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.     

Demonstration of a highly-sensitive portable double-flash kinetic spectrophotometer for measurement of electron transfer reactions in intact plants

A highly sensitive, portable spectrophotometer for use in measuring flash-induced absorbance changes in intact leaves is demonstrated. The design of the instrument is modified for whole plant use from that suggested by Joliot and Joliot (Biochim. Biophys. Acta 765, 210–218). The spectrophotometer uses trifurcated light guides to deliver measuring and actinic beams to two comparable areas of the leaf. The measuring beam is provided by a series of short, relatively intense light pulses from a xenon flashlamp in place of the constant weak measuring beam used in conventional machines. The use of a flash measuring beam and differential detection allows for a high signal-to-noise ratio (noise levels of 10^-5A) without significant actinic effects. The time resolution of the instrument is 2 sec and the noise level is independent of the experimental time range. The instrument is battery or mains powered, computer operated, and has a liquid crystal display for computer-user interface and dialogue, and to show the kinetic traces graphically. Wavelength selection is provided by interchangeable interference filters. The instrument can communicate with a laboratory-based computer, receiving programming information and sending experimental data to be processed and plotted. The instrument is demonstrated by following the kinetics of the electrochromic shift, the change in redox states of cytochrome f and the b cytochromes in an intact cucumber leaf, and in the same leaf after infiltration with DCMU.     

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.     

Low-angle light-scattering instrument for DNA solutions

A light-scattering instrument capable of measurements on native DNA at angles as low as 10° is described. The major features of the instrument, which make it capable of low-angle measurements, are the use of an intense light source in which the incident beam is monitored directly and the use of a long, rectangular sample cell in which the scattered light can be measured at low angles with no interference from the incident beam. Methods for calibrating the instrument and for determining scattering correction factors are described. Procedures for the preparation and use of various calibration standards are given.     

LED array spectrophotometer for measurement of time resolved difference spectra in the 530–600 nm wavelength region

A new type of computer controlled spectrophotometer is described which is based on an array of independent, monochromatic pulsed light sources consisting of light emitting diodes (LED) equipped with narrow band interference filters. The LEDs are sequentially pulsed at a high repetition rate. The absorbance information at specific wavelengths is sampled in the s-time range, using a computer-controlled, highly selective technique of synchronous amplification. A first prototype of this LED Array Spectrophotometer allows simultaneous recording of kinetic changes at 16 different wavelengths in the range from 530 to 600 nm, with a time resolution of 1 ms/point. Special features of the new type of spectrophotometer are: Weak integrated measuring light intensity, high signal/noise ratio even with scattering samples like intact leaves, active baseline adjustment by LED current regulation, computer control of system operation and data analysis. To deconvolute the complex absorbance changes in the cytochrome -band region, standard spectra of the major components are stored in computer memory and used for curve fitting of difference spectra and kinetic changes. As an example of application, the light-induced absorbance changes in a heat-pretreated spinach leaf are analysed. The system effectively separates specific absorbance changes of C550, cyt f, cyt b ₅₅₉ and cyt b ₅₆₃ from a large background of non-specific changes.Abbreviations DCMU 3-(3, 4-dichlorophenyl-)1, 1-dimethylurea - DAD diaminodurol - DNP-INT 2, 4-dinitrophenylether of 2-iodo-4-nitrothymol - ANT-2p 2-(3-chloro-4-trifluoromethyl)-anilino-3, 5-dinitrothiophene - RAM random access memory - LED light emitting diode - HBW half bandwidth     

Dark-interval relaxation kinetics (DIRK) of absorbance changes as a quantitative probe of steady-state electron transfer

We introduce a new, non-invasive technique to measure linear electron transfer in intact leaves under steady-state illumination. Dark-interval relaxation kinetic or ‘DIRK’ analysis is based on measurements of the initial rates of relaxation of steady-state absorbance signals upon a rapid light-dark transition. We show that estimates of electron flux by DIRK analysis of absorbance signals, reflecting redox changes in the photosynthetic electron transfer chain, can yield quantitative information about photosynthetic flux when the light-dependent partitioning of electrons among redox components of the electron transfer chain are considered. This concept is modeled in computer simulations and then demonstrated in vivo with tobacco plants under non-photorespiratory conditions resulting in linear relationships between DIRK analysis and gross carbon assimilation (A_G). Estimation based on DIRK analysis of the number of electrons transferred through the photosynthetic apparatus for each CO₂ fixed was within 20% of the theoretical value. Possible errors and future improvements are discussed. We conclude that the DIRK method represents a useful tool to address issues such as plant stress and photosynthetic regulation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Moderate heat stress of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> leaves causes chloroplast swelling and plastoglobule formation

Photosynthesis is inhibited by heat stress. This inhibition is rapidly reversible when heat stress is moderate but irreversible at higher temperature. Absorbance changes can be used to detect a variety of biophysical parameters in intact leaves. We found that moderate heat stress caused a large reduction of the apparent absorbance of green light in light-adapted, intact Arabidopsis thaliana leaves. Three mechanisms that can affect green light absorbance of leaves, namely, zeaxanthin accumulation (absorbance peak at 505 nm), the electrochromic shift (ECS) of carotenoid absorption spectra (peak at 518 nm), and light scattering (peak at 535 nm) were investigated. The change of green light absorbance caused by heat treatment was not caused by changes of zeaxanthin content nor by the ECS. The formation of non-photochemical quenching (NPQ), chloroplast movements, and chloroplast swelling and shrinkage can all affect light scattering inside leaves. The formation of NPQ under high temperature was not well correlated with the heat-induced absorbance change, and light microscopy revealed no appreciable changes of chloroplast location because of heat treatment. Transmission electron microscopy results showed swollen chloroplasts and increased number of plastoglobules in heat-treated leaves, indicating that the structural changes of chloroplasts and thylakoids are significant results of moderate heat stress and may explain the reduced apparent absorbance of green light under moderately high temperature.     

Light-induced spectral absorbance changes in relation to photosynthesis and the epoxidation state of xanthophyll cycle components in cotton leaves

When cotton (Gossypium hirsutum L., cv Acaia SJC-1) leaves kept in weak light were suddenly exposed to strong red actinic light a spectral absorbance change took place having the following prominent characteristics. (a) It was irreversible within the first four minute period after darkening. (b) The difference in leaf absorbance between illuminated and predarkened leaves had a major peak at 505 nanometers, a minor peak at 465 nanometers, a shoulder around 515 nanometers, and minor troughs at 455 and 480 nanometers. (c) On the basis of its spectral and kinetic characteristics this absorbance change can be readily distinguished from the much faster electrochromic shift which has a peak at 515 nanometers, from the slow, so-called light-scattering change which has a broad peak centered around 535 nanometers and is reversed upon darkening, and from absorbance changes associated with light-induced chloroplast rearrangements. (d) The extent and time course of this absorbance change closely matched that of the deepoxidation of violaxanthin to zeaxanthin in the same leaves. (e) Both the absorbance change and the ability to form zeaxanthin were completely blocked in leaves to which dithiothreitol (DTT) had been provided through the cut petlole. DTT treatment also caused strong inhibition of that component of the 535-nanometer absorbance change which is reversed in less than 4 minutes upon darkening and considered to be caused by increased light scattering. Moreover, DTT inhibited a large part of nonphotochemical quenching of chlorophyll fluorescence in the presence of excessive light. However, DTT had no detectable effect on the photon yield of photosynthesis measured under strictly rate-limiting photon flux densities or on the light-saturated photosynthetic capacity, at least in the short term. We conclude that it is possible to monitor light-induced violaxanthin de-epoxidation in green intact leaves by measurement of the absorbance change at 505 nanometers. Determination of absorbance changes in conjunction with measurements of photosynthesis in the presence and absence of DTT provide a system well suited for future studies of meachanisms of dissipation of excessive excitation energy in intact leaves.     

Growth behavior of microorganisms using UV-Vis spectroscopy: Escherichia coli

Multiwavelength transmission spectra of microorganisms and cell suspensions consist of combined absorption and scattering phenomena resulting from the interaction of light with microorganisms or cells typically suspended in a nonabsorbing media. The distribution of intensities as a function of wavelength depends on the size, shape, and optical properties of the sample. The optical properties are functions of the chemical composition and the state of aggregation, or association, of the chromophoric groups contained in the microorganisms. This article explores the growth behavior of Escherichia coli from the perspective of multiwavelength UV-Vis spectroscopy. Experimentally, it is demonstrated that the spectral signatures of the microorganism evolve as a function of time. It is also demonstrated that the spectral changes observed during growth are consistent with data reported elsewhere. From the theoretical point of view, it is demonstrated that the spectral signatures can be adequately represented with an interpretation model based on light-scattering theory. The parameters from the interpretation model reflect changes in size and chemical composition known to take place in the microorganisms during growth.     

Twin-beam laser velocimeter for the investigation of spermatozoon motility

Previous laser light-scattering studies of spermatozoon motility have been hampered by the large, asymmetric shape of spermatozoa, which causes difficulties in the interpretation of intensity fluctuations in the light scattered from a single laser beam. This paper describes an experimental arrangement for measuring the distribution of transit times for swimming spermatozoa using two slightly separated, focused laser beams. The theory of operation of the instrument is developed to enable the analysis of the experimentally obtained cross-correlation functions. The effects of the pronounced spermatozoon asymmetry and associated intensity modulation in the scattered light are also investigated and shown to be negligible for the twin beam experimental arrangement, provided that the swimming speed distribution has a coefficient of variation (sigma/upsilon greater than 0.1. Results obtained using this apparatus are presented for the velocity distribution of spermatozoa from a variety of bulls.     

Light-induced absorbance changes in Phycomyces: evidence for cryptochrome-associated flavosemiquinones

Light-induced absorbance changes (LIACs), which are associated with early photochemical events of blue-light transduction, were detected in growing zones of Phycomyces sporangiophores. The novel LIACs meet all the essential requirements for a spectrophotometric photoreceptor assay which was previously unavailaible for blue-light receptors (cryptochromes). In-vivo absorption spectra of growing zones were derived from reflection spectra which were measured with a novel rapid-scan spectrophotometer. To detect photoreceptor-associated absorbance changes white mutants were employed which lack the interfering bulk pigment β-carotene. Blue and white light, not however red light, induced in these strains absorbance changes near 460–490 and 600–620 nm. The LIACs were absent in light-insensitive mutants with defects in the genes madA, madB and madC. Because these genes affect photosensory adaptation and the blue-light receptor itself, the novel in-vivo LIACs must be associated with photochemical events which occur early in the transduction chain. The spectral characteristics of the LIACs are in accordance with a blue- and red-light absorbing flavosemiquinone which is generated upon light absorption by an oxidized flavin receptor. It is proposed that the flavosemiquinone functions itself as photoreceptor which mediates several red-light responses of Phycomyces. Received: 28 September 1998 / Accepted: 25 November 1998     

Modified spectrophotometer for multi-dimensional circular dichroism/fluorescence data acquisition in titration experiments: application to the pH and guanidine-HCI induced unfolding of apomyoglobin.

In a previous paper (Ramsay and Eftink, Biophys. J. 66:516-523) we reported the development of a modified spectrophotometer that can make nearly simultaneous circular dichroism (CD) and fluorescence measurements. This arrangement allows multiple data sets to be collected during a single experiment, resulting in a saving of time and material, and improved correlation between the different types of measurements. The usefulness of the instrument was shown by thermal melting experiments on several different protein systems. This CD/fluorometer spectrophotometer has been further modified by interfacing with a syringe pump and a pH meter. This arrangement allows ligand, pH, and chemical denaturation titration experiments to be performed while monitoring changes in the sample's CD, absorbance, fluorescence, and light scattering properties. Our data acquisition program also has an ability to check whether the signals have approached equilibrium before the data is recorded. For performing pH titrations we have developed a procedure which uses the signal from a pH meter in a feedback circuit in order to collect data at evenly spaced pH intervals. We demonstrate the use of this instrument with studies of the unfolding of sperm whale apomyoglobin, as induced by acid pH and by the addition of guanidine-HCI.     

Selective absorption and scattering of light by solutions of macromolecules and by particulate suspensions

For particulate suspensions and for solutions that scatter light measurably the total absorbance A generally contains contributions due to specific absorption (Aa) and scattering of light (As). The quantity As is closely related to the turbidity tau. In general, spectrophotometry of such systems requires proper modification of the spectrophotometer used in order to permit accurate determination of the absorbance A and of the derived quantities Aa and As. Apparent deviation from Beer's law in such systems is often due to inappropriate experimental technique. After a discussion of the parameters that determine the intensity of light scattered by solutes, an account is given of the experimental precautions to be taken for determination of the absorbance of light scattering suspensions and solutions and of techniques for correcting absorbance spectra for scattering of light. Measurement of the turbidity is briefly confronted with determination of the scattering ratio i90 degrees/Io and the impact of erroneous turbidity measurements on derived molecular parameters is discussed.     

The indirect nature of interaction of glucose transport with the system transporting galactosides

The membrane transport systems for galactosides and glucose derivatives interact in enteric microorganisms. Stop-flow experiments with a double wavelength spectrophotometer and a flow-through cuvette (designed to minimize light-scattering effects) were used to measure the speed of interaction in Escherichia coli. The in vivo hydrolysis of ortho-nitrophenol-beta-D-galactopyranoside was measured by comparing the light transmitted by cell suspensions at 420 nm with that at 500 nm. Measurements at the latter wavelength corrected for residual scattering effects. The stop-flow experiment allowed the study of the early kinetics of transport and hydrolysis. It was found with strain ML308 that there was a significant lag in the achievement of steady-state inhibition by glucose and its derivative methyl-alpha-D-glucopyranoside (alpha MG). This strain constitutively produces high levels of permease and beta-galactosidase. The absorbancy increases at 420 nm are limited by transport because the beta-galactosidase is present inside the cells in excess. From earlier results, it was not surprising that inhibition is delayed with low concentrations of the glucose compounds, but the new double wavelength technique showed no kinetic component of rapid inhibition. This result therefore excludes competition for some membrane-bound component and is consistent with the production of the dephosphorylated form of the soluble Enzyme IIIglu that binds and inhibits the permease system in the membrane.     

The influence of photometer design on optical-conformational changes.

When cells and large subcellular structures suffer a change in volume or internal structure, their light-scattering properties are normally altered. These optical-conformation changes are potential sources of information about conformation and processes which alter it. Classical light-scattering theory for spherical particles is used to determine how the transmittance or extinction of a cell suspension should respond when such a conformational change occurs and the measurements are made with a conventional photometer. This extends an earlier study of transmittances measured with an “ideal” photometer. The photocell of an ideal instrument collects only the directly transmitted light. In a conventional instrument it also collects the light scattered at small angles, which is usually most of the scattered light. Extinction (optical density, absorbance) of suspensions of spherical cells was computed for several photometer designs. It is found that γ, the angle of acceptance of the photocell, has a significant influence on the extent and even the nature of the photometric response to a given conformational change. Earlier, it was shown that a decrease in cell volume or increase in internal structure will increase extinction for cells of many sizes. Now it is found that a large γ-value increases these effects. An approach to the interpretation of transmitted light fluxes in terms of theoretical predictions is outlined.     

Application of orthogonal light scattering for routine screening of lymphocyte samples

Orthogonal and forward light-scattering properties of lymphocytes were measured from patients with different lymphocytic diseases in order to determine the potential value of light scattering as a screening device. Monitoring of orthogonal light scattering of lymphocytes of a B-cell chronic lymphocytic leukemia patient during splenic irradiation (SI) revealed the selective decrease of malignant cells and the fact that the major part of the residual lymphocytes were cytotoxic lymphocytes. By combining forward and orthogonal light scattering it was shown that lymphocytes from a patient with T gamma lymphocytosis were abnormal. Orthogonal light scattering also showed an increase in cytotoxic lymphocytes in a patient with mononucleosis infectiosa and in a splenectomized patient. Orthogonal light scattering of lymphocyte subpopulations showed that the leu8+ population of a patient with mononucleosis infectiosa was bidisperse. For elderly donors the occurrence of CD3+, CD4+, CD8+, and HNK-1+ lymphocytes with a large orthogonal light scattering varied considerably. The CD8+ lymphocytes of these donors consisted mainly of cytotoxic lymphocytes. These results show that determination of light-scattering properties of lymphocytes may yield important diagnostic information and can indicate when further investigation of the lymphocytes by means of immunofluorescence is necessary.     

Molecular weight of T7 and calf thymus DNA by low-angle light scattering

A low-angle light-scattering instrument has been used to measure molecular weights of native calf-thymus and T7 DNA. Molecular weights obtained by extrapolation of angular data to 0° from measurements above 30° are less than molecular weights from extrapolation of data taken at low angles (below 30°). The low-angle molecular weights determined for calf-thymus DNA and for T7 DNA agree well with estimates of weight-average molecular weight obtained with other techniques. The low-angle light-scattering molecular weight for calf-thymus DNA is higher than previously reported values by light scattering at angles above 30°. A concentration dependence in the scattering from DNA solutions is also observed.     

A simple and accurate method for quantification of magnetosomes in magnetotactic bacteria by common spectrophotometer

A simple apparatus for measuring the magnetism of magnetotactic bacteria was developed with a common laboratory spectrophotometer, which was based on measuring the change in light scattering resulting from cell alignment in a magnetic field. A multiple coils were built around the cuvette holder of the spectrophotometer to compensate geomagnetic field and to generate two mutually perpendicular magnetic fields. In addition, we defined a novel magnetism parameter, R(mag), by modifying the definition of C(mag) to a normalized parameter with the culture absorbance obtained without application of magnetic field. The number of magnetosomes in each cell was determined by transmission electron microscopy to assess the relationship between the two magnetism parameters and the distribution of magnetosomes in the cells. We found that both R(mag) and C(mag) were linearly correlated rather with the percentage of magnetosome-containing bacteria than with the average magnetosome numbers, and R(mag) exhibited a better linearity than C(mag) with respect to the percentage of magnetosome-containing bacteria.     

Light-scattering polarization measurements as a new parameter in flow cytometry

Polarization measurement of orthogonal light scattering is introduced as a new optical parameter in flow cytometry. In the experimental setup, the electrical field of the incident laser beam is polarized in the direction of the sample flow. The intensity of the orthogonal light scattering polarized along the direction of the incoming laser beam is called depolarized orthogonal light scattering. Theoretical analysis shows that for small values of the detection aperture, the measured depolarization is caused by anisotropic cell structures and multiple scattering processes inside the cell. Measurements of the orthogonal depolarized light scattering in combination with the normal orthogonal light scattering of human leucocytes revealed two populations of granulocytes. By means of cell sorting it was shown that the granulocytes with a relatively high depolarization are eosinophilic granulocytes. Similar experiments with human lymphocytes revealed a minor subpopulation of yet-unidentified lymphocytes with a relative large orthogonal light-scattering depolarization. The results were obtained with an argon ion laser tuned at different wavelengths as well as with a 630-nm helium neon laser. These results show that measurement of depolarized orthogonal light scattering is a useful new parameter for flow-cytometric cell differentiation.