Fluorescence microscopy of nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated ion channels abundantly expressed in the central nervous system. Changes in the assembly and trafficking of nAChRs are pertinent to disease states including nicotine dependence, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), and Parkinson’s disease (PD). Here we investigate the application of high resolution fluorescence techniques for the study of nAChR assembly and trafficking. We also describe the construction and validation of a fluorescent α5 subunit and subsequent experiments to elucidate the cellular mechanisms through which α5 subunits are expressed, assembled into mature receptors, and trafficked to the cell surface. The effects of a known single nucleotide polymorphism (D398N) in the intracellular loop of α5 are also examined.

Additionally, this report describes the development of a combined total internal reflection fluorescence (TIRF) and lifetime imaging (FLIM) technique and the first application of this methodology for elucidation of stochiometric composition of nAChRs. Many distinct subunit combinations can form functional receptors. Receptor composition and stoichiometry confers unique biophysical and pharmacological properties to each receptor sub-type. Understanding the nature of assembly and expression of each receptor subtype yields important information about the molecular processes that may underlie the mechanisms through which nAChR contribute to disease and addiction states.

Spectral technique for measuring hyperfine structure of atoms

A new spectral technique for measuring the hyperfine structure of atoms is reported. A divergent atomic beam and a divergent laser beam are crossed. Because of the Doppler effect, the hyperfine structure of atomic levels will be directly displayed in the interaction region in the form of spatially resolved fluorescence arc bands. By measuring the spatial-fluorescence intensity distribution, it is possible to obtain the hyperfine splittings of atomic levels. Basic principles and experimental results are given.

Effect of air annealing on the spectral properties of Ce : Y3Al5O12 single crystals grown by the temperature gradient technique

Five absorption hands, at 227, 300 340, 370 and 457nm, were observed in the optical absorption spectrum of Ce:Y3Al5O12 (Ce:YAG) crystals grown by the temperature gradient technique (TGT). The absorption bands at 227, 340, and 457 nm were identified Lis belonging to the Ce3+ -ion in the YAG crystal. A near UV optical emission band at 398nm was observed. with an excitation spectrum containing two bands, at 235 and 370nm. No fluorescence was detected under 300 nm excitation. The pair of absorption bands at 235 and 370 nm and the absorption band at 300 nm were attributed to the F- and F+-type color centers, respectively. The color centers model was also applied to explain the spectral changes in the Ce:YAG (TGT) crystal, including the reduction in the Ce 31 -ion absorption intensity, after annealing in an oxidizing atmosphere (air). (C) 2004 Elsevier B.V. All rights reserved.

Single crystal beta-Ga2O3: Cr grown by floating zone technique and its optical properties

beta-Ga2O3: Cr single crystals were grown by floating zone technique. Absorption spectra and fluorescence spectra were measured at room temperature. The values of field splitting parameter Dq and Racah parameter B were obtained by the peak values of absorption spectra. The value 10Dq/B=23.14 manifests that in beta-Ga2O3 crystals Cr3+ ions are influenced by low energy crystal field. After high temperature annealing in air, the Cr3+ intrinsic emission was enhanced and the green luminescence disappeared. The strong and broad 691 nm emission was obtained at 420 nm excitation due to the electron transition occurred from T-4(2) to (4)A(2). The studies manifest that the beta-Ga2O3 crystals have the potential application for tunable laser.

Fluorescence cross-correlation spectroscopy to study hemodynamics

[EN] In the recent years a series of optical correlation techniques have been developed in order to be able to measure flow velocity with high spatial resolution while being non-invasive in order to be employed in-vivo on biological organisms. The technique employed in my thesis work, scanning laser image correlation (SLIC), is a powerful approach for the detection of flow motions because it overcomes some limitations of the classical spectroscopy techniques. SLIC method consists in repeated laser scans over a linear pattern and on the cross correlation of the signal emitted by the excited fluorophores in different positions along the scan line. Therefore, the resulting measurements for flow velocity are really accurate.

Autonomous extraction of optimal flame fronts in OH planar laser-induced fluorescence images.

The location of a flame front is often taken as the point of maximum OH gradient. Planar laser-induced fluorescence of OH can be used to obtain the flame front by extracting the points of maximum gradient. This operation is typically performed using an edge detection algorithm. The choice of operating parameters a priori poses significant problems of robustness when handling images with a range of signal-to-noise ratios. A statistical method of parameter selection originating in the image processing literature is detailed, and its merit for this application is demonstrated. A reduced search space method is proposed to decrease computational cost and render the technique viable for large data sets. This gives nearly identical output to the full method. These methods demonstrate substantial decreases in data rejection compared to the use of a priori parameters. These methods are viable for any application where maximum gradient contours must be accurately extracted from images of species or temperature, even at very low signal-to-noise ratios.

An improved technique to measure axial variation in wall flow along the channels of a diesel particulate filter

A technique to measure wall flow variation in Diesel Particle Filters (DPFs) is described. In a recent paper, it was shown how the flow distribution in DPFs could be measured in a non-destructive manner. This involved measuring the progressive dilution of a tracer gas introduced at the "outlet" channel upstream end. In the present paper, a significant further improvement to this technique is described, in which only a single probe is required, rather than the two of the previous technique. The single, traversable, probe consists of a controllable flow sink, and slightly downstream, a tracer gas supply. By controlling the sink flow rate such that a very small concentration of tracer gas is aspirated into it, the total flow up to that location in the channel is determined. Typical results showing the axial variation in the wall flow for known wall blockage cases are presented. It is suggested that this technique could be used to interpret the soot loading in the filter channels in a non-intrusive way.

Measurement of liquid film thickness by optical fluorescence and its application to an oscillating piston positive displacement flowmeter

The movement of the circular piston in an oscillating piston positive displacement flowmeter is important in understanding the operation of the flowmeter, and the leakage of liquid past the piston plays a key role in the performance of the meter. The clearances between the piston and the chamber are small, typically less than 60 νm. In order to measure this film thickness a fluorescent dye was added to the water passing through the meter, which was illuminated with UV light. Visible light images were captured with a digital camera and analysed to give a measure of the film thickness with an uncertainty of less than 7%. It is known that this method lacks precision unless careful calibration is undertaken. Methods to achieve this are discussed in the paper. The grey level values for a range of film thicknesses were calibrated in situ with six dye concentrations to select the most appropriate one for the range of liquid film thickness. Data obtained for the oscillating piston flowmeter demonstrate the value of the fluorescence technique. The method is useful, inexpensive and straightforward and can be extended to other applications where measurement of liquid film thickness is required. © 2011 IOP Publishing Ltd.

Development of a time and space resolved sampling probe diagnostic for engine exhaust hydrocarbons

In order to understand how unburned hydrocarbons emerge from SI engines and, in particular, how non-fuel hydrocarbons are formed and oxidized, a new gas sampling technique has been developed. A sampling unit, based on a combination of techniques used in the Fast Flame Ionization Detector (FFID) and wall-mounted sampling valves, was designed and built to capture a sample of exhaust gas during a specific period of the exhaust process and from a specific location within the exhaust port. The sampling unit consists of a transfer tube with one end in the exhaust port and the other connected to a three-way valve that leads, on one side, to a FFID and, on the other, to a vacuum chamber with a high-speed solenoid valve. Exhaust gas, drawn by the pressure drop into the vacuum chamber, impinges on the face of the solenoid valve and flows radially outward. Once per cycle during a specified crank angle interval, the solenoid valve opens and traps exhaust gas in a storage unit, from which gas chromatography (GC) measurements are made. The port end of the transfer tube can be moved to different locations longitudinally or radially, thus allowing spatial resolution and capturing any concentration differences between port walls and the center of the flow stream. Further, the solenoid valve's opening and closing times can be adjusted to allow sampling over a window as small as 0.6 ms during any portion of the cycle, allowing resolution of a crank angle interval as small as 15°CA. Cycle averaged total HC concentration measured by the FFID and that measured by the sampling unit are in good agreement, while the sampling unit goes one step further than the FFID by providing species concentrations. Comparison with previous measurements using wall-mounted sampling valves suggests that this sampling unit is fully capable of providing species concentration information as a function of air/fuel ratio, load, and engine speed at specific crank angles. © Copyright 1996 Society of Automotive Engineers, Inc.

Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced. and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order: PM18S02 > PM28S02 > PM28S01 > PM18S01, and that of the probes specific to T. pulchella was TP18S02 > TP28S01 > TP28S02 > TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02; TP18S02 and TP28S01, and the protocol, were also useful for the detection of algae in natural samples.

Ni silicide nanowires analysis by atom probe tomography

The Ni silicide formed at low temperature on Si nanowire has been analyzed by atom probe tomography (APT) thanks to a special technique for sample preparation. A method of preparation has been developed using the focused ion beam (FIB) for the APT analysis of nanowires (NWs). This method allow for the measurement of the radial distribution when a NW is cut, buried in a protective metal matrix, and finally mounted on the APT support post. This method was used for phosphorous doped Si NWs with or without a silicide shell, and allows obtaining the concentration and distribution of chemical elements in three-dimensions (3D) in the radial direction of the NWs. The distribution of atoms in the NWs has been measured including dopants and Au contamination. These measurements show that δ-Ni2Si phase is formed on Si NW, Au is found as cluster at the Ni/δ-Ni2Si interface and P is segregated at the δ-Ni2Si/ Si NW interface. The results obtained on NWs after silicidation were compared with the silicide on the Si substrate, showing that the same silicide phase δ-Ni2Si formed in both cases (NWs and substrate). © 2013 Elsevier B.V. All rights reserved.

Atom probe tomography for advanced metallization

In microelectronics, the increase in complexity and the reduction of devices dimensions make essential the development of new characterization tools and methodologies. Indeed advanced characterization methods with very high spatial resolution are needed to analyze the redistribution at the nanoscale in devices and interconnections. The atom probe tomography has become an essential analysis to study materials at the nanometer scale. This instrument is the only analytical microscope capable to produce 3D maps of the distribution of the chemical species with an atomic resolution inside a material. This technique has benefit from several instrumental improvements during last years. In particular, the use of laser for the analysis of semiconductors and insulating materials offers new perspectives for characterization. The capability of APT to map out elements at the atomic scale with high sensitivity in devices meets the characterization requirements of semiconductor devices such as the determination of elemental distributions for each device region. In this paper, several examples will show how APT can be used to characterize and understand materials and process for advanced metallization. The possibilities and performances of APT (chemical analysis of all the elements, atomic resolution, planes determination, crystallographic information...) will be described as well as some of its limitations (sample preparation, complex evaporation, detection limit, ...). The examples illustrate different aspect of metallization: dopant profiling and clustering, metallic impurities segregation on dislocation, silicide formation and alloying, high K/metal gate optimization, SiGe quantum dots, as well as analysis of transistors and nanowires. © 2013 Elsevier B.V. All rights reserved.

Ultrafast carrier dynamics in undoped and p-doped InAs/GaAs quantum dots characterized by pump-probe reflection measurements

We investigate the dependence of the differential reflection on the structure parameters of quantum dot (QD) heterostructures in pump-probe reflection measurements by both numerical simulations based on the finite-difference time-domain technique and theoretical calculations based on the theory of dielectric films. It is revealed that the value and sign of the differential reflection strongly depend on the thickness of the cap layer and the QD layer. In addition, a comparison between the carrier dynamics in undoped and p-doped InAs/GaAs QDs is carried out by pump-probe reflection measurements. The carrier capture time from the GaAs barrier into the InAs wetting layer and that from the InAs wetting layer into the InAs QDs are extracted by appropriately fitting differential reflection spectra. Moreover, the dependence of the carrier dynamics on the injected carrier density is identified. A detailed analysis of the carrier dynamics in the undoped and p-doped QDs based on the differential reflection spectra is presented, and its difference with that derived from the time-resolved photoluminescence is discussed. (C) 2008 American Institute of Physics.

Preparation and characterization of Si sheets by renewed SSP technique

Silicon sheets from powder (SSP) ribbons have been prepared by modified SSP technique using electronic-grade (9N purity) silicon powder. The surface morphology, crystallographic quality, composition and electric properties of the SSP ribbons were investigated by surface profiler, X-ray diffraction (XRD), scanning electron microscopy (SEM), metallurgical microscope, Auger electron spectroscopy (AES) and four-point probe apparatus, respectively. The results show that the SSP ribbon made from electronic-grade silicon powder is a suitable candidate for the substrates of crystalline silicon thin film (CSiTF) solar cells, which could meet the primary requirements of CSiTF solar cell process on the substrates, including surface smoothness, crystallographic quality, purity and electric conductivity, etc. (C) 2004 Elsevier B.V. All rights reserved.

Research on the ultrafast fluorescence property of thylakoid membranes of the wild-type and mutant rice

A high yielding rice variety mutant (Oryza sativa L., Zhenhui 249) with low chlorophyll b (Chl b) has been discovered in natural fields. It has a quality character controlled by a pair of recessive genes (nuclear gene). The partial loss of Chl b in content affects the efficiency of light harvest in a light harvest complex (LHC), thus producing the difference of the exciting energy transfer and the efficiency of photochemistry conversion between the mutant and wild-type rice in photosynthetic unit. The efficiency of utilizing light energy is higher in the mutant than that in the wildtype rice relatively. For further discussion of the above-mentioned difference and learning about the mechanism of the increase in the photochemical efficiency of the mutant, the pico-second resolution fluorescence spectrum measurement with delay-frame-scanning single photon counting technique is adopted. Thylakoid membranes of the mutant and the wild-type rice are excited by an Ar+ laser with a pulse width of 120 ps, repetition rate of 4 MHz and wavelength of 514 nm. Compared with the time and spectrum property of exciting fluorescence, conclusions of those ultrafast dynamic experiments are: 1) The speeds of the exciting energy transferred in photo-system I are faster than that in photo-system II in both samples. 2) The speeds of the exciting energy transfer of mutant sample are faster than those of the wildtype. This might be one of the major reasons why the efficiency of photosynthesis is higher in mutant than that in the wild-type rice.