Real-time monitoring of intracellular mRNA hybridization inside single living cells

A molecular beacon, an oligonucleotide probe with inherent signal transduction mechanisms, is an optimal tool for visualizing real-time mRNA hybridization in single living cells. Each molecular beacon (MB) consists of a single-stranded DNA molecule in a stem-loop conformation with a fluorophore linked to the 5' end and a quencher at the 3' end. In this study, we demonstrate real-time monitoring of mRNA-DNA hybridization inside living cells using molecular beacons. A MB specific for beta-actin mRNA has been designed and synthesized. After microinjection into the cytoplasm of single living kangaroo rat kidney cells (PtK2 cells), the MB hybridizes with beta-actin mRNA as shown by fluorescence measurements over time. Hybridization dynamics have been followed. Strict control experiments have been carried out to confirm that the fluorescence signal increase is indeed due to the hybridization of mRNA inside single living cells. Variation in the MB/mRNA hybridization fluorescent signal has been observed for different PtK2 cells, which indicates the amount of mRNA in different cells is different. We have also monitored the beta-1 andrenergic receptor mRNA inside the PtK2 cells. These studies demonstrate the feasibility of using MBs and the ultrasensitivity achieved in our fluorescence imaging system for real-time detection of mRNA hybridization and for the visualization of oligonucleotide/mRNA interactions inside single living cells.     

Simultaneously monitoring gene expression kinetics and genetic noise in single cells by optical well arrays

An optical fiber based well array platform was used for simultaneous, dynamic gene expression monitoring from hundreds of individual live Escherichia coli cells carrying promoter-fluorescent reporter gene fusions. High information content about gene expression kinetics and cell-to-cell gene expression variability can be collected from a single experiment. These data are invaluable for investigating gene regulation and gene networks as well as for systems biology applications.     

Simultaneous quantification of multiple magnetic nanoparticles

Distinct magnetic nanoparticle designs can have unique spectral responses to an AC magnetic field in a technique called the magnetic spectroscopy of Brownian motion (MSB). The spectra of the particles have been measured using desktop spectrometers and in vivo measurements. If multiple particle types are present in a region of interest, the unique spectral signatures allow for the simultaneous quantification of the various particles. We demonstrate such a potential experimentally with up to three particle types. This ability to concurrently detect multiple particles will enable new biomedical applications.     

Electromigration of cadmium in contaminated soils driven by single and multiple primary cells

This study tentatively used an iron (Fe) and carbon (C) primary cell, instead of dc electric power, to drive the electromigration of cadmium in contaminated soils. The addition of acid to C compartment increased the electric potential, while the addition of acid to Fe compartment had a slight influence on the potential. It was feasible using the primary cell to drive the electromigration of cadmium in kaolin. The electromigration efficiencies were highly related to the soil pH. Lower pH led to greater migration efficiency. The mechanisms involved the desorption of cadmium from soils to pore solution and the electromigration of cadmium in the pore solution. The desorption was critical to the electromigration process. The series of primary cells could expand the treatment area, but the electromigration efficiencies of cadmium in each cell were less than that achieved by single primary cell. Since the potential gradient produced by the primary cell was rather low, the electromigration rate of pollutants was very low and remediation duration was long. The application would be acceptable in some specific sites, such as acidic soils or artificially controlled acid conditions so that heavy metals have been desorbed from soils.     

Simultaneous formation of machine and human cells in group technology: a multiple objective approach

Since the advent of group technology (GT) as a primary manufacturing tool for reducing setup times and improving production efficiencies, its central theme has been the grouping of similar parts into part families and machines into machine cells. Although the formation of machine-part manufacturing cells is the essence of GT, its full benefits cannot be gained without forming ‘human’ cells in such a way that machine operators with similar expertise and skills are brought together to produce similar part families. Nevertheless, much of the existing GT literature overlooks the behavioural issues associated with a group of workers in the machine cell. This paper addresses such issues by simultaneously forming both machine and compatible human cells. In so doing, we develop a multiple objective model that enables us to analyse the tradeoff between economic and behavioural benefits.     

Analysis of single quantum-dot mobility inside 1D nanochannel devices

We visualized individual quantum dots using a combination of a confining nanochannel and an ultra-sensitive microscope system, equipped with a high numerical aperture lens and a highly sensitive camera. The diffusion coefficients of the confined quantum dots were determined from the experimentally recorded trajectories according to the classical diffusion theory for Brownian motion in two dimensions. The calculated diffusion coefficients were three times smaller than those in bulk solution. These observations confirm and extend the results of Eichmann et al (2008 Langmuir 24 714-21) to smaller particle diameters and more narrow confinement. A detailed analysis shows that the observed reduction in mobility cannot be explained by conventional hydrodynamic theory.     

Detection of a single electron produced inside bulk superfluid helium

The HERON project is an effort to develop a detector for low-energy solar neutrinos in real time by observing their elastic scattering from electrons using superfluid helium as the target material. By applying appropriate electric fields, the recoil electron can be separated from the positive ion, drifted upward to the liquid–vacuum interface, transmitted through the surface with the aid of a vortex ring, and detected using a calorimeter. By studying the correlation of the 16 eV photon signal produced by scintillation and the single-electron signal, we can locate a neutrino event in a large detector and distinguish it from the background events involving multiple Compton scattering.     

On monitoring of multiple non-linear profiles

Most state-of-the-art profile monitoring methods involve studies of one profile. However, a process may contain several sensors or probes that generate multiple profiles over time. Quality characteristics presented in multiple profiles may be related multiple aspects of product or process quality. Existing charting methods for simultaneous monitoring of each multiple profile may result in high false alarm rates. Or worse, they cannot correctly detect potential relationship changes among profiles. In this study, we propose two approaches to detect process shifts in multiple non-linear profiles. A simulation study was conducted to evaluate the performance of the proposed approaches in terms of average run length under different process shift scenarios. Pros and cons of the proposed methods are discussed. A guideline for choosing the proposed methods is introduced. In addition, a hybrid method combining the salient points of both approaches is explored. Finally, a real-world data-set from a vulcanisation process is used to demonstrate the implementation of the proposed methods.     

Transport, location, and quantal release monitoring of single cells on a microfluidic device

A novel microfluidic device has been developed for on-chip transport, location, and quantal release monitoring of single cells. The microfluidic device consists of a plate of PDMS containing channels for introducing cells and stimulants and a glass substrate into which a cell micro-chamber was etched. The two tightly reversibly sealed plates can be separated for respective cleaning, which significantly extends the lifetime of the microchip that is frequently clogged in cell analysis experiments. Using hydraulic pressure, single cells were transported and located on the microfluidic chip. After location of a single PC12 cell on the microfluidic chip, the cell was stimulated by nicotine that was also introduced through the micro-channels, and the quantum release of dopamine from the cell was amperometricly detected with our designed carbon fiber microelectrode. The results have demonstrated the convenience and efficiency of using the microfluidic chip for monitoring of quantal release from single cells and have offered a facile method for the analysis of single cells on microfluidic devices.     

The density control chart: a general approach for constructing a single chart for simultaneously monitoring multiple parameters

The majority of the existing literature on simultaneous control charts, i.e. control charting mechanisms that monitor multiple population parameters such as mean and variance on a single chart, assume that the process is normally distributed. In order to adjust and maintain the overall type-I error probability, these existing charts rely largely on the property that the sample mean and sample variance are independent under the normality assumption. Furthermore, the existing charting procedures cannot be readily extended to non-normal processes. In this article, we propose and study a general charting mechanism which can be used to construct simultaneous control charts for normal and non-normal processes. The proposed control chart, which we call the density control chart, is essentially based on the premise that if a sample of observations is from an in-control process, then another sample of observations is no less likely to be also from the in-control process if the likelihood of the latter is no less than the likelihood of the former. The density control chart is developed for normal and non-normal processes where the distribution of the plotting statistic of the density control chart can be explicitly derived. Real examples are given and discussed in these cases. We also discuss how the density control chart can be constructed in cases when the distribution of the plotting statistic cannot be determined. A discussion of potential future research is also given.     

Simultaneous optical and electrochemical recording of single nanoparticle electrochemistry

Single nanoparticle collisions have become popular for studying the electrochemical activity of single nanoparticles by determining the transient current during stochastic collisions with the electrode surface.However,if only the electrochemical current is measured,it remains challenging to identify and characterize the individual particle that is responsible for a specific current peak in a collision event;this hampers the understanding of the structure-activity relationship.Herein,we report simultaneous optical and electrochemical recording of a single nanoparticle collision;the electrochemical signal corresponds with the activity of a single nanoparticle,and the optical signal reveals the size and location of the same nanoparticle.Consequently,the structure (optical signal)-activity (electrochemical signal) relationship can be elucidated at the single nanopartide level;this has implications for various applications induding batteries,electrocatalysts,and electrochemical sensors.In addition,our previous studies have suggested an optical-to-electrochemical conversion model to independently calculate the electron transfer rate of single nanoparticles from the optical signal.The simultaneous optical and electrochemical recording achieved in the present work enables direct and quantitative validation of the optical-to-electrochemical conversion model.     

Creation of multiple nanodots by single ions

In the search to develop tools that are able to modify surfaces on the nanometre scale, the use of heavy ions with energies of several tens of MeV is becoming more attractive. Low-energy ions are mostly stopped by nuclei, which causes the energy to be dissipated over a large volume. In the high-energy regime, however, the ions are stopped by electronic excitations, and the extremely local (approximately 10 nm3) nature of the energy deposition leads to the creation of nanosized 'hillocks' or nanodots under normal incidence. Usually, each nanodot results from the impact of a single ion, and the dots are randomly distributed. Here we demonstrate that multiple, equally spaced dots, each separated by a few tens of nanometres, can be created if a single high-energy xenon ion strikes the surface at a grazing angle. By varying this angle, the number of dots, as well as their spacing, can be controlled.     

Simultaneous buckling design of stiffened shells with multiple cutouts

Buckling is usually initiated from a local region near the cutout for cylindrical stiffened shells under axial compression, and then the evolution of buckling waves is governed by the combined effects of local and global stiffness, which limit the load-carrying capacity. Therefore, a simultaneous buckling pattern is crucial for improving the structural efficiency. In this study, a multi-step optimization strategy for the integrated design of near and far fields away from cutouts is proposed, and the convergence criterion of buckling optimization is improved as a deformation-based index. The numerical implementation of the asymptotic homogenization method is utilized to construct an efficient finite element model for post-buckling analysis. A 5?m diameter stiffened shell in a launch vehicle demonstrates that the proposed framework can provide a simultaneous buckling design with high structural efficiency in an efficient manner. Both the buckling deformations and stress of the optimum design are more uniform compared to other optimum designs.     

单个冰球蓄冷过程的数值计算与性能研究

通过对冰球蓄冷过程的分析,在第三类边界条件下考虑过冷度对冰球蓄冷过程的影响,建立冰球蓄冷过程的数值仿真模型,并用实验数据验证模型的正确性。数值计算分析载冷剂温度、对流换热系数、冰球球径以及材质对蓄冷量和凝固完成时间等的影响规律,为冰球蓄冷过程的进一步优化设计和运行提供指导。     

Simultaneous determination of conductance and thermopower of single molecule junctions

We report the first concurrent determination of conductance (G) and thermopower (S) of single-molecule junctions via direct measurement of electrical and thermoelectric currents using a scanning tunneling microscope-based break-junction technique. We explore several amine-Au and pyridine-Au linked molecules that are predicted to conduct through either the highest occupied molecular orbital (HOMO) or the lowest unoccupied molecular orbital (LUMO), respectively. We find that the Seebeck coefficient is negative for pyridine-Au linked LUMO-conducting junctions and positive for amine-Au linked HOMO-conducting junctions. Within the accessible temperature gradients (<30 K), we do not observe a strong dependence of the junction Seebeck coefficient on temperature. From histograms of thousands of junctions, we use the most probable Seebeck coefficient to determine a power factor, GS(2), for each junction studied, and find that GS(2) increases with G. Finally, we find that conductance and Seebeck coefficient values are in good quantitative agreement with our self-energy corrected density functional theory calculations.     

Optical nanosensors for chemical analysis inside single living cells. 2. Sensors for pH and calcium and the intracellular application of PEBBLE sensors

Optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced for intracellular measurements of pH and calcium. Five varieties of pH-sensitive sensors and three different calcium-selective sensors are presented and discussed. Each sensor combines an ion-selective fluorescent indicator and an ion-insensitive internal standard entrapped within an acrylamide polymeric matrix. Calibrations and linear ranges are presented for each sensor. The photobleaching of dyes incorporated into PEBBLEs is comparable to that of the respective free dye that is incorporated within the matrix. These PEBBLE sensors are fully reversible over many measurements. The leaching of fluorescent indicator from the polymer is less than 50% over a 48-h period (note that a typical application time is only a few hours). The PEBBLE sensors have also been applied to intracellular analysis of the calcium flux in the cytoplasm of neural cells during the mitochondrial permeability transition. Specifically, a distinct difference is noted between cells of different types (astrocyte vs neuron-derived cells) with respect to their response to the toxicant m-dinitrobenzene (DNB). Use of PEBBLE sensors permits the quantitative discrimination of subtle differences between the ability of human SY5Y neuroblastoma and C6 glioma to respond to challenge with DNB. Specifically, measurement of intracellular calcium, the precursor to cell death, has been achieved.     

Measurement of gene expression from single adherent cells and spheroids collected using fast electrical lysis

The cytosol of a single adherent cell was collected by the electrical cell lysis method with a Pt-ring capillary probe, and the cellular messenger RNA (mRNA) was analyzed at a single-cell level. The ring electrode probe was positioned 20 microm above the cultured cells that formed a monolayer on an indium-tin oxide (ITO) electrode, and an electric pulse with a magnitude of 40 V was applied for 10 micros between the probe and the ITO electrodes in an isotonic sucrose solution. Immediately after the electric pulse, less than 1 microL of the lysed solution was collected using a micro-injector followed by RNA purification and first strand cDNA synthesis. Real-time PCR was performed to quantify the copy numbers of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression inside the single cell. The average copy numbers of GAPDH mRNA collected by the electrical cell lysis method were found to be comparable to those obtained by a simple capillary suction method. Although single-cell analysis has already been demonstrated, we have shown for the first time that the fast electrical cell lysis can be used for quantitative mRNA analysis at the single-cell level. This electrical cell lysis method was further applied for the analysis of mRNA obtained from single spheroids-the aggregated cellular masses formed during the three-dimensional culture -- as a model system to isolate small cellular clusters from tissues and organs.     

Computational fluid flow solution over endothelial cells inside the capillary vascular system

Computational blood flow analysis through glycocalyx located over the endothelial cells in human capillary vascular system is performed. Stable numerical schemes are developed for obtaining approximate solutions to the mixed problem to the partial differential equation with variable space operator to model the blood flow. Numerical techniques are developed by applying a procedure of the solution to the linear difference equation with matrix coefficients. The flow equations inside the core flow region and porous region are established. Discretization is done and the flow velocities in both regions are calculated. The effect of the flow over the glycocalyx is investigated. The wall shear stresses and the drag force over the glycocalyx are calculated. Copyright © 2007 John Wiley & Sons, Ltd.     

The mechanical properties and test of a single ZnO nanorod inside scanning electron microscopy

The bending and tensile tests of the ZnO nanorods were carried out by controlling a force sensor and a nano-manipulator inside a scanning electron microscope (SEM). The force sensor was mounted on the nano-manipulator, was controlled with the nano-manipulate. The load response during the mechanical test for the ZnO nanorod was obtained by using the force sensor which is formed as a cantilever. The elastic modulus of the ZnO nanorods after the tensile and bending tests were calculated and compared. The elastic modulus of ZnO nanorods was depended on a size and an aspect ratio of the ZnO nanorods. The difference of the elastic modulus of ZnO nanorods was obtained with a difference of test methods performed along crystal facets direction of the ZnO nanorods. The average elastic modulus calculated after the tensile test was approximately 57.15 GPa. In case of the bending test, the average elastic modulus was approximately 29.37 GPa.