Difference equation sensor characterization algorithms for two-thermocouple probes

The characterization of thermocouple sensors for temperature measurement in variable flow environments is a challenging problem. In this paper, novel difference equation-based algorithms are presented that allow in situ characterization of temperature measurement probes consisting of two-thermocouple sensors with differing time constants. Linear and non-linear least squares formulations of the characterization problem are introduced and compared in terms of their computational complexity, robustness to noise and statistical properties. With the aid of this analysis, least squares optimization procedures that yield unbiased estimates are identified. The main contribution of the paper is the development of a linear two-parameter generalized total least squares formulation of the sensor characterization problem. Monte-Carlo simulation results are used to support the analysis.

Absolute Calibration of Atomic Density Measurements by Laser-Induced Fluorescence Spectroscopy with Two-Photon Excitation (TALIF)

The two-photon resonances of atomic hydrogen (? = 2 × 205.1 nm), atomic nitrogen (? = 2 × 206.6 nm) and atomic oxygen (? = 2 × 225.6 nm) are investigated together with two selected transitions in krypton (? = 2×204.2 nm) and xenon (? = 2×225.5 nm). The natural lifetimes of the excited states, quenching coefficients for the most important collisions partners, and the relevant ratios of the two-photon excitation cross sections are measured. These data can be applied to provide a calibration for two-photon laser-induced fluorescence measurements based on comparisons with spectrally neighbouring noble gas resonances.

Laser-Induced Fluorescence Measurements of Absolute Atomic Densities: Concepts and Limitations

The potential of laser-induced fluorescence spectroscopy of atoms is reviewed with emphasis on the determination of absolute densities. Examples of experiments with single-photon and two-photon excitation are presented. Calibration methods applicable with the different schemes are discussed. A new method is presented that has the potential to allow absolute measurement in plasmas of elevated pressure where collisional depletion of the excited state is present.

Absolute Atomic Oxygen Density Measurements by Two-Photon Absorption Laser-induced Fluorescence Spectroscopy in an RF-Excited Atmospheric Pressure Plasma Jet

The atmospheric pressure plasma jet is a capacitively coupled radio frequency discharge (13.56 MHz) running with a high helium flux (2m3 h-1) between concentric electrodes. Small amounts (0.5%) of admixed molecular oxygen do not disturb the homogeneous plasma discharge. The jet effluent leaving the discharge through the ring-shaped nozzle contains high concentrations of radicals at a low gas temperature—the key property for a variety of applications aiming at treatment of thermally sensitive surfaces. We report on absolute atomic oxygen density measurements by two-photon absorption laser-induced fluorescence (TALIF) spectroscopy in the jet effluent. Calibration is performed with the aid of a comparative TALIF measurement with xenon. An excitation scheme (different from the one earlier published) providing spectral matching of both the two-photon resonances and the fluorescence transitions is applied.

Expedited Solid-Phase Synthesis of Fluorescently Labeled and Biotinylated Aminoalkane Diphenyl Phosphonate Affinity Probes for Chymotrypsin- and Elastase-Like Serine Proteases

In this study, we report on a novel, expedited solid-phase approach for the synthesis of biotinylated and fluorescently tagged irreversible affinity based probes for the chymotrypsin and elastase-like serine proteases. The novel solid-phase biotinylation or fluorescent labeling of the aminoalkane diphenyl phosphonate warhead using commercially available Biotin-PEG-NovaTag or EDANS NovaTag resin permits rapid, facile synthesis of these reagents. We demonstrate the kinetic evaluation and utilization of a number of these irreversible inactivators for chymotrypsin-like (chymotrypsin/human cathepsin G) and elastase-like serine proteases. Encouragingly, these compounds display comparable potency against their target proteases as their N-benzyloxycarbonyl (Cbz)-protected parent compounds, from which they were derived, and function as efficient active site-directed inactivators of their target proteases. We subsequently applied the biotinylated reagents for the sensitive detection of protease species via Western blot, showing that the inactivation of the protease was specifically mediated through the active site serine. Furthermore, we also demonstrate the successful detection of serine protease species with the fluorescently labeled derivatives “in-gel”, thus avoiding the need for downstream Western blotting. Finally, we also show the utility of biotinylated and pegylated affinity probes for the isolation/enrichment of serine protease species, via capture with immobilized streptavidin, and their subsequent identification via de novo sequencing. Given their selectivity of action against the serine proteases, we believe that these reagents can be exploited for the direct, rapid, and selective identification of these enzymes from biological milieu containing multiple protease subclasses.

Interactions between the budding yeast IQGAP homologue Iqg1p and its targets revealed by a split-EGFP bimolecular fluorescence complementation assay.

A split-EGFP based bimolecular fluorescence complementation (BiFC) assay has been used to detect interactions between the Saccharomyces cerevisiae cytoskeletal scaffolding protein Iqg1p and three targets: myosin essential light chain (Mlc1p), calmodulin (Cmd1p) and the small GTPase Cdc42p. The format of the BiFC assay used ensures that the proteins are expressed at wild type levels thereby avoiding artefacts due to overexpression. This is the first direct in vivo detection of these interactions; in each case, the complex is localised to discrete regions of the yeast cytoplasm. The labelling with EGFP fragments results in changes in growth kinetics, cell size and budding frequency. This is partly due to the reassembled EGFP locking the complexes into essentially permanent interactions. The consequences of this for Iqg1p interactions and BiFC assays in general are discussed. (c) 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Quantitative mapping of aqueous microfluidic temperature with sub-degree resolution using fluorescence lifetime imaging microscopy

The use of a water-soluble, thermo-responsive polymer as a highly sensitive fluorescence-lifetime probe of microfluidic temperature is demonstrated. The fluorescence lifetime of poly(N-isopropylacrylamide) labelled with a benzofurazan fluorophore is shown to have a steep dependence on temperature around the polymer phase transition and the photophysical origin of this response is established. The use of this unusual fluorescent probe in conjunction with fluorescence lifetime imaging microscopy (FLIM) enables the spatial variation of temperature in a microfluidic device to be mapped, on the micron scale, with a resolution of less than 0.1 degrees C. This represents an increase in temperature resolution of an order of magnitude over that achieved previously by FLIM of temperature-sensitive dyes

Fluorescence (FISH) and chromogenic (CISH) in situ hybridisation in prostate carcinoma cell lines: comparison and use of virtual microscopy

Chromogenic in situ hybridisation (CISH) has become an attractive alternative to fluorescence in situ hybridisation (FISH) due to its permanent stain which is more familiar to pathologists and because it can be viewed using light microscopy, The aim of the present study is to examine reproducibility in the assessment of abnormal chromosome number by CISH in comparison to FISH. Using three prostate cell lines - PNTIA (derived from normal epithelium), LNCAP and DU145 (derived from prostatic carcinoma), chromosomes 7 and 8 were counted in 40 nuclei in FISH preparations (x100 oil immersion) and 100 nuclei in CISH preparations (x40) by two independent observers. The CISH slides were examined using standard fight microscopy and virtual microscopy. Reproducibitity was examined using paired Student's t-test (P