CCD Detection Research Articles

Electrophoretic mobility measurements of fluorescent dyes using on‐chip capillary electrophoresis

We present an experimental study of the effect of pH, ionic strength, and concentrations of the electroosmotic flow (EOF)-suppressing polymer polyvinylpyrrolidone (PVP) on the electrophoretic mobilities of commonly used fluorescent dyes (fluorescein, Rhodamine 6G, and Alexa Fluor 488). We performed on-chip capillary zone electrophoresis experiments to directly quantify the effective electrophoretic mobility. We use Rhodamine B as a fluorescent neutral marker (to quantify EOF) and CCD detection. We also report relevant acid dissociation constants and analyte diffusivities based on our absolute estimate (as per Nernst-Einstein diffusion). We perform well-controlled experiments in a pH range of 3-11 and ionic strengths ranging from 30 to 90 mM. We account for the influence of ionic strength on the electrophoretic transport of sample analytes through the Onsager and Fuoss theory extended for finite radii ions to obtain the absolute mobility of the fluorophores. Lastly, we briefly explore the effect of PVP on adsorption-desorption dynamics of all three analytes, with particular attention to cationic R6G.

Study on the Relation between Electrospinning Fiber Diameter Distribution and Taylor Cone Dynamics

Electrospinning is an effective and versatile technology to fabricate ultrafine nanofiber,however further development is urgently limited due to the low uniformity distribution and unpredictable feature of the fiber deposited. For that sake, experiment has been done and analyzed to address these problems. Relation between process physics-Taylor cone and fiber diameter characters has been discovered. Furthermore, the feature parameters of the Taylor cone are extracted effectively by CCD detection and image processing. The above experiments results and processing data is analysis and examed by the steady jet theory. This paper offers significant theoretical guidance and technical support to the online control of electrospinning fiber diameter.

Compact crystal spectrometers for ion microbeam WDX–PIXE applications

AbstractThe use of a CCD for detection of X‐rays and a focused or collimated beam of ionising radiation enables the design of a compact, wavelength‐dispersive X‐ray (WDX) spectrometer, with increased solid angle and tolerable loss of energy resolution. Experimental investigation of WDX spectrometer designs is rather expensive and time consuming, so in order to compare various designs, a numerical X‐ray tracing procedure (XTRACE) has been developed for the calculation of instrumental functions and solid angles. XTRACE has been applied to a range of candidate designs for downsized WDX spectrometers with CCD detection and flat, Von Hamos, Johann, Johansson and spherical crystals. Most of the crystal types studied for use in downsized WDX‐PIXE spectrometers are sensitive to sample surface rotation, which may affect the reproducibility of experimental conditions. Implications for construction of downsized WDX spectrometers for PIXE application using an ion microbeam are given. Copyright © 2011 John Wiley & Sons, Ltd.

Image Scanning Microscopy

A new microscopy technique is introduced, image scanning microscopy (ISM), which combines conventional confocal-laser scanning microscopy with fast wide-field CCD detection. The technique allows for doubling the lateral optical resolution in fluorescence imaging. The physical principle behind ISM is similar to structured illumination microscopy, by combining the resolving power of confocal-laser scanning microscopy with that of a wide-field imaging microscopy. This Letter describes the theoretical foundation and experimental realization of ISM.

Probing membrane proteins: Monitoring proton-translocation quantitatively

Proton-translocating membrane-bound proteins create and maintain pH gradients across membranes, playing important bioenergetic and regulatory roles. Some of these proteins are implied in ageing and neurodegeneration, others in tumor biology and drug resistance. Unfortunately, membrane proteins are notoriously difficult to study. They are vastly underrepresented in the growing body of protein structures, and recent proteomics advances have mostly involved soluble proteins.We present a system for quantitatively monitoring proton-translocation by membrane proteins. The protein is reconstituted into large vesicles, which provide a well defined environment. Optionally, the protein can be first fused to a cytochrome, facilitating unidirectional reconstitution1. The pH inside and outside the vesicles is monitored simultaneously using novel membrane-impermeable nanoprobes2,3. Using CCD detection, full spectral coverage of the nanoprobes and any protein-bound chromophores is delivered at up to ms time resolution. The proteoliposomes are then subjected to computer-controlled, semi-automated titrations of substrate and/or pH changes4.Key improvements over similar methodologies previously employed are the advantageous characteristics of the nanoprobes, the precision and speed of the titration system, and the CCD detection technology. Combined, these features ensure robust, fast, quantitative results on proton-pumping stoichiometries across membranes.Using this method, we characterize the lipid bilayer of the vesicles in terms of passive ion leakage and stability. We then investigate subunits of respiratory Complex I, and their antiporter homologues, reconstituted into liposomes (see Sindra Peterson Arskold's poster).1. Gustavsson, T., Eek, M., Leiding, T., Peterson Arskold, S., and Hagerhall, C. (2008) Submitted to Biochimica et Biophysica Acta Bioenergetics.2. Finikova, O., Galkin, A., Rozhkov, V., Cordero, M., Hagerhall, C., and Vinogradov, S. (2003) J. Am. Chem. Soc. 125, 4882-4893.3. Leiding, T., Gorecki, K., Vinogradov, S., Hagerhall, C., and Peterson Arskold, S. (2008) Submitted to Analytical Biochemistry.4. Autonomous Science Machines TM.

Stabilization of the spectrum position in the main stellar spectrograph of the 6-m telescope of the Special Astrophysical Observatory of the Russian Academy of Sciences

New results on the stability analysis study of the Main Stellar Spectrograph of the 6-m telescope of the Special Astrophysical Observatory of the Russian Academy of Sciences when it operated jointly with the CCD detection system on a laboratory source are reported. The spectrum is found to drift along the dispersion direction for several tenths of a pixel (several hundredths of angstrom) over a one-hour time scale. To prevent this drift (i.e., the spectrum shift)—a plane parallel glass plate is mounted; tilt angle of this plate varies as a function of the misalignment between the current and reference spectra of a hollow-cathode tube laboratory source. Operation of the spectrum stabilizer is described. The correction time is of about 1 minute, allowing the position of the spectrum to be maintained to within ±0.0006A.

Gradient Elution Isotachophoresis for Enrichment and Separation of Biomolecules

A novel format for performing capillary isotachophoresis (ITP) is described -- gradient elution ITP (GEITP). GEITP merges the recently described electrophoretic separation technique of gradient elution moving boundary electrophoresis (GEMBE) with an ITP enrichment step. GEMBE utilizes a combination of continuous sample injection with a pressure-controlled counterflow; as the counterflow is reduced, analytes are sequentially eluted onto the separation column and detected as boundary interfaces. By incorporating leading electrolytes into the counterflow and terminating electrolytes into the sample matrix, an ionic interface can be formed near the capillary inlet. The discontinuous buffer system forms highly enriched analyte zones outside of the capillary, which are then eluted onto the separation capillary as the counterflow is reduced. Separation of fluorescent analytes was achieved either through discrete electrolyte spacers added to the sample or by using ampholyte mixtures to form a continuum of spacers. As the ITP process occurs off-column, extremely short length separations can be achieved, as demonstrated by a separation in 30 microm. The effects of various parameters on the GEITP enrichment process are investigated, including initial counterflow rates, electric field, leading electrolyte concentration, and counterflow acceleration, which is an adjustable parameter allowing for highly flexible separations. Typical enhancements in limits of detection and sensitivity were greater than 10,000-fold and were achieved in less than 2 min, yielding low-picomolar detection limits using arc lamp illumination and low-cost CCD detection. An optimized system afforded greater than 100,000-fold improvement in detection of carboxyfluorescein in 8 min. Specific examples of enrichment and separation demonstrated include the following: small dye molecules, DNA, amino acid mixtures, and protein mixtures.

Three-dimensional in vivo fluorescence diffuse optical tomography of breast cancer in humans

We present three-dimensional (3D) in vivo images of human breast cancer based on fluorescence diffuse optical tomography (FDOT). To our knowledge, this work represents the first reported 3D fluorescence tomography of human breast cancer in vivo. In our protocol, the fluorophore Indocyanine Green (ICG) is injected intravenously. Fluorescence excitation and detection are accomplished in the soft-compression, parallel-plane, transmission geometry using laser sources at 786 nm and spectrally filtered CCD detection. Phantom and in vivo studies confirm the signals are due to ICG fluorescence, rather than tissue autofluorescence and excitation light leakage. Fluorescence images of breast tumors were in good agreement with those of MRI, and with DOT based on endogenous contrast. Tumorto- normal tissue contrast based on ICG fluorescence was two-to-four-fold higher than contrast based on hemoglobin and scattering parameters. In total the measurements demonstrate that FDOT of breast cancer is feasible and promising.

Modeling and simulation of luminescence detection platforms

Motivated by the design of an integrated CMOS-based detection platform, a simulation model for CCD and CMOS imager-based luminescence detection systems is developed. The model comprises four parts. The first portion models the process of photon flux generation from luminescence probes using ATP-based and luciferase label-based assay kinetics. An optics simulator is then used to compute the incident photon flux on the imaging plane for a given photon flux and system geometry. Subsequently, the output image is computed using a detailed imaging sensor model that accounts for photodetector spectral response, dark current, conversion gain, and various noise sources. Finally, signal processing algorithms are applied to the image to enhance detection reliability and hence increase the overall system throughput. To validate the model, simulation results are compared to experimental results obtained from a CCD-based system that was built to emulate the integrated CMOS-based platform.

Optimizing low-light microscopy with back-illuminated electron multiplying charge-coupled device: enhanced sensitivity, speed, and resolution

The back-illuminated electron multiplying charge-coupled device (EMCCD) camera is having a profound influence on the field of low-light dynamic cellular microscopy, combining highest possible photon collection efficiency with the ability to virtually eliminate the readout noise detection limit. We report here the use of this camera, in 512 x 512 frame-transfer chip format at 10-MHz pixel readout speed, in optimizing a demanding ultra-low-light intracellular calcium flux microscopy setup. The arrangement employed includes a spinning confocal Nipkow disk, which, while facilitating the need to both generate images at very rapid frame rates and minimize background photons, yields very weak signals. The challenge for the camera lies not just in detecting as many of these scarce photons as possible, but also in operating at a frame rate that meets the temporal resolution requirements of many low-light microscopy approaches, a particular demand of smooth muscle calcium flux microscopy. Results presented illustrate both the significant sensitivity improvement offered by this technology over the previous standard in ultra-low-light CCD detection, the GenIII+intensified charge-coupled device (ICCD), and also portray the advanced temporal and spatial resolution capabilities of the EMCCD.

An improved microstrip plasma for optical emission spectrometry of gaseous species

A modified compact 2.45 GHz microstrip plasma (MSP) operated with Ar as working gas at atmospheric pressure has been characterized and examined for its suitability for the determination of Hg as gaseous species by optical emission spectrometry. As a formerly described MSP the new device is provided on a sapphire substrate. The areas of plasma stability in terms of gas flow rates and microwave power for both MSPs with respect to plasma form and reflected power were investigated. Power levels of 5–40 W and Ar flow rates of 15–60 l/h were used. The modified MSP, which extends out of the channel in the sapphire substrate, was used for the recording of emission spectra for Hg vapor at different working conditions. Using optimized parameters a detection limit for Hg of less than 10 ng Hg/l Ar is obtained. The attainable excitation temperatures in the modified MSP at different microwave power were determined under the use of Fe as thermometric species and introducing ferrocene into the plasma. They were found to be at the order of 6000–7000 K for a power of 10–40 W and a gas flow of 15 l/h. It was shown that the modified MSP source can be combined with both a conventional monochromator with photomultiplier detection and a miniaturized spectrometer with CCD detection, whereby space–angle limitations are not stringent.

Application of two-way decomposition methods in the simultaneous determination of cobalt, nickel and iron by coupling stopped-flow techniques and CCD detection

The simultaneous kinetic determination of cobalt, nickel, and iron with principal component regression (PCR) and partial least squares (PLS) methods is discussed. PCR and PLS extract information from kinetic differences between the complexes of these metals after their reaction with a common ligand. The coupling of stopped-flow (SF) with CCD detection resulted in massive data quantities making the use of data-reduction techniques not only interesting, but also necessary. Initial rates of the reactions were calculated using an algorithm, whose description is given somewhere else [Anal. Chim. Acta 423 (2000) 277]. Both methods were successfully applied to the simultaneous determination of these metals in alloys, and a comparative study of the results was undertaken.

Magnetic anisotropy of aligned magnetic stripe domains in FePd studied by soft x-ray resonant magnetic scattering, magnetic force microscopy and micromagnetic modeling

FePd thin-film samples with different perpendicular magnetic anisotropies have been studied with magnetic force microscopy (MFM), micromagnetic calculations and soft x-ray resonant magnetic scattering (SXRMS). The competition between perpendicular magnetic anisotropy (PMA) and shape anisotropy leads to the formation of highly ordered stripe domain patterns with a magnetization component perpendicular to the film plane. The magnetic stripes with a period of ∼100 nm, which are seen in the MFM images and can be modeled by micromagnetic calculations, give rise to magnetic peaks in the diffraction pattern. Closure domains occur in samples with a low to medium PMA, while a high PMA inhibits their formation. The in-plane magnetization component of the closure domains is not observable with MFM. In the presence of closure domains the interference between the scattering amplitudes in SXRMS from perpendicular and in-plane magnetized domains gives rise to a circular dichroism in the transverse geometry, where the scattering plane is along the stripes. We also recorded the magnetic speckle pattern from an 8 μm FePd wire using coherent x-rays and CCD detection. A high degree of coherence was obtained as evidenced from the observed intensity fluctuations. The speckle pattern can in principle provide information about the local disorder of the magnetic stripe domains.

Magnetic anisotropy of aligned magnetic stripe domains in FePd studied by soft x-ray resonant magnetic scattering, magnetic force microscopy and micromagnetic modeling*1

FePd thin-film samples with different perpendicular magnetic anisotropies have been studied with magnetic force microscopy (MFM), micromagnetic calculations and soft x-ray resonant magnetic scattering (SXRMS). The competition between perpendicular magnetic anisotropy (PMA) and shape anisotropy leads to the formation of highly ordered stripe domain patterns with a magnetization component perpendicular to the film plane. The magnetic stripes with a period of ∼100 nm, which are seen in the MFM images and can be modeled by micromagnetic calculations, give rise to magnetic peaks in the diffraction pattern . Closure domains occur in samples with a low to medium PMA, while a high PMA inhibits their formation. The in-plane magnetization component of the closure domains is not observable with MFM. In the presence of closure domains the interference between the scattering amplitudes in SXRMS from perpendicular and in-plane magnetized domains gives rise to a circular dichroism in the transverse geometry, where the scattering plane is along the stripes. We also recorded the magnetic speckle pattern from an 8 μm FePd wire using coherent x-rays and CCD detection. A high degree of coherence was obtained as evidenced from the observed intensity fluctuations. The speckle pattern can in principle provide information about the local disorder of the magnetic stripe domains.

High-speed depth-sectioned wide-field imaging using low-coherence photorefractive holographic microscopy

Low-coherence photorefractive holography has the potential to acquire wide-field coherence-gated images at frame rates approaching 1000 frames/s, including through scattering media. We present a quantitative analysis of the system optimization and limits of performance for coherence-gated imaging through scattering media using photorefractive holography and compare this performance to direct CCD detection. We show that, for high optical quality recording photorefractive multiple quantum well devices, photorefractive holography has the potential to provide a higher dynamic range than is possible with direct CCD-based detection.

Table-top X-ray microscopy : Sources, optics and applications

We have developed the first operative compact sub-visible-resolution x-ray microscope for the water-window region (λ = 2.3 - 4A nm). The microscope is based on a 100 Hz liquid-jet-target laser-plasma x-ray source, normal-incidence multilayer condenser optics, diffractive zone plate optics and CCD detection. In the present article we emphasize the system's aspects and summarize the recent progress on the components, all aiming at the reduction of the exposure time of a few seconds, i.e., similar to bending-magnet based microscopes. This primarily includes improved laser-plasma source, improved condenser optics using Cr/Sc multilayers, and improved image handling capability using wavelet algorithms. Such compact short-exposure time microscopes would significantly increase the applicability of the technology.

Determination of ultratrace elements in natural waters by solid-phase extraction and atomic spectrometry methods

A study was carried out on the preconcentration of ultratrace amounts of cadmium, lead, manganese, copper and iron from high-salinity aqueous samples and determination by atomic spectrometry methods. Sample volume, amount of resin, loading flow rate, and elution volume were optimized in order to obtain the simultaneous preconcentration of all the analytes. Quantitative recoveries were obtained by using 200 mg of iminodiacetic resin with a loading flow rate of 2 mL min(-1), elution volume of 3 mL and sample volume of 50-450 mL. Only copper in seawater samples was not completely retained by the resin (60-70% recovery), due to unfavorable competition of iminodiacetic-active groups with organically bound metal.To quantify the metals in the eluates, two atomic spectrometry techniques were compared: electrothermal atomization atomic absorption spectrometry (ETAAS) and inductively coupled plasma-optical emission spectrometry (ICP-OES) with simultaneous CCD detection system. Both techniques are suitable for sample analysis with detection limits of 1.0, 4.7, 3.3, 6.8, and 53 ng L(-1) using ETAAS and 12, 122, 3.4, 17, and 21 ng L(-1) using ICP-OES for Cd, Pb, Mn, Cu, and Fe, respectively. Relative standard deviations of the procedures ranged from 1.7 to 14% at the sub-microg L(-1) concentration level. The accuracy of both methods was verified by analyzing various certified reference materials (river water, estuarine water, coastal and off-shore seawater).

Inclusion of carvone enantiomers in cyclomaltoheptaose (β-cyclodextrin): thermal behaviour and H→D and D→H exchange

The inclusion compounds of carvone enantiomers in cylcomaltoheptaose (β-cyclodextrin, βCD) are studied at defined temperatures above room temperature and in relation to H→D and D→H exchanges. Loss of water molecules and release of carvone molecules from the βCD cavity are caused by increase of temperature above room temperature and are measured by the integrated intensities of the OH and CH Raman stretching bands, respectively. In turn, H→D and D→H exchanges are monitored by the integrated intensities of the OH and OD Raman stretching bands, respectively. All of these processes were followed in real time with a Raman spectrometer equipped with CCD detection. The results indicate that distinct carvone enantiomers lead to the formation of different βCD inclusion hydrates that have different water content and hydration structures. In particular, the results suggest that SCarv–βCD has a greater water content, dehydrates strongly for temperatures above room temperature, and exchanges protons faster than the RCarv–βCD complex.

Isotropic Raman line shapes near gas–liquid critical points: The shift, width, and asymmetry of coupled and uncoupled states of fluid nitrogen

In order to improve the experimental database about the additional nonrotational broadening of vibrational line shapes observable when a simple fluid approaches its gas–liquid critical point, we improved the pioneering measurements of Clouter and Kiefte [for their own review see Phys. Rev. A 33, 2749 (1986)] on the critical behavior of the polarized Raman line of fluid nitrogen by using the isotopic mixture (14N2).975−(14N15N).025, giving special attention to the fact that the isotropic line shape of liquid N2 (ν̃≈2327 cm−1) is affected by intermolecular vibrational resonance couplings. Using a highest-resolution double monochromator and modern CCD detection techniques, we were able to follow the temperature dependencies of the line shape parameters (i.e., shift, width, and asymmetry) of the coupled N142 and, depending on the S/N ratio available, of the uncoupled N1415N in the range 45 K≲T≲300 K with up to mK resolution (1) in the β-solid phase, (2) in the coexisting liquid and gas phases, and (3) along the critical isochore. Comparing the line shifts of both isotopic species, clear evidence was found that vibrational resonance couplings are present in all dense phases studied, the line position ν̃0 being more density than temperature dependent. Additionally, the existence of (negative) cross correlations between resonant and nonresonant dephasing mechanisms has been confirmed by the change in sign observed for the small but non-negligible difference in the linewidths between coupled N142 and uncoupled N1415N around 90 K. The λ-shaped dependencies of the width parameters, observed when moving along the coexistence line through the critical point, Tcrit=126.192 K, and along the critical isochore, is much more evident in the line asymmetry than in the usually considered linewidth. Clear proof was found that, in accordance with theoretical predictions, the linewidth converges to a constant maximum value regardless if the critical point is reached along the coexistence line or along the critical isochore, i.e., it does not diverge approaching the critical temperature up to our closest value |T/Tcrit−1|≈10−5.

An Inexpensive Laser Raman Spectrometer Based on CCD Detection

We describe an inexpensive modular Raman spectrometer system that can be assembled from commercial components for under $5000. Three typical applications are presented: a demonstration of a vibrational isotope effect, a Raman polarization experiment, and a resonance Raman experiment. This spectrometer system should make it easier to include the important topic of Raman spectroscopy in the undergraduate physical and analytical chemistry laboratory curricula.