A hyphenated optical trap capillary electrophoresis laser induced native fluorescence system for single-cell chemical analysis

Single-cell measurements allow a unique glimpse into cell-to-cell heterogeneity; even small changes in selected cells can have a profound impact on an organism's physiology. Here an integrated approach to single-cell chemical sampling and assay are described. Capillary electrophoresis (CE) with laser-induced native fluorescence (LINF) has the sensitivity to characterize natively fluorescent indoles and catechols within individual cells. While the separation and detection approaches are well established, the sampling and injection of individually selected cells requires new approaches. We describe an optimized system that interfaces a single-beam optical trap with CE and multichannel LINF detection. A cell is localized within the trap and then the capillary inlet is positioned near the cell using a computer-controlled micromanipulator. Hydrodynamic injection allows cell lysis to occur within the capillary inlet, followed by the CE separation and LINF detection. The use of multiple emission wavelengths allows improved analyte identification based on differences in analyte fluorescence emission profiles and migration time. The system enables injections of individual rat pinealocytes and quantification of their endogenous indoles, including serotonin, N-acetyl-serotonin, 5-hydroxyindole-3-acetic acid, tryptophol and others. The amounts detected in individual cells incubated in 5-hydroxytryptophan ranged from 10(-14) mol to 10(-16) mol, an order of magnitude higher than observed in untreated pinealocytes.     

Neurotransmitter sampling and storage for capillary electrophoresis analysis

Quantitative analysis of signaling molecules from single cells and cellular materials requires careful validation of the analytical methods. Strategies have been investigated that enable single neurons and neuronal tissues to be stored before being assayed for many low-weight, biologically active molecules, such as serotonin, dopamine, and citrulline. Both metacerebral cell and pedal ganglia homogenates isolated from Pleiuohbrain-Chae californica have been studied by capillary electrophoresis with two complimentary laser-induced fluorescence detection methods. For homogenized ganglia samples, several cellular analytes (such as arginine and citrulline) are unaffected by standing at room temperature for days. Many other analytes in the biological matrix, including the catecholamines and indolamines, degrade by 20% within 10 h at room temperature. Rapidly freezing samples or preserving them with ascorbic acid preserves more than 80% of the dopamine and about 70% of the serotonin even after five days. In addition, serotonin and dopamine remain completely stable for at least five days by combining the ascorbic acid preservation and freezing at -20 degrees C. The timing of preservation is critical in maintaining the original composition of the biological samples. Using our optimum storage protocol of freezing the sample within 2 h after isolation, we can store frozen homogenate ganglia samples for more than four weeks before assay while still obtaining losses less than 10% of the original serotonin and dopamine. The nanoliter-volume single cell samples, however, must be analyzed within 4 h to obtain losses of less than 10% for serotonin related metabolites.     

Asymptotic distribution theory of statistical functionals: The compact derivative approach for robust estimators

Summary Derivatives of statistical functionals have been used to derive the asymptotic distributions ofL-,M- andR-estimators. This approach is often heuristic because the types of derivatives chosen have serious limitations. The Gateaux derivative is too weak and the Fréchet derivative is too strong. In between lies the compact derivative. This paper obtains strong results in a rigorous manner using the compact derivative onC ₀(R). This choice of space allows results for a broader class of functionals than previous choices, and the fact that is often tight provides the compact set required. A major result is the derivation of the compact derivative of the inverse c.d.f. when the range space is endowed with the uniform norm. It has applications to the asymptotic theory ofL-,M- andR-estimators. We illustrate the power of this result by applications toL-estimators in settings including the one sample problem, data grouped by quantiles, and censored survival time data.     

Dynamics Analysis of Ground Contact Pressure of English Pointer Dogs

In this study force sensing resistors were used to determine thepressure on the central area of each of the weight bearing pads of thefore and hind paws of dogs at the walk. Six adult normal healthy EnglishPointer dogs were used in this project. Pressure data were collected byaffixing a force sensing resistor to the central area of the groundcontact surface of each weight bearing paw pad of the right fore andhind limbs. Pressure signal data from stance phase during walking wereanalyzed. Within paw pads, the pressure graphs were consistent in formand magnitude. Within paws, there were significant pressure differencesamong pads on both fore and hind limbs. The coefficient of restitution,the embedding dimension, and the Lyapunov exponents were calculated. Theability to measure and analyze pressure on individual paw pads providesinsight into soft tissue stresses on the palmar/plantar surface of thepaw. Pressure at a wound site on the pads has a detrimental effect onwound healing and a better understanding these stresses will be ofbenefit when suturing and bandaging pad wounds. Such information isespecially important in athletic and working dogs, e.g. search andrescue dogs.     

Nodal Bases for the Serendipity Family of Finite Elements

Using the notion of multivariate lower set interpolation, we construct nodal basis functions for the serendipity family of finite elements, of any order and any dimension. For the purpose of computation, we also show how to express these functions as linear combinations of tensor-product polynomials.     

Neurotransmitter sampling and storage for capillary electrophoresis analysis

Quantitative analysis of signaling molecules from single cells and cellular materials requires careful validation of the analytical methods. Strategies have been investigated that enable single neurons and neuronal tissues to be stored before being assayed for many low-weight, biologically active molecules, such as serotonin, dopamine, and citrulline. Both metacerebral cell and pedal ganglia homogenates isolated from Pleurobranchaea californica have been studied by capillary electrophoresis with two complimentary laser-induced fluorescence detection methods. For homogenized ganglia samples, several cellular analytes (such as arginine and citrulline) are unaffected by standing at room temperature for days. Many other analytes in the biological matrix, including the catecholamines and indolamines, degrade by 20% within 10 h at room temperature. Rapidly freezing samples or preserving them with ascorbic acid preserves more than 80% of the dopamine and about 70% of the serotonin even after five days. In addition, serotonin and dopamine remain completely stable for at least five days by combining the ascorbic acid preservation and freezing at –20 °C. The timing of preservation is critical in maintaining the original composition of the biological samples. Using our optimum storage protocol of freezing the sample within 2 h after isolation, we can store frozen homogenate ganglia samples for more than four weeks before assay while still obtaining losses less than 10% of the original serotonin and dopamine. The nanoliter-volume single cell samples, however, must be analyzed within 4 h to obtain losses of less than 10% for serotonin related metabolites. Received: 23 August 2000 / Revised: 2 November 2000 / Accepted: 7 November 2000     

Jones phase microscopy of transparent and anisotropic samples

We developed an interferometric microscopy technique, referred to as Jones phase microscopy, capable of extracting the spatially resolved Jones polarization matrix associated with transparent and anisotropic samples. This is a generalization of quantitative phase imaging, which is recovered from one diagonal element of the measured matrix. The principle of the technique is demonstrated with measurements of a liquid crystal spatial light modulator and the potential for live cell imaging with experiments on live neurons in culture.