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The optics of vitreous surgery.

We calculated the refractive power of the human eye in the various optical configurations that may occur during vitreous surgery. We found that a biconcave corneal contact lens with an anterior surface refractive power of -63 to -98 diopters allowed the fundus to be seen when the vitreous cavity was filled with air, as did a weak minus lens attached to the bottom of the operating microscope. Our tests showed that the unsymmetrical biconcave lenses were superior.

Contact Lenses↗

Obtaining a high-quality retinal image with a biconvex intraocular lens.

We investigated the quality of the retinal image provided by a conventional, plano-convex intraocular lens by calculating the point spread function using a wide-angle model of the eye. We then calculated the ratio of the front and back curvatures for a biconvex lens with the conventionally used refractive index (1.491) using Seidel's aberration formulas. The point spread function for this intraocular lens was better than that of the conventional plano-convex lens. THe calculated ratio, which depends on the refractive index and the depth of the anterior chamber, was 3.16 (refractive index, 1.491; anterior chamber, 3.5 mm). As the depth of the anterior chamber decreases, the best ratio increases whereas the total power of the intraocular lens decreases. If an asymmetric intraocular lens is used, the greater curvature must face the cornea.

Anterior Chamber↗

Scleral canal size and optic nerve head drusen.

From projected optic disk photographs we measured the size of the scleral canal in two samples of emmetropic patients: one of patients with unilateral pseudopapilledema and drusen and the other of the general normal population. Measurements on the non-drusen-containing optic disk of patients with unilateral drusen were taken to reflect the scleral canal size of the fellow, affected eye. For both trained (t = 6.642) and untrained (t = 4.274) observers, the average diameters of the non-drusen-containing optic disks of patients with unilateral drusen were significantly smaller than those of the optic disks of normal patients (P = .0005, one-tailed independent t-test). The association of a small scleral canal with vascular anomalies, frequently noted in optic disks of patients with drusen, indicates a mesodermal dysgenesis of the optic nerve head.

Adult↗

Differential refractometric determination of binding of sodium dodecyl sulfate to protein using high-performance gel chromatography.

When sodium dodecyl sulfate (SDS) is added to a high-performance gel chromatographic column equilibrated with a buffer solution containing SDS at a level above the critical micelle concentration, the surplus SDS migrates as micelles giving a sharp peak. The presence of an unfolded protein in the sample solution gives a polypeptide peak in advance of the SDS micelle peak. As the result of SDS binding to the polypeptide, the SDS micelle peak is attenuated in comparison to that in the absence of protein. Thus the amount of SDS bound to the polypeptide can be determined accurately and simply from the decrease in the area of the SDS micelle peak. This approach is particularly useful for precise determination of bound SDS, which is pertinent to understanding the state of the protein polypeptide-SDS complex under the conditions of SDS-polyacrylamide gel electrophoresis.

Chromatography, High Pressure Liquid↗

Detection of receptor-ligand interactions using surface plasmon resonance: model studies employing the HIV-1 gp120/CD4 interaction.

Surface plasmon resonance (SPR), a label-free, real time optical detection principle, has been investigated for its potential to detect and quantitate macromolecular ligand-ligate interactions. As model systems, the interactions of the HIV-1 envelope glycoprotein, gp120, and the monoclonal antibody L-71, with a soluble form of the T-cell receptor CD4 (sCD4), were investigated. In an effort to demonstrate potential analytical applications of this technology, operational characteristics of the SPR instrumentation (BIAcore, Pharmacia) including stability of the sensing surface and reproducibility in the measurement of such macromolecular interactions were investigated. In addition, the ability to detect and quantitate sCD4 directly from unfractionated cell culture supernatants, such as Streptomyces lividans, was investigated. The results demonstrate that SPR has potential in quantitating macromolecular interactions in both purified and crude samples and that the reproducibility in, and sensitivity of, such determinations is comparable to other techniques.

Antibodies, Monoclonal↗

Immobilization chemistries suitable for use in the BIAcore surface plasmon resonance detector.

Surface plasmon resonance detectors, such as the BIAcore instrument produced by Pharmacia, show promise for the detection and quantitation of macromolecular interactions in a label-free mode. Such detectors rely on the covalent immobilization of one of the interacting species onto the sensing surface. To date, the only published chemistry for this purpose is reaction of primary amino-containing ligands with an N-hydroxysuccinimide (NHS) ester-activated surface. In an effort to increase the versatility of the BIAcore with respect to immobilizing ligands, we undertook an investigation of activation chemistries compatible with this system. Using readily available reagents, we demonstrated that the carboxylated dextran-coated sensing surface could be easily converted to functions other than NHS-esters, including amine-activated, hydrazine-activated, and sulfhydryl-activated surfaces. In addition, use was made of the streptavidin/biotin interaction to probe chemical modifications of the sensing surface, by employing specifically modified biotin derivatives.

Avidin↗

The thickness of monoolein lipid bilayers as determined from reflectance measurements.

Measurements of the reflectance of monoolein/n-alkane and monoolein/squalene lipid bilayers have been made. The total thickness of the bilayer was calculated from the dependence of reflectance on the refractive index of the aqueous salt or sucrose solution surrounding the bilayer. The total thickness was then compared to the thickness of the hydrocarbon chain region as determined from capacitance measurements. From this comparison, we found that the thickness of each polar region of the bilayers in salt solutions was 0.5 +/- 0.1 nm, independent of the hydrocarbon solvent used. When the aqueous solutions contained sucrose, each polar region was approx. 0.9 nm thick. When n-tetradecane and n-hexadecane were used as solvents, microlenses of solvent trapped in the monoolein bilayer increased the reflectance. After about one hour, the coalescence of microlenses into larger lenses allowed the reflectance of the bilayer alone to be measured. The use of reflectance to measure the thickness of monoolein bilayers appears to be consistent with other methods and to give useful information about the structure of lipid bilayers.

Chemical Phenomena↗

Determination of solute reflection coefficients in kidney brush-border membrane vesicles by light scattering: influence of the refractive index.

Solute reflection coefficients sigma of cell membrane vesicles or liposomes are commonly determined by comparison of the water flow induced by a gradient of the studied solute and that of a reference molecule using light scattering techniques. However, variations in scattered light which are mainly related to change in vesicle volume are also influenced by the refractive index of the surrounding medium. Therefore comparing kinetics of vesicle shrinkage induced by hyperosmotic solutions which have different refractive indexes might lead to an under or over estimation of sigma. We determined sigma NaCl in rat kidney brush-border membrane vesicles by two different approaches using mannitol, a poorly permeant molecule, as reference. (1) The refractive index of the hyperosmotic NaCl solution was adjusted to that of mannitol by addition of polyvinyl pyrrolidone (Mr 40,000), without a significant increase in osmolality. Thereby the change in scattered light intensity induced by osmotic vesicle shrinkage due to gradients of NaCl and mannitol were comparable and led to a sigma NaCl value close to one instead of the previously published value of 0.53. (2) The reflection coefficient was calculated from the lifetime of vesicle shrinkage which is not refractive index-dependent. Again sigma NaCl was not different from one. These results suggest that the water proteic pathways found in the luminal membrane of proximal tubules are not shared by salts.

Animals↗

Phase equilibria and formation of vesicles of dioleoylphosphatidylcholine in glycerol/water mixtures.

The lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) forms a lamellar liquid crystalline phase (L alpha) in arbitrary mixtures of glycerol and water. The phase has been characterized by means of X-ray diffraction, 31P-NMR spectroscopy and differential scanning calorimetry (DSC). In the L alpha state, and for DOPC concentrations greater than 50% (w/w), the thickness of the lipid bilayer decreases, while the area of the polar head group increases with increasing glycerol concentration. The phase transition from gel to L alpha state occurs in the range of 240 to 260 K. Contrary to a previous (McDaniel, R.V., McIntosh, T.J. and Simon, S.A. (1983) Biochim. Biophys. Acta 731, 97) study of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) we find that in the gel state, the thickness of the DOPC lipid bilayer is greater than that in the L alpha state. This suggests that in the gel state, the lipid acyl chains of DOPC are in extended configuration. The lamellar phase reaches its maximum swelling at about 50% (w/w) of DOPC. At lower DOPC concentrations a two-phase system is formed where the lamellar phase exists in equilibrium with excess of solvent. Unilamellar vesicles can be prepared from a diluted suspension of the lamellar phase either by using the sonicator or extruder technique. We show this by means of 31P-NMR, EPR and fluorescence spectroscopy. The mean radius of the vesicles, prepared by a sonicator, has been determined at different glycerol/water mixtures. It is found to decrease continuously from 100 A at 100% water to a minimum of 75 A at about 50% water in the solvent mixture. By further decreasing the water content in the solution, the radius rapidly increases, and a mean radius of 450 A is estimated at a water content of 10%. The rotational relaxation times of a fluorescent probe and two EPR spin probes, solubilized in DOPC vesicles, have been measured at different glycerol/water mixtures. It is found that the rotational rates are always much slower in the systems containing glycerol.

Calorimetry, Differential Scanning↗

Automation of a cryopreservation apparatus.

An organ preservation apparatus was interfaced to a process-control computer and automated. The flexibility of the equipment was demonstrated by running simultaneous dual sets of experiments under multiple and variable conditions. The computer-control program was written in a new high-level language, Real Time Extended Basic (RTEB), which allowed interactive calibration of the instruments, accurate control of variables, and modification of parameters during run time. A series of preliminary tests in the areas of normothermia, hypothermia, cooling of isolated rat hearts, and cryoprotectant toxicity were performed successfully. This work describes the apparatus and computer program in use.

Analog-Digital Conversion↗