Estimation of flattening coefficient for absorption and circular dichroism using simulation

The absorbance and circular dichroism (CD) of suspensions is lower than if the same amount of chromophore were uniformly distributed throughout the medium. Several mathematical treatments of this absorption flattening phenomenon have been presented using various assumptions and approximations. This article demonstrates an alternative simulation approach that allows relaxation of assumptions. On current desktop computers, the algorithm runs quickly with enough particles and light paths considered to get answers that are usually accurate to better than 3%. Results from the simulation agree with the most popular analytical model for 0.01 volume fraction of particles, showing that the extent of flattening depends mainly on the absorbance through a particle diameter. Unlike previous models, the simulation can show that flattening is significantly lower when volume fraction increases to 0.1 but is higher when the particles have a size distribution. The simulation can predict the slope of the nearly linear relationship between flattening of CD and the absorbance of the suspension. This provides a method to correct experimental CD data where volume fraction and particle size are known.     

Spherical scatters in the lens of Acrossocheilus hexagonolepis as revealed by scanning electron microscopy and UV-visible spectroscopy

Scanning electron microscopy revealed the presence of spherical granules 0.25-0.75 micron in diameter in the lens fibres of the hill stream fish Acrossocheilus hexagonolepis. The density of the granules was approximately 8,500 per mm2. The size of the particles and their distribution pattern suggested they functioned in spherical scattering of the light. The absorbance and transmittance of light at different wavelengths obtained from the spectroscopic analysis of the lens indicated that a small percentage of light was neither absorbed nor transmitted. Since reflection usually does not take place from the ocular refractive structure, it may be that this proportion of the light was scattered from the lens.     

Experimental test of absorption flattening correction for circular dichroism of particle suspensions

There are well established theoretical models for correction for absorption flattening of circular dichroism (CD) measurements on particle suspensions. However, these have not been directly tested experimentally. We describe a test system with the chiral tris(ethylenediamine)Co(III) complex dissolved in water trapped inside sephadex particles, suspended in 1-butanol. Independent measurements of particle size distribution, volume fraction, and the absorbance of the suspension are used to calculate the required CD correction. The corrected CD signal is found to agree rather well with that for the same amount of Co-complex dispersed uniformly throughout the sample cell. This holds for different particle volume fractions and Co-complex concentrations inside the particles. The correction seems to work despite a substantial scattering contribution to the absorbance, which is not considered in the theoretical models.     

Size and shape determination of fixed chylomicrons and emulsions with fluid or solid surfaces by three-dimensional analysis of shadows

Chylomicrons and chylomicron-sized emulsions are spherical particles in suspension but their shape and apparent size may be distorted by electron microscopy processing. To assess adsorption to grids, flattening, and shrinkage, chylomicrons and emulsions were fixed with osmium tetroxide and together with polystyrene beads were shadowed with platinum. Vertical profiles projected from particle shadows indicated that the chylomicrons and emulsions were slightly shrunken, slightly truncated, oblate spheroids while the polystyrene beads were spheres. Particle diameters were corrected by assuming that volumes of oblate spheroids on the grid surface were equal to volumes of spheres in the original lipid suspension. Because of the compensating effects of shrinkage (decreases diameter) and flattening (increases diameter) the differences between the means of measured diameters and corrected diameters were less than or equal to 5%.     

A novel method for the follow-up of shape changes in erythrocyte and other particles

The absorbance of a suspension of asymmetrical particles fluctuates when the suspension is stirred in a circular motion. The effect is due to the fact that particles move in spiral orbits and present alternatively their short or long axis to the narrow beam of light used for measurement.Changes in the shape of erythrocytes such as flattening and swelling could be detected and quantitated by measuring the change in the amplitude of the fluctuating absorbance. The diagnostic value of this method is under investigation.     

Effects of Epidermal Cell Shape and Pigmentation on Optical Properties of Antirrhinum Petals at Visible and Ultraviolet Wavelengths

We used the Mixta+ and mixta- lines of Antirrhinum majus as a model system to investigate the effects of epidermal cell shape and pigmentation on tissue optical properties in the visible and ultraviolet (UV) spectral regions. Adaxial epidermal cells of Mixta+ flowers have a conical-papillate shape; in the mixta- line the cells are slightly domed. Mixta+ cells contained significantly more anthocyanin and other flavonoids than mixta- cells when plants were grown under either high- or low-UV conditions. Mixta+ cells focused light (3.5-4.7 times incident) within their pigmented interiors, whereas mixta- cells focused light (2.1-2.7 times incident) in the unpigmented mesophyll. UV light penetrated the epidermis (commonly 20-50% transmittance at 312 nm) mainly through the unpigmented peripheral regions of the cells that were similar for the two lines, so that overall penetration through Mixta+ and mixta- epidermises was equal. However, maximum UV absorption in the central region of epidermal cells was slightly greater in Mixta+ than mixta-, and intact Mixta+ flowers reflected less light in the spectral regions with intermediate flavonoid absorbance. In both cases, about 50 to 75% of the difference could be attributed to cell shape and resulting changes in the optical pathlength or focusing.     

Circular dichroism studies of subtilisin Carlsberg immobilised on micron sized silica particles

Immobilised enzymes are widely used in industry, but the reasons for loss of activity of such biocatalysts are usually not known. We have used circular dichroism (CD) to investigate the structure of one such system, i.e., subtilisin Carlsberg (SC) immobilised on silica gel particles (60 microm). A number of technical problems have to be overcome in order to obtain appropriate data from which conclusions can be drawn. A rotating cell holder has been developed to avoid sedimentation of the silica particles during the collection of spectra. By moving the cell holder as close as possible to the detector window, the effects of differential scattering can be minimised. However, the effects of absorption flattening limit the extent to which reliable quantitative information on secondary structure content can be obtained from far UV CD studies. We have used an empirical approach based on absorbance units derived from the high-tension voltage to correct for absorption flattening effects. After applying the correction there was satisfactory agreement with the solution spectra. Comparison of the fresh and used (inactive) SC-silica gel spectra in organic media reveals substantial change in the secondary structure. Additional evidence for loss of native conformation is provided by the significant decrease in the near UV CD spectrum. These results for the first time clearly demonstrate the origin of enzyme instability in the immobilised state.     

Abrasion of suspended biofilm pellets in airlift reactors: importance of shape, structure, and particle concentrations

The detachment of biomass from suspended biofilm pellets in three-phase internal loop airlift reactors was investigated under nongrowth conditions and in the presence of bare carrier particles. In different sets of experiments, the concentrations of biofilm pellets and bare carrier particles were varied independently. Gas hold-up, bubble size, and general flow pattern were strongly influenced by changes in volume fractions of biofilm pellets and bare carrier particles. In spite of this, the rate of biomass detachment was found to be linear with both the concentration of biofilm pellets and the bare carrier concentration up to a solids hold-up of 30%. This implies that the detachment rate was dominated by collisions between biofilm pellets and bare carrier particles. These collisions caused an on-going abrasion of the biofilm pellets, leading to a reduction in pellet volume. Breakage of the biofilm pellets was negligible. The biofilm pellets were essentially ellipsoidal, which made three-dimensional size determination necessary. Calculating particle volumes from two-dimensional image analysis measurements and assuming a spherical shape led to serious errors. The abrasion rate was not equal on all sides of the biofilm pellets, resulting in an increasing flattening of the pellets. This flattening was oriented with the basalt carrier inside the biofilm and independent of the absolute abrasion rate. These observations suggest that the collisions causing abrasion are somehow oriented. The internal structure of the biofilms showed two layers, a cell-dense outer layer and an interior with a low biomass density. Taking this density gradient into account, the washout of detached biomass matched observed changes in volume of the biofilm pellets. No gradient in biofilm strength with biofilm depth was indicated. (c) 1997 John Wiley & Sons, Inc.     

Ultraviolet absorption and epidermal-transmittance spectra in foliage

We examined the UV absorption spectra and the epidermal-transmittance spectra (280–350 nm) of foliage from 42 plant species. Sun foliage was sampled from naturally growing individuals of seven species in each of six life forms comprising two evergreen groups (gymnosperms and angiosperms) and four deciduous angiosperm groups (trees, shrubs and vines, herbaceous dicotyledons and grasses). There were large differences in absorption spectra of whole-leaf extracts among species. While absorbance declined with increasing wavelength in most woody species, there was a trough in absorbance around 300 nm in many herbaceous species. Absorption spectra were negatively correlated with epidermal-transmittance spectra in 31 of the 42 species. Relationships between absorbance and transmittance did not follow the theoretical exponential function. Species rankings of UV-screening effectiveness were similar when we assessed it by using epidermal transmittance at single wavelengths (300 or 320 nm) or different UV-action spectra to weight epidermal-transmittance spectra and estimate the levels of biologically effective UV reaching the mesophyll. Thus, differences in absolute epidermal transmittance among species appeared to overshadow spectral differences. Nevertheless, the differences we found in the internal UV spectral regime in foliage suggest that whole-plant action spectra will differ among species. While species rankings of UV-screening effectiveness were similar when different action spectra were used, the absolute amounts of biologically effective UV reaching the mesophyll of species varied considerably when different action spectra were used.     

Differential absorption flattening optical effects are significant in the circular dichroism spectra of large membrane fragments

The circular dichroism spectra of membrane particles are distorted by effects of differential absorption flattening, which are a consequence of the nonrandom distribution of chromophores in these samples. We have shown that this phenomenon is not significant in small unilamellar vesicles with high lipid to protein ratios [Mao, D., & Wallace, B. A. (1984) Biochemistry 23, 2667-2673]. It has recently been claimed [Glaeser, R. M., & Jap, B. K. (1985) Biochemistry 24, 6398-6401] that absorption flattening effects are also inconsequential in large membrane fragments with high protein concentrations, such as purple membrane sheets. This paper will demonstrate that absorption flattening is significant in these samples and that it causes substantial distortion of the calculated protein secondary structures derived from the uncorrected circular dichroism data.     

A gimbal-mounted pressurization chamber for macroscopic and microscopic assessment of ocular tissues

The biomechanical model of glaucoma considers intraocular pressure-related stress and resultant strain on load bearing connective tissues of the optic nerve and surrounding peripapillary sclera as one major causative influence that effects cellular, vascular, and axonal components of the optic nerve. By this reasoning, the quantification of variations in the microstructural architecture and macromechanical response of scleral shells in glaucomatous compared to healthy populations provides an insight into any variations that exist between patient populations. While scleral shells have been tested mechanically in planar and pressure-inflation scenarios the link between the macroscopic biomechanical response and the underlying microstructure has not been determined to date. A potential roadblock to determining how the microstructure changes based on pressure is the ability to mount the spherical scleral shells in a method that does not induce unwanted stresses to the samples (for instance, in the flattening of the spherical specimens), and then capturing macroscopic and microscopic changes under pressure. Often what is done is a macroscopic test followed by sample fixation and then imaging to determine microstructural organization. We introduce a novel device and method, which allows spherical samples to be pressurized and macroscopic and microstructural behavior quantified on fully hydrated ocular specimens. The samples are pressurized and a series of markers on the surface of the sclera imaged from several different perspectives and reconstructed between pressure points to allow for mapping of nonhomogenous strain. Pictures are taken from different perspectives through the use of mounting the pressurization scheme in a gimbal that allows for positioning the sample in several different spherical coordinate system configurations. This ability to move the sclera in space about the center of the globe, coupled with an upright multiphoton microscope, allows for collecting collagen, and elastin signal in a rapid automated fashion so the entire globe can be imaged.     

Lipid transport required to make lipids of photosynthetic membranes

This study presents a mathematical model, which expresses the absorbance of a photosynthetic sample as a non-linear polynomial of selected reference absorbance. The non-linearity is explained by inhomogeneities of a product of pigment concentration and light path length in the sample. The quadratic term of the polynomial reflects the extent of inhomogeneities, and the cubic term is related to deviation of the product distribution from a symmetric one. The model was tested by measurements of suspension of unstacked tobacco thylakoid membranes of different chlorophyll concentrations in cuvettes of different thicknesses. The absorbance was calculated from the diffuse transmittance and reflectance of sample, illuminated by perpendicular collimated light. The evaluated quantity was a sensitivity defined as the relative difference between the sample absorbance and the reference absorbance to the reference absorbance. The non-linearity of sample absorbance was demonstrated by a characteristic deviation of the sensitivity spectrum from a constant value. The absorbance non-linearity decreased on an increase of the product of pigment concentration and cuvette thickness. The model suggests that the sieve and detour effects influence the absorbance in a similar way. The model may be of interest in modeling of leaf or canopy optics including light absorption and scattering.     

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.     

Circular dichroism analyses of membrane proteins: an examination of differential light scattering and absorption flattening effects in large membrane vesicles and membrane sheets

The circular dichroism spectra of membrane suspensions are distorted by differential light scattering and absorption flattening effects, which arise as a consequence of the large size of the membrane particles relative to the wavelength of light and the high concentration of proteins in the membranes. In this paper, the consequences of these phenomena on the protein spectra of large membrane particles are discussed, and methods for eliminating them are examined. The distortions due to differential light scattering are relatively small in membrane systems, and can be compensated for by use of a large detector acceptance angle geometry. Several methods for correcting for differential flattening, which introduces a substantial distortion, have been evaluated, and a new method, the flattening quotient approach, which produces by far the best results, is described. Since the secondary structures calculated from circular dichroism spectra are highly dependent on accurate spectral shape and magnitude, this method for correcting the spectra may find general application in circular dichroism studies of membrane proteins.     

Core-shell Au nanoparticle formation with DNA-polymer hybrid coatings using aqueous ATRP

We report here a direct surface-grafting approach to forming DNA-containing polymer shells outside of Au nanoparticles using aqueous atom transfer radical polymerization (ATRP). In this approach, DNA molecules were immobilized on Au particles to introduce ATRP initiators on the surface. The same DNA molecules also acted as particle stabilizers through electrostatic repulsion and allowed particles to stay suspended in water. The immobilized ATRP initiators prompted polymer chain growth under certain conditions to form thick polymer shells outside of the particles. The formation of DNA-polymer hybrids outside of Au nanoparticles was characterized using absorption spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), and gel electrophoresis. The presence of thick polymer shells improved particle stability in high ionic strength media, whereas particles with the DNA coating only aggregated. A visible color difference between these two particle solutions was clearly observed, providing the basis for DNA sensing in homogeneous solutions.     

Comparative analysis of Cryptococcus neoformans acid-resistant particles generated from pigmented cells grown in different laccase substrates

Cryptococcus neoformans produces pigments in vitro in the presence of exogenous substrate. We characterized acid-resistant particles isolated from pigmented cells grown in L-dopa, methyl-dopa, (-)-epinephrine or (-)-norepinephrine. The goals of this study were to determine whether pigments made from each of these substrates were melanins and the consequences of pigmentation on related cell characteristics. The greatest yield of acid-resistant particles occurred with methyl-dopa followed by L-dopa. Electron microscopy indicated that L-dopa and methyl-dopa produced particles with thicker shells. The mAb 6D2 reacted with all particles, but a lower reactivity was observed with epinephrine-derived particles. ESR analysis revealed that epinephrine-derived particles failed to produce a stable free radical signal typical of melanins. Growth of C. neoformans in different substrates affected cell and capsule size but not capsule induction. Hence, the type of pigment produced by C. neoformans is dependent on the substrate and not all pigments meet the criteria for melanins.     

Spectral transmission through the cuticle of the American cockroach,Periplaneta americana

Small pieces of cuticle were removed from the following areas of the cockroach, P. americana, to determine the relative transmittance qualities across the visible and infrared regions of the spectrum: wing tip; abdominal tergite and sternite; light and dark pigmented areas of the pronotum; vertex; two pairs of wing tips plus underlying tergite. The transmittance across the spectral areas investigated showed significant differences in μW/cm² between the cuticular areas assayed. A single wing tip possessed high transmittance qualities whereas the vertex and lesser pigmented cuticle showed considerably lower per cent transmission.     

Absorption flattening in the circular dichroism spectra of small membrane fragments

The inhomogeneous distribution of chromophore occurring in a particulate suspension can result in a reduction in the apparent molar ellipticity recorded in circular dichroism (CD) spectra. The possibility of such a systematic error has often been a matter of concern when CD spectra of cell membrane proteins are recorded. The recent publication of CD spectra for bacteriorhodopsin in native and sonicated membranes, in detergent-solubilized form, and reconstituted into small unilamellar vesicles [Mao, D., & Wallace, B. A. (1984) Biochemistry 23, 2667-2673] gives a unique opportunity to apply the theoretical analysis of Gordon and Holzwarth [Gordon, D. J., & Holzwarth, G. (1971) Arch. Biochem. Biophys. 142, 481-488] so as to provide a definitive answer to the question of whether absorption flattening is significant for membrane particles. We show here that the data of Mao and Wallace can be combined with the theoretical analysis of Gordon and Holzwarth to rule out significant absorption flattening effects over the range 200-240 nm for submicrometer-sized membranes. In addition, the results show that absorption flattening can be disregarded even at 190 nm for membranous material in the size range below 100 nm. The demonstration that there are no major flattening effects in the CD spectra of bacteriorhodopsin, particularly in the region of 200-240 nm, means that the experimental spectra are incompatible with the proposal that this transmembrane protein contains seven transmembrane helices.     

Virus-like particles associated with mass mortalities of the pen shell Atrina pectinata in Japan

Mass mortalities of the pen shell Atrina pectinata occurred in the fishing grounds of Ariake Bay, in southwestern Japan, during late spring and summer in 2003 and 2004. Histological examination revealed extensive necrosis in the epithelial cells of the kidney and gill, and impairment of the endothelial cells of the mantle arteria. Although cestode larvae belonging to the genus Tylocephalum were found in the mantle, adductor muscle, kidney, and digestive gland, their prevalence and the intensity of infection were low. Examinations of moribund pen shells for Haplosporidium spp. infection using PCR analysis and for Perkinsus spp. infection using Ray's fluid thioglycollate medium were negative. Unenveloped virus-like particles were detected by transmission electron microscopy in the cytoplasm of affected kidney and gill cells of moribund pen shells. They were icosahedral spherical and 50 to 55 nm in diameter. These virus-like particles found in moribund pen shells are different from those described in other marine mollusks, and may be the causative agent of the mass mortalities of pen shells.