 mm depth. In contrast, the amount of light at 750 nm increased during the initial 0.5 mm to 2.9 times incident light and then declined linearly to 0.5 times incident light at the dark side of the leaf (4.5 mm). The implications of the magnitude of the contribution of light scatter to the light gradient is also discussed.     

Scanning near-field fluorescence resonance energy transfer microscopy

A new microscopic technique is demonstrated that combines attributes from both near-field scanning optical microscopy (NSOM) and fluorescence resonance energy transfer (FRET). The method relies on attaching the acceptor dye of a FRET pair to the end of a near-field fiber optic probe. Light exiting the NSOM probe, which is nonresonant with the acceptor dye, excites the donor dye introduced into a sample. As the tip approaches the sample containing the donor dye, energy transfer from the excited donor to the tip-bound acceptor produces a red-shifted fluorescence. By monitoring this red-shifted acceptor emission, a dramatic reduction in the sample volume probed by the uncoated NSOM tip is observed. This technique is demonstrated by imaging the fluorescence from a multilayer film created using the Langmuir-Blodgett (LB) technique. The film consists of L-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers containing the donor dye, fluorescein, separated by a spacer group of three arachidic acid layers. A DPPC monolayer containing the acceptor dye, rhodamine, was also transferred onto an NSOM tip using the LB technique. Using this modified probe, fluorescence images of the multilayer film reveal distinct differences between images collected monitoring either the donor or acceptor emission. The latter results from energy transfer from the sample to the NSOM probe. This method is shown to provide enhanced depth sensitivity in fluorescence measurements, which may be particularly informative in studies on thick specimens such as cells. The technique also provides a mechanism for obtaining high spatial resolution without the need for a metal coating around the NSOM probe and should work equally well with nonwaveguide probes such as atomic force microscopy tips. This may lead to dramatically improved spatial resolution in fluorescence imaging.     

Antibody recognition imaging by force microscopy.

We have developed a method that combines dynamic force microscopy with the simultaneous molecular recognition of an antigen by an antibody, during imaging. A magnetically oscillated atomic force microscopy tip carrying a tethered antibody was scanned over a surface to which lysozyme was bound. By oscillating the probe at an amplitude of only a few nanometers, the antibody was kept in close proximity to the surface, allowing fast and efficient antigen recognition and gentle interaction between tip and sample. Antigenic sites were evident from reduction of the oscillation amplitude, as a result of antibody-antigen recognition during the lateral scan. Lysozyme molecules bound to the surface were recognized by the antibody on the scanning tip with a few nanometers lateral resolution. In principle, any ligand can be tethered to the tip; thus, this technique could potentially be used for nanometer-scale epitope mapping of biomolecules and localizing receptor sites during biological processes.     

A helium gas probe for use in cryosurgery

The design and testing of a prototype cryosurgical probe utilizing helium gas precooled with liquid nitrogen are described. An 8-mm-diameter probe produced an ice ball with a diameter of 28 mm after 10 min freezing using a helium gas flow rate of 42 liter/min. This indicated a surface heat transfer coefficient of 0.34 W/cm² °K and temperature of ?138 °C at the probe tip. Improved performance figures can be achieved using higher gas pressures and flow rates. A helium gas flow system schematic for use with this new type of cryoprobe is also presented. It is claimed that this system will overcome the problems of developing both multiple-tipped probes and small-diameter needle probes for use in cryoanalgesia.     

Enlarged gold-tipped silicon microprobe arrays and signal compensation for multi-site electroretinogram recordings in the isolated carp retina

In order to record multi-site electroretinogram (ERG) responses in isolated carp retinae, we utilized three-dimensional (3D), extracellular, 3.5-μm-diameter silicon (Si) probe arrays fabricated by the selective vapor-liquid-solid (VLS) growth method. Neural recordings with the Si microprobe exhibit low signal-to-noise (S/N) ratios of recorded responses due to the high-electrical-impedance characteristics of the small recording area at the probe tip. To increase the S/N ratio, we designed and fabricated enlarged gold (Au) tipped Si microprobes (10-μm-diameter Au tip and 3.5-μm-diameter probe body). In addition, we demonstrated that the signal attenuation and phase delay of ERG responses recorded via the Si probe can be compensated by the inverse filtering method. We conclude that the reduction of probe impedance and the compensation of recorded signals are useful approaches to obtain distortion-free recording of neural signals with high-impedance microelectrodes.     

Atomic force microscopy-based single virus particle spectroscopy

Atomic force microscopy-based single virus particle force spectroscopy was developed using dielectrophoresis for fixing virions at the tip of an atomic force microscope (AFM) probe. Electron microscopic visualization was found to be necessary to prove the deposition of virus particles on the tip of the AFM probe, while fixation of single virions by incubating the tip with a virus suspension proved impossible. Force spectroscopy measurements were performed for the vaccinia virus, influenza virus, and bacteriophage AP22. ForceReader special software was designed for analyzing the force–distance curves.     

Probing membrane proteins using atomic force microscopy

To gain insights into how biological molecules function, advanced technologies enabling imaging, sensing, and actuating single molecules are required. The atomic force microscope (AFM) would be one of novel potential tools for these tasks. In this study, techniques and efforts using AFM to probe biomolecules are introduced and reviewed. The state-of-art techniques for characterizing specific single receptor using the functionalized AFM tip are discussed. An example of studying the angiotensin II type 1 (AT1) receptors expressed in sensory neuronal cells by AFM with a functionalized tip is given. Perspectives for identifying and characterizing specific individual membrane proteins using AFM in living cells are provided. Given that many diseases have their roots at the molecular scale and are best understood as a malfunctioning biological nanomachines, the prospects of these unique techniques in basic biomedical research or in clinical practice are beyond our imagination.     

Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be measured to ~10 pN by a laser and sectored detector, which can be converted to image topography. Amplitude modulation or “tapping mode” AFM involves the probe making intermittent contact with the surface while oscillating at its resonant frequency to produce an image. Used in conjunction with a fluid cell, tapping-mode AFM enables the imaging of biological macromolecules such as proteins in physiologically relevant conditions. Tapping-mode AFM requires manual tuning of the probe and frequent adjustments of a multitude of scanning parameters which can be challenging for inexperienced users. To obtain high-quality images, these adjustments are the most time consuming.PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) produces an image by measuring a force response curve for every point of contact with the sample. With ScanAsyst software, PF-QNM can be automated. This software adjusts the set-point, drive frequency, scan rate, gains, and other important scanning parameters automatically for a given sample. Not only does this process protect both fragile probes and samples, it significantly reduces the time required to obtain high resolution images. PF-QNM is compatible for AFM imaging in fluid; therefore, it has extensive application for imaging biologically relevant materials.The method presented in this paper describes the application of PF-QNM to obtain images of a bacterial red-light photoreceptor, RpBphP3 (P3), from photosynthetic R. palustris in its light-adapted state. Using this method, individual protein dimers of P3 and aggregates of dimers have been observed on a mica surface in the presence of an imaging buffer. With appropriate adjustments to surface and/or solution concentration, this method may be generally applied to other biologically relevant macromolecules and soft materials.     

Rigid-body fitting to atomic force microscopy images for inferring probe shape and biomolecular structure

Atomic force microscopy (AFM) can visualize functional biomolecules near the physiological condition, but the observed data are limited to the surface height of specimens. Since the AFM images highly depend on the probe tip shape, for successful inference of molecular structures from the measurement, the knowledge of the probe shape is required, but is often missing. Here, we developed a method of the rigid-body fitting to AFM images, which simultaneously finds the shape of the probe tip and the placement of the molecular structure via an exhaustive search. First, we examined four similarity scores via twin-experiments for four test proteins, finding that the cosine similarity score generally worked best, whereas the pixel-RMSD and the correlation coefficient were also useful. We then applied the method to two experimental high-speed-AFM images inferring the probe shape and the molecular placement. The results suggest that the appropriate similarity score can differ between target systems. For an actin filament image, the cosine similarity apparently worked best. For an image of the flagellar protein FlhA_C, we found the correlation coefficient gave better results. This difference may partly be attributed to the flexibility in the target molecule, ignored in the rigid-body fitting. The inferred tip shape and placement results can be further refined by other methods, such as the flexible fitting molecular dynamics simulations. The developed software is publicly available.     

Methods for measuring acoustic power of an ultrasonic neurosurgical device

Measurement of the acoustic power in high-energy ultrasonic devices is complex due to occurrence of the strong cavitation in front of the sonotrode tip. In our research we used three methods for characterization of our new ultrasonic probe for neuroendoscopic procedures. The first method is based on the electromechanical characterization of the device measuring the displacement of the sonotrode tip and input electrical impedance around excitation frequency with different amounts of the applied electrical power The second method is based on measuring the spatial pressure magnitude distribution of an ultrasound surgical device produced in an anechoic tank. The acoustic reciprocity principle is used to determinate the derived acoustic power of equivalent ultrasound sources at frequency components present in the spectrum of radiated ultrasonic waves. The third method is based on measuring the total absorbed acoustic power in the restricted volume of water using the calorimetric method. In the electromechanical characterization, calculated electroacoustic efficiency factor from equivalent electrical circuits is between 40-60%, the same as one obtained measuring the derived acoustic power in an anechoic tank when there is no cavitation. When cavitation activity is present in the front of the sonotrode tip the bubble cloud has a significant influence on the derived acoustic power and decreases electroacoustic efficiency. The measured output acoustic power using calorimetric method is greater then derived acoustic power, due to a large amount of heat energy released in the cavitation process.     

Involvement of EphA2 in the formation of the tail notochord via interaction with ephrinA1

Eph receptors have been implicated in cell-to-cell interaction during embryogenesis. We generated EphA2 mutant mice using a gene trap method. Homozygous mutant mice developed short and kinky tails. In situ hybridization using a Brachyury probe found the notochord to be abnormally bifurcated at the caudal end between 11.5 and 12.5 days post coitum. EphA2 was expressed at the tip of the tail notochord, while one of its ligands, ephrinA1, was at the tail bud in normal mice. In contrast, EphA2-deficient notochordal cells were spread broadly into the tail bud. These observations suggest that EphA2 and its ligands are involved in the positioning of the tail notochord through repulsive signals between cells expressing these molecules on the surface.     

In vivo microdialysis--a new approach to the analysis of neurotransmitters in the brain

Microdialysis is a new technique to monitor levels of chemical compounds in the extracellular space over time. It involves the implantation of a microdialysis probe into the brain tissue. The probe is similar to a push-pull probe but the perfusate is contained inside a semi-permeable membrane located at the tip of the probe. Substances in the extracellular fluid will diffuse into the perfusate while substances included in the perfusate will diffuse into the tissue. This principle opens up a wealth of possibilities to monitor chemical events within the brain and to study the working mechanism of various drugs. The perfusate may be analysed by a number of different techniques. In this paper we give a short summary of various HPLC techniques that have proven particularly useful.     

Combinatorial peptide on-resin analysis: optimization of static nanoelectrospray ionization technique for sequence determination.

The optimizations of static nanoelectrospray parameters to determine peptide or mimetic sequences released from resin were explored. Several different manufacturers of probe tips were utilized and a method was developed for the direct analysis of bead-bound peptides by nanoelectrospray. The method involved minimum sample handling to assure maximum recovery from individual beads. Parameters that were explored included an inside and outside wash of the probe tip, the distance from the probe housing to the probe tip, source temperature, drying gas flow, individual tips and presence of beads. The same soluble synthetic peptide was used in all comparisons, which had a molecular weight of 717 amu. The discovery of the sequence of a bead-bound peptide was achieved. The parameters that were found to effect signal were outside wash, presence of bead and distance. There was the need for pneumatic assist to initiate electrospray on some occasions, although this generally resulted in unsatisfactory performance.     

Design of an MRI-compatible robotic stereotactic device for minimally invasive interventions in the breast

The objective of this work was to develop a robotic device to perform biopsy and therapeutic interventions in the breast with real-time magnetic resonance imaging (MRI) guidance. The device was designed to allow for (i) stabilization of the breast by compression, (ii) definition of the interventional probe trajectory by setting the height and pitch of a probe insertion apparatus, and (iii) positioning of an interventional probe by setting the depth of insertion. The apparatus is fitted with five computer-controlled degrees of freedom for delivering an interventional procedure. The entire device is constructed of MR compatible materials, i.e. nonmagnetic and non-conductive, to eliminate artifacts and distortion of the MR images. The apparatus is remotely controlled by means of ultrasonic motors and a graphical user interface, providing real-time MR-guided planning and monitoring of the operation. Joint motion measurements found probe placement in less than 50 s and sub-millimeter repeatability of the probe tip for same-direction point-to-point movements. However, backlash in the rotation joint may incur probe tip positional errors of up to 5 mm at a distance of 40 mm from the rotation axis, which may occur for women with large breasts. The imprecision caused by this backlash becomes negligible as the probe tip nears the rotation axis. Real-time MR-guidance will allow the physician to correct this error Compatibility of the device within the MR environment was successfully tested on a 4 Tesla MR human scanner     

Viewing and Inducing Symmetry Breaking at the Single‐Molecule Limit

Symmetry breaking by photons, electrons, and molecular interactions lies at the heart of many important problems as varied as the origin of homochiral life to enantioselective drug production. Herein we report a system in which symmetry breaking can be induced and measured in situ at the single‐molecule level using scanning tunneling microscopy. We demonstrate that electrical excitation of a prochiral molecule on an achiral surface produces large enantiomeric excesses in the chiral adsorbed state of up to 39%. The degree of symmetry breaking was monitored as a function of scanning probe tip state, and the results revealed that enantiomeric excesses are correlated with the intrinsic chirality in scanning probe tips themselves, as evidenced by height differences between single molecule enantiomers. While this work has consequences for the study of two‐dimensional chirality, more importantly, it offers a new method for interrogating the coupling of photons, electrons, and combinations of physical fields to achiral starting systems in a reproducible manner. This will allow the mechanism of chirality transfer to be studied in a system in which enantiomeric excesses are quantified accurately by counting individual molecules. Chirality 24:1051–1054, 2012. © 2012 Wiley Periodicals, Inc.     

Measurement of cell adhesion force by vertical forcible detachment using an arrowhead nanoneedle and atomic force microscopy

The properties of substrates and extracellular matrices (ECM) are important factors governing the functions and fates of mammalian adherent cells. For example, substrate stiffness often affects cell differentiation. At focal adhesions, clustered–integrin bindings link cells mechanically to the ECM. In order to quantitate the affinity between cell and substrate, the cell adhesion force must be measured for single cells. In this study, forcible detachment of a single cell in the vertical direction using AFM was carried out, allowing breakage of the integrin–substrate bindings. An AFM tip was fabricated into an arrowhead shape to detach the cell from the substrate. Peak force observed in the recorded force curve during probe retraction was defined as the adhesion force, and was analyzed for various types of cells. Some of the cell types adhered so strongly that they could not be picked up because of plasma membrane breakage by the arrowhead probe. To address this problem, a technique to reinforce the cellular membrane with layer-by-layer nanofilms composed of fibronectin and gelatin helped to improve insertion efficiency and to prevent cell membrane rupture during the detachment process, allowing successful detachment of the cells. This method for detaching cells, involving cellular membrane reinforcement, may be beneficial for evaluating true cell adhesion forces in various cell types.     

Metallic Probe with Integrated Symmetry-Breaking Nanoslits for Enhanced Nanofocusing

To isolate the focused near-field excitation at the probe apex from the contribution of the far-field illumination, a novel metallic probe which consisted of a taper cylinder tip and a couple of curved nanoslits on the probe base was designed. The local field enhancement and confinement performance varying with the structure parameters of the probe and the type of substrates were investigated by means of finite-difference time-domain simulation. With the optimized probe working in probe-substrate coupling mode, the local electric field intensity at the probe apex can be effectively enhanced by more than 500 times compared to the incident field.