A diffused-optics flash kinetic spectrophotometer (DOFS) for measurements of absorbance changes in intact plants in the steady-state

Measurements of steady-state light-induced absorbance changes in intact plants are often hindered by interference from large changes in the light-scattering properties of the chloroplasts. In this work we present a new instrument, the diffused-optics flash spectrophotometer (DOFS), which reduces the magnitude of light scattering interference to manageable levels. In this spectrophotometer, the conventional light path is replaced with a set of light-scrambling chambers formed from a highly light-scattering plastic. The main scrambling chamber acts both to homogeneously diffuse as well as to split the measuring beam into sample and reference channels. Since the measuring beam has no defined incident angle, it is essentially 'pre-scattered', and further scattering changes that occur in the sample have minimal effect on the apparent absorbance changes. The combination of a pulsed probe light and differential optics and electronics provides a high signal-to-noise ratio, stable baseline and high time resolution. We also introduce a technique to account for residual scattering changes. Sets of measurements are made with the instrument in optical configurations that are differentially sensitive to light-scattering changes but yield nearly identical absorbance changes. The difference in apparent absorbance spectra taken with the two configurations reveals the spectral shape of the scattering changes without interference from absorbance signals. Spectra of the scattering contributions are then used to eliminate residual scattering interference from kinetic traces. We suggest that DOFS is ideally suited for study of steady-state electron transfer reactions in intact plants.     

Optical solid-state detection of organophosphates using organophosphorus hydrolase

We have developed a sensor surface for optical detection of organophosphates based on reversible inhibition of organophosphorus hydrolase (OPH) by copper complexed meso-tri(4-sulfonato phenyl) mono(4-carboxy phenyl) porphyrin (CuC1TPP). OPH immobilized onto glass microscope slides retains catalytic activity for more than 232 days. CuC1TPP is a reversible, competitive inhibitor of OPH, binding at the active site of the immobilized enzyme. The absorbance spectrum of the porphyrin-enzyme complex is measured via planar waveguide evanescent wave absorbance spectroscopy using a blue LED as a light source and an Ocean Optics USB2000 as the spectrophotometer. The characteristics of the absorbance spectrum of CuC1TPP are specific and different when the porphyrin is bound to the enzyme or is bound non-specifically to the surface of the slide. Addition of a substrate of OPH such as one of the organophosphates paraoxon, coumaphos, diazinon, or malathion displaces the porphyrin from the enzyme resulting in reduced absorbance intensity at 412 nm. Absorbance changes at 412 nm show log-linear dependence on substrate concentration. Paraoxon concentrations between 7 parts per trillion (ppt) and 14 parts per million (ppm) were investigated and a 3:1 S/N detection limit of 7 ppt was determined. Concentrations of 700 ppt to 40 ppm were investigated for diazinon, malathion, and coumaphos with detection limits of 800 ppt, 1 part per billion, and 250 ppt, respectively. This optical technique does not require the addition of reagents or solutions other than the sample and absorbance spectra can be collected in less than 6 s.     

Arsenazo III-Ca2+. Effect of pH, ionic strength, and arsenazo III concentration on equilibrium binding evaluated with Ca2+ ion-sensitive electrodes and absorbance measurements.

Equilibrium binding properties of the metallochromic indicator Arsenazo III (AIII) were characterized by Ca2+ and acid/base titration. Free calcium was measured directly with Ca2+ ion-sensitive electrodes. Absorbance changes were measured by both a conventional scanning spectrophotometer and a dual wavelength spectrophotometer. Acid/base titration of AIII in conjunction with Ca2+ ion-sensitive electrode measurement and absorbance changes indicate that pH can change AIII absorbance through a change of the K(D) for Ca-AIII formation, and, in addition, that there is a pH-specific component that is not dependent on Ca-AIII formation. THe dissociation constant (K(D)) of AIII varied not only with pH, but with ionic strength and AIII concentration. Studies conducted to examine AIII-Ca stoichiometry resulted in different initial conclusions, depending on the method of analysis. Log delta A-log AIII relations were in accord with previously published results, which indicate that more than one AII binds to one Ca2+ ion. But Job plots. Scatchard analysis, and Hill plots all indicated 1:1 binding. THe method of absorbance measurement, i.e., scanning or dual wavelength did not influence the results. these findings were reconciled on the basis of changes in K(D) with AIII concentration and ionic strength. In 200 mM KCl, K(D) of AIII-Ca varies by a factor of 7 between 10(-4.3) and 10(-3) M AIII. Thus, a disproportionately large amount of Ca-AIII is formed as AIII concentration is increased, which results in slopes greater than unity for log delta A-log AIII relations.     

New multichannel kinetic spectrophotometer–fluorimeter with pulsed measuring beam for photosynthesis research

A multichannel kinetic spectrophotometer–fluorimeter with pulsed measuring beam and differential optics has been constructed for measurements of light-induced absorbance and fluorescence yield changes in isolated chlorophyll-proteins, thylakoids and intact cells including algae and photosynthetic bacteria. The measuring beam, provided by a short (2 μs) pulse from a xenon flash lamp, is divided into a sample and reference channel by a broad band beam splitter. The spectrum in each channel is analyzed separately by a photodiode array. The use of flash measuring beam and differential detection yields high signal-to-noise ratio (noise level of 2 × 10⁻⁴ in absorbance units per single flash) with negligible actinic effect. The instrument covers a spectral range between 300 and 1050 nm with a spectral resolution of 2.1, 6.4 or 12.8 nm dependent on the type of grating used. The optical design of the instrument enables measuring of the difference spectra during an actinic irradiation of samples with continuous light and/or saturation flashes. The time resolution of the spectrophotometer is limited by the length of Xe flash lamp pulses to 2 μs.     

Quantitative analysis of a tetraribonucleotide mixture by ultraviolet spectrophotometry

Using the least squares method we have calculated the proportions of each nucleotide of a mixture of AMP, CMP, GMP and UMP, after measuring the absorbance of the mixture every ten nanometers from 230 to 290 nm at pH 12.7 and 2. The method is very simple and rapid (the calculations are made in less than one minute), does not require a highly sensitive spectrophotometer to obtain reasonably precise results and, in contrast to the other methods of the literature, it can be applied to quantities as little as 50 micrograms of nucleotides.     

A stopped-flow assay for glycogen phosphorylase appropriate to measure catalytic activity at high enzyme concentrations

Glycogen phosphorylase (EC 2.4.1.1) may be assayed in the glycogen degradation direction by a continuous spectrophotometric method. The formation of glucose 1-phosphate from glycogen and phosphate produces a controlled change of pH which can be measured by the changes in absorbance of phenol red added to the system. The procedure may be conveniently applied to a stopped-flow spectrophotometer to measure the rate of the reaction. Therefore the activity of the enzyme may be determined at low conventional concentrations and, by the same technique, at high enzyme concentrations approaching those supposed to exist in vivo.     

Absorption spectra indicate conformational alteration of myoglobin adsorbed on polydimethylsiloxane.

To assess the effects of adsorption on protein structure, ultraviolet optical absorption spectra of myoglobin (Mb) bound to polydimethylsiloxane (PDMS) were measured. A flow cell, which enabled adsorption under controlled hydrodynamic conditions, was used in conjunction with a conventional spectrophotometer to obtain the spectra. Adsorption to PDMS reduced significantly the absorbance in the Soret region of the Mb spectrum, whereas the spectrum in the region near 280 nm was essentially unaffected. This result showed that disruption of the native structure of Mb occurs following interaction with PDMS. Furthermore, the change in the absorption spectrum may indicate loss of heme from the heme pocket of the adsorbed protein. Mb structure was altered from its solution configuration within fifteen min of contact with the surface. Exchange of adsorbed Mb with Mb in solution had little or no effect on the absorption spectrum of the surface-confined protein, indicating that exchange occurs only between conformationally altered species or between native species.     

Sensing minute changes in biological cell monolayers with THz differential time-domain spectroscopy

We used terahertz differential time-domain spectroscopy (THz-DTDS) to measure minute changes of bovine lung microvessel endothelial cells (BLMVEC) in response to vascular endothelial growth factor (VEGF). These changes were reflected by alterations in THz wave attenuations and THz dielectric properties of the treated cells. The VEGF-induced THz attenuations of cell monolayers correlated well with changes in transendothelial resistance, as measured using electric cell-substrate impedance sensing (ECIS). However, the morphological differences that gave rise to these changes were not observed with standard optical phase contrast microscopy. We conclude that THz-DTDS is a highly sensitive, non-invasive, powerful new tool to measure minute changes in the morphology of live, cultured cell monolayers. This method enables spectroscopic investigations of cells in the THz band, providing information unavailable through other conventional methods such as optical phase contrast microscopy and ECIS.     

Dual wavelength/split beam spectrophotometry: computer acquisition of absorbance spectra.

The adaptation of a commercially available dual wavelength/split beam spectrophotometer for the real time acquisition by a minicomputer of absorbance spectra is described. The computer software and interface are designed so that: a) The normal operation of the spectrophotometer is unaltered; b) the adaptation of the spectrophotometer for computer acquisition of spectra is relatively simple and only a rudimentary knowledge of electronics and computer programming is required.The utility of this adaptation of a dual wavelength/split beam spectrophotometer for baseline correction, enhancement of the sensitivity of the spectrophotometer and subsequent analysis of recorded absorbance spectra is described.     

Shrinkage of growing Escherichia coli cells by osmotic challenge.

The immediate response of growing Escherichia coli to changing external osmotic pressure was studied with stopped-flow turbidimetric measurements with a narrow-beam spectrophotometer. It is shown theoretically that in such a photometer rod-shaped bacteria have an apparent absorbance which is proportional to the inverse of the surface area. The apparent optical density, corrected for effects of alteration of the index of refraction of the medium, increased continuously as the external osmotic pressure was raised. Because of the short time scale of the measurements, the turbidity increases could result either from shrinkage of the cells or from plasmolysis, or both, but not from growth or metabolic adaptation. With low concentrations of pentose such that the external osmotic pressure was not greater than that inside the cells, plasmolysis would not occur and, consequently, only shrinkage of the previously stretched sacculus remains to account for the observed optical effects. Taking the osmotic pressure of the growing cells as 5 atmospheres (506 kPa), the turbidity changes correspond to the murein fabric having been stretched 20% beyond its unstressed equilibrium area during growth under the conditions used.     

Identification and quantitation of heparins by microelectrophoresis on agarose gel

The microelectrophoresis procedure on agarose devised by Jaques, Ballieux, Dietrich, and Kavanagh (1) for the identification and measurement of heparin provides a quick, versatile, and adaptable procedure. Differentiation of heparin and related polyanions can be accomplished at various stages of the procedure—migration, fixation, staining, destaining, evaluation. Tests such as solubility in solvents, precipitation by chemical reagents, action of enzymes, may be applied to minute amounts of materials.A linear relationship was observed between absorbance and heparin concentration applied and between log of spot area and log concentration so that total optical density of spots can be used to estimate heparin. Visual comparison of microelectrophoresis slides with a series of reference slides with 0.02 to 0.6 units of heparin gave a coefficient of variation of 15.5%. Difficulty was found in providing a densitometer with sufficient sensitivity and reproducibility. Special slide holders were designed to hold the microelectrophoresis slide in the Chromoscan densitometer and in the Beckman DK-2 spectrophotometer. By specifying carriage position for the slide, slit size in the optical system, baseline adjustment, background correction, light filter, reproducibility of measurement could be achieved with the Chromoscan densitometer with 10% maximum error with low counts. The Coefficient of Variation was 6.2% when all precautions were taken.     

A rapid-scanning spectrophotometer designed for biological tissues in vitro or in vivo

Rapid and sensitive detection of optical signals from biological materials has been very useful in studies of cell physiology and biochemistry. A rapid-scanning spectrophotometer is described here that has the following advantages: (i) it can be used in transmission or reflection modes, (ii) it can rapidly accumulate spectra and simultaneous kinetic data, (iii) it has high accuracy and sensitivity, and (iv) it can analyze and store large amounts of spectral information. Evaluations described here are aimed toward the measurement of reduction/oxidation shifts of mitochondrial cytochromes in tissues, but the flexibility of the optical components makes this spectrophotometer adaptable to the study of light absorption changes of intrinsic or extrinsic optically active molecules in a variety of light scattering preparations, tissues, and organs in vitro or in vivo.     

A double-beam rapid-scanning stopped-flow spectrophotometer.

A double-beam rapid-wavelength-scanning stopped-flow spectrophotometer system based on the Norcon model 501 spectrometer was construced, which enables u.v.-or visible absorbance spectra to be recorded at the rate of 800/s after the rapid mixing (within 3ms) of two reactant solutions. Each spectrum spans about 200nm in 1ms. It is possible to record difference spectra during reactions with half-lives less than 10ms involving absorbance changes of less than 0.1 absorbance unit. Analogue circuitry is used to produce spectra of absorbance against wavelength. Up to 32 such spectra can be recorded at pre-selected times during a reaction and stored in an 8Kx8-bit-word hard-wired data-capture system to be subsequently displaned individually or simultaneously. Time-courses at different wavelengths can also be displayed. By averaging up to 216 spectra it is possible to record spectra under conditions of low signal-to-noise ratios...     

A label-free biosensor based on gold nanoshell monolayers for monitoring biomolecular interactions in diluted whole blood

Gold nanoshells (GNSs) were self-assembled on the surface of transparent glasses modified with 3-aminopropyltrimethoxysilane (APTES) to form GNS self-assembled monolayers (SAMs). Because the localized surface plasmon resonance (LSPR) of GNSs can be controlled in the near-infrared (NIR) region of the spectrum, where the optical transmission through tissue and whole blood is optimal, GNSs would be used as an effective signal transduction in whole blood. Accordingly, after modified with cystamine and biotin-NHS (N-hydroxy succinimide), GNS SAMs were used as a novel optical biosensor for real-time detection of streptavidin-biotin interactions in diluted human whole blood within short assay time, without any sample purification/separation. An UV-vis-NIR spectrophotometer was used to monitor the absorbance changes at 730 nm as a function of time for different concentrations of streptavidin in 20% whole blood, and the results showed that the biosensor displayed low detection limit of approximately 3 microg/mL and wide dynamic range of approximately 3-50 microg/mL. This approach provides an opportunity to construct LSPR biosensor for protein sensing and cellular analysis in diluted whole blood.     

Optical probe responses on sarcoplasmic reticulum: oxacarbocyanines as probes of membrane potential.

The relationship between Ca2+ fluxes and the ion diffusion potential was analyzed on sarcoplasmic reticulum membranes using oxacarbocyanine dyes as optical probes for membrane potential. 3.3'-Diethyloxodicarbocyanine responds to ATP-induced Ca2+ uptake by isolated sarcoplasmic reticulum vesicles with a decrease in absorbance at 600 nm. The optical change is reversed during Ca2+ release from sarcoplasmic reticulum induced by KCl or by ADP and inorganic phosphate. The absorbance changes are largely attributable to the binding of accumulated Ca2+ to the membrane. There is no indication that sustained changes in membrane diffusion potential would accompany pump-mediated Ca2+ fluxes. A large change in the absorbance of 3,3'-diethyloxodicarbocyanine was observed on sarcoplasmic reticulum vesicles under the influence of membrane potential generated by valinomycin in the presence of a K+ gradient or by ionophore A23187 in the presence of a Ca2+ gradient. The maximum of the potential-dependent absorbance change is at 575--580 nm. The potentials generated by valinomycin or ionophore A23187 are short-lived due to the high permeability of sarcoplasmic reticulum membranes for cations and anions. There is no correlation between the direction and magnitude of the artifically imposed membrane potential and the rate of Ca2+ uptake or release by isolated sarcoplasmic reticulum vesicles.     

A portable,non-focusing optics spectrophotometer (NoFOSpec) for measurements of steady-state absorbance changes in intact plants

Kinetically-resolved absorbance measurements during extended, or steady-state illumination are typically hindered by large, light-induced changes in the light-scattering properties of the material. In this work, a new type of portable spectrophotometer, the Non-Focusing Optical Spectrophotometer (NoFOSpec), is introduced, which reduces interference from light-scattering changes and is in a form suitable for fieldwork. The instrument employs a non-focusing optical component, called a compound parabolic concentrator (CPC), to simultaneously concentrate and homogeneously diffuse measuring and actinic light (from light-emitting diode sources) onto the leaf sample. Light passing through the sample is then collected and processed using a subsequent series of CPCs leading to a photodiode detector. The instrument is designed to be compact, lightweight and rugged for field work. The pulsed measuring beam allows for high sensitivity (typically < 100 ppm noise) and time resolution (∼ 10 μs) measurements in the visible and near infrared spectral regions. These attributes allow high-resolution measurements of signals associated with energization of the thylakoid membrane (the electrochromic shifting of carotenoid pigments), as well as electron transfer, e.g., the 820-nm changes associated with electron transfer through Photosystem I (PS I). In addition, the instrument can be used as a kinetic fluorimeter, e.g., to measure saturation-pulse fluorescence changes indicative of Photosystem II (PS II) quantum efficiency. The instrument is demonstrated by estimating electron and proton fluxes through the photosynthetic apparatus in an intact tobacco leaf, using respectively the saturation-pulse fluorescence changes and dark-interval relaxation kinetics (DIRK) of the electrochromic shift. A linear relationship was found, confirming our earlier results with the laboratory-based diffused-optics flash spectrophotometer, indicating a constant H⁺/e⁻ stoichiometry for linear electron transfer, and suggesting that cyclic electron flow around PS I is either negligible or proportional to linear electron flow. This type of measurement should be useful under field conditions for estimating the extent of PS I cyclic electron transfer, which is proposed to operate under stressed conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Multidimensional spectroscopic data correlation in the conformation transition of biological macromolecules

A computerized spectrophotometer system which is capable of simultaneously obtaining three spectral dimensions, that is, the absorbance, the circular dichroism and the fluorescence intensity, from one sample solution has been developed for the purpose of attaining a higher resolving power in the study of conformational transitions of biological macromolecules. Measurement conditions, such as the wavelength, the temperature, pH or the concentration of reagents in the sample solution, can be scanned according to a sequence that is set just prior to the measurement. A computer-driven micro-injector and a pH electrode directly immersed in the sample solution make it possible to obtain a titration curve in parallel to the optical measurement. All the data taken are stored on a magnetic disk for later retrieval. They can be processed and displayed in any required form. The helix-coil transitions of a polynucleotide caused by temperature and those of a polypeptide caused by pH, and the denaturation of proteins caused by guanidine hydrochloride, were studied by this measuring system. The continuous plotting of transition profiles and the correlation diagrams among different spectral dimensions has proved to be a good way of demonstrating the existence of different modes of transition.     

Novel Covellite CuS Single-Crystal Thin Films for Optoelectronic Applications

Copper sulfide (CuS) thin films have been used in many applications such as solar cells, photo-thermal, electro-conductive, and microwave shielding. In this work, copper sulfide thin films were deposited on glass and silicon substrates by thermal evaporation of in situ synthesized CuS powder. XRD analysis of these films revealed a single-crystal structure, AFM measurements indicated the films have a surface roughness (14.1 nm) and agglomerates of multiple monocrystalline particles with average size (66 nm), and the optical properties were investigated by UV-Vis spectrophotometer showing the films have high transmission (>80%) in the visible region and low absorbance with wide energy gap (3.813 eV). This novel structure with outstanding optical properties makes it very promising optical materials in optoelectronics.

     

Ionic Dependence of Membrane Potential and Glutamate Receptor-Linked Responses in Synaptoneurosomes as Measured with a Cyanine Dye, DiS-C2-(5)

Membrane potentials of particles present in a subcellular brain preparation, called synaptoneurosomes, have been monitored by measurement of changes in the absorbance of a cyanine dye, DiS-C2-5. The membrane potential of the particles seems to be dependent on both Cl- and K+ diffusion potentials, as judged from dependence of the absorbance changes on the K+ equilibrium potential across the membrane in the presence of Ba2+ or when Cl- was replaced with gluconate. The apparent high Cl- permeability of the membrane preparation was reduced in the presence of picrotoxin, a finding suggesting endogenous activation of receptor-linked Cl- channels. Glutamate and kainate caused depolarization of the membranes present in the preparation. This effect was only seen if K+ channels had been blocked in the presence of Ba2+ or 4-aminopyridine. No responses were observed with other glutamate receptor agonists (quisqualate or N-methyl-D-aspartate). The membrane potential of particles present in conventional synaptosomal preparations neither had a high Cl- permeability nor reacted to glutamate or kainate in the present conditions. The results suggest that synaptoneurosome preparations may be used for functional studies on postsynaptic neurotransmitter receptor-linked membrane potential changes with optical probes of membrane potential.