Imaging properties of bright-field and annular-dark-field scanning confocal electron microscopy: II. point spread function analysis.

The imaging properties of bright field and annular dark field scanning confocal electron microscopy (BF-SCEM and ADF-SCEM) are discussed based on their point spread functions (PSFs) in comparison with multislice simulations. Although the PSFs of BF-SCEM and ADF-SCEM show similar hourglass shapes, their numerical distributions are quite different: BF-SCEM PSF is always positive and shows a center of symmetry whereas the ADF-SCEM PSF is complex and has Hermitian symmetry. These PSF properties explain the large elongation effect in BF-SCEM for laterally extended object and almost no-elongation in ADF-SCEM, illustrating the importance of the numerical analysis of PSFs. The Hermitian symmetry of the ADF-SCEM PSF results in an interesting "edge enhancement effect" at the interface. Simulation using the PSF and the multislice method verified this effect at GaAs surfaces and InAs interfaces embedded in GaAs. This unique feature of ADF-SCEM can potentially be useful for depth sectioning. It is also pointed out that a PSF imaging model cannot be applicable for BF-SCEM of a phase object, when the system is symmetric and aberration free.

Microstructures and rheological properties of tilapia fish-scale collagen hydrogels with aligned fibrils fabricated under magnetic fields.

Tilapia fish-scale type I atelocollagen hydrogels with aligned fibril structures were fabricated under a strong magnetic field of 6 or 12 T using two different methods. In the first method, a solution of acid-soluble collagen was neutralized with phosphate buffer saline and maintained in the magnetic field at 28°C for 3h. Under these conditions fibrogenesis occurs, and a hydrogel is formed. The hydrogel was subsequently crosslinked with ethyl-dimethylcarbodiimide (EDC). In the second method, the hydrogels were formed as described above, but in the absence of an applied magnetic field. Only after being crosslinked with EDC were these gels exposed to the magnetic field (28°C for 3h). Both methods led to alignment of the collagen fibrils perpendicular to the magnetic direction, the extent of which depended on the duration of magnetic treatment. Even after EDC treatment, collagen fibrils can align, indicating that crosslinking has taken place within fibrils. Both sorts of aligned hydrogels exhibited similar rheological properties with higher storage and loss moduli than were observed with unoriented gels. The hydrogels treated at 6 T had the best rheological properties. The decrease in tangent angle phase delta indicated that the ratio of elasticity to viscosity was greater in the crosslinked than in the non-crosslinked hydrogels. Atomic force microscopy images showed that magnetic treatment had no effect on the nanostructure of collagen fibrils. Differential scanning calorimetry measurements indicated that collagen hydrogels with and without magnetic treatment had the same denaturation temperature, 48°C, while EDC crosslinking increased the denaturation temperature to 62°C.

Imaging properties of bright-field and annular-dark-field scanning confocal electron microscopy.

Imaging properties of scanning confocal electron microscopy (SCEM) were studied by calculating simple model systems using the multislice method. A simple geometrical explanation was given, particularly for the difference between bright field (BF) and annular dark field (ADF) SCEM. It is demonstrated that the BF-SCEM image contrast consists of two features. One gradually changes over a wide defocus range and depends on the lateral size of the object. Another appears only near the focus and is independent of sample size. On the contrary, ADF-SCEM image contrast does not depend on the lateral size of the object. Therefore, the ADF-SCEM will provide more readily interpretable image contrast.

Bloch wave-based calculation of imaging properties of high-resolution scanning confocal electron microscopy.

An efficient, Bloch wave-based method is presented for simulation of high-resolution scanning confocal electron microscopy (SCEM) images. The latter are predicted to have coherent nature, i.e. to exhibit atomic contrast reversals depending on the lens defocus settings and sample thickness. The optimal defocus settings are suggested and the 3D imaging capabilities of SCEM are analyzed in detail. In particular, by monitoring average image intensity as a function of the probe focus depth, it should be possible to accurately measure the depth of a heavy-atom layer embedded in a light-element matrix.

Layer-doubling method in ADF-STEM image simulation.

A layer-doubling method developed in LEED calculation is applied to the ADF-STEM image simulation. This approach makes it possible to simulate image intensities of systems having a repeated slab structure, such as embedded precipitates or defects, with a much higher efficiency because it does not require the diagonalization of repeated slabs. As a simple example of this method, channeling effects are calculated for a system with embedded crystalline displaced slabs for various different slab thicknesses.

New scheme for calculation of annular dark-field STEM image including both elastically diffracted and TDS waves.

A new scheme of calculation of high-angle annular dark-field STEM image, capable of including both elastically diffracted and thermal diffuse scattering waves, has been presented by a combination of Pennycook's and Nakamura's methods. The new scheme has been demonstrated for image simulations of Si(011) as functions of thickness, defocus values and detector angles. In the present method, the TDS electron intensities are treated in the same way as in Pennycook's method, having a clear physical picture of its origin and reflecting the atom configuration in the systems. For the case of Si(011), it has been confirmed that at the detector angle of 60 to 160 mrad, which is usually applied, the image becomes highly incoherent, and even the image formed only from SOLZ beams becomes incoherent at the detector angle. At a low detector angle, however, the image has coherent features indicating the necessity of a simulation for individual systems.

High-angle annular dark-field STEM observation of Xe nanocrystals embedded in Al.

High-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) observation of Xe precipitates embedded in crystalline membranes has been made using electron probes of atomic dimensions and HAADF-STEM images of Xe precipitates qualitatively different from conventional TEM observation results have been obtained. Multislice-based HAADF-STEM simulation has been made and it has been revealed that the intensity of images of Xe atoms at positions displaced from Al matrix columns decreases rapidly as the thickness increases. Even in a thin specimen, the off-site Xe atoms of the precipitate at deep locations, were not observable. Therefore, different images are expected for specimens of different thicknesses or depths of these precipitates. These results indicate that the observation of precipitates in crystalline membranes requires some care.

High-resolution transmission electron microscopy observation of the cross-sectional structure of reconstructed silicon (5,5,12) surface.

The cross-sectional reconstructed structure of Si (5,5,12) surface was, for the first time, observed using ultrahigh vacuum high-resolution transmission electron microscopy (UHV-HRTEM) profile view method. In the high-index region, two units of the (337) surface combine with one units of the (225) surface to complete one unit cell of (5,5,12) surface. The (337) unit at one side of the (225) unit is distinctly different from that at another side of (225) unit. The observed HRTEM images do not agree with the previous structural models of the (5,5,12) surface. We propose a new structure model of this surface using a TEM image simulation. Our model is agreement with the previously reported scanning tunnelling microscope images.

Properties of the membranes containing the particulate methane monooxygenase from Methylosinus trichosporium OB3b.

The particulate methane monooxygenase (pMMO) from Methylosinus trichosporium OB3b was partially purified and characterized by measuring the effects of reducing agents and additives, and the stability of pMMO was studied. Duroquinol was a suitable reducing agent, and pMMO was stabilized by bovine serum albumin (BSA). Among the additives, the copper (II) ion stimulated pMMO at low concentration and inhibited at high concentration. The optimum conditions for pMMO activity were as follows: 45 degrees C, pH 6.5 and 55 mM 3-morpholinopropanesulfonic acid (MOPS) buffer, and the rate of propene epoxide formation was 13.6 nmol min-1 mg-1 protein. ESR spectra indicate that the copper cluster in the membrane fraction is reduced by duroquinol and oxidized by dioxygen. The result suggests that the copper cluster is contained in the active site of pMMO.

Ouabain-insensitive, vanadate-sensitive K(+)-ATPase of rat distal colon is partly similar to gastric H+,K(+)-ATPase.

A membrane fraction from rat distal colon contained both ouabain-sensitive and -insensitive K(+)-ATPase activities, which were measured under Na(+)-free conditions. About 38% of the ouabain-insensitive K(+)-ATPase activity was inhibited by vanadate. It was determined whether the ouabain-insensitive, vanadate-sensitive K(+)-ATPase in the colon is similar or identical to gastric H+,K(+)-ATPase. This colonic K(+)-ATPase activity was inhibited completely by monoclonal antibody HK4001, which inhibits the hog gastric H+,K(+)-ATPase activity but not Na+,K(+)-ATPase or Ca(2+)-ATPase. The colonic ATPase activity was inhibited partly by SCH 28080, which is a specific reversible inhibitor of gastric H+,K(+)-ATPase. The colonic ATPase activity was stimulated by low concentrations of K+ (its half-maximal stimulating concentration was 1 mM) and inhibited by high concentrations of K+ (its half-maximal inhibiting concentration was 10 mM), indicating that high and low K+ affinity sites are present in the colonic enzyme as in gastric H+,K(+)-ATPase and that this enzyme is not fully operative under normal physiological conditions. Two other monoclonal antibodies, which inhibit the gastric H+,K(+)-ATPase activity, did not inhibit the colonic K(+)-ATPase activity. The present results suggest that the colonic ouabain-insensitive K(+)-ATPase is partly similar but not identical to the gastric H+,K(+)-ATPase.

The presence of H+,K(+)-ATPase in the crypt of rabbit distal colon demonstrated with monoclonal antibodies against gastric H+,K(+)-ATPase.

Indirect evidence indicates the presence of an active H+/K+ antiporter for the secretion of acid in the distal colon. It was examined whether the H+/K+ antiporter in the rabbit distal colon was hydrogen-potassium-stimulated adenosine triphosphatase (H+,K(+)-ATPase), which acts as a proton pump in the gastric mucosa. For this purpose, four monoclonal antibodies against hog gastric H+,K(+)-ATPase were raised. Three monoclonal antibodies dose-dependently inhibited the ouabain-insensitive gastric ATP-ase activity. Antibody HK4001 completely inhibited the ATPase activity. In indirect immunofluorescence studies, all four monoclonal antibodies stained H+,K(+)-ATPase in gastric mucosae of various animal species. Two monoclonal antibodies including antibody HK4001 cross-reacted with H+,K(+)-ATPase located in crypts of the transverse and descending colon and rectum of rabbits. Because the other two antibodies did not cross-react with the H+,K(+)-ATPase in the colon, this colonic enzyme is similar but not identical to gastric H+,K(+)-ATPase. On the other hand, HK4001 and SCH 28080 did not inhibit ouabain-sensitive K(+)-dependent ATPase activity in the guinea pig distal colon, and the antibodies did not stain the enzyme in the tissue. Therefore, ouabain-sensitive H+/K+ antiporter in the guinea pig is not similar to ouabain-insensitive rabbit colonic H+,K(+)-ATPase.

Antihypertensive and diuretic effects of NZ-105, a novel dihydropyridine derivative.

Studies on the antihypertensive and diuretic actions of NZ-105, a new dihydropyridine derivative, were performed in comparison with nicardipine. NZ-105 and nicardipine (5, 10 and 20 mg/kg, p.o.) dose-dependently decreased systolic blood pressure in three types of experimentally hypertensive rats, including spontaneously hypertensive rats, renal hypertensive rats and deoxycorticosterone acetate-salt hypertensive rats and normotensive Wistar-strain rats. The hypotensive effects were larger in hypertensive rats than in normotensive Wistar rats. The hypotensive actions of NZ-105 were very slow in onset and long-lasting in all models, e.g., the hypotension by NZ-105 (10 mg/kg, p.o.) reached a peak (-52 mmHg) at 3 hr and lasted for more than 9 hr in spontaneously hypertensive rats. The hypotensive action in spontaneously hypertensive rats was reproducible after repeated dosing twice a day for 29 days. The hypotensive action after i.v. injection of NZ-105 (0.1 mg/kg) in spontaneously hypertensive rats was also slow in onset (peak time: 10 min) and long-lasting (more than 120 min). The hypotensive potency of NZ-105 was about the same as that of nicardipine, but the increment in heart rate was smaller than in the case of nicardipine. Both NZ-105 and nicardipine showed diuretic and natriuretic actions in spontaneously hypertensive rats. After repeated administration, these actions of NZ-105 were unchanged, whereas those of nicardipine were reduced. These results suggest that NZ-105 is a useful antihypertensive drug with concomitant diuretic effects.

Detection of Neisseria gonorrhoeae by ultraviolet illumination of NADH generated by an immobilized antibody-gonococcal enzyme complex.

Frozen, cervical swabs were placed in a lysis buffer containing an immobilized antibody to the gonococcal enzyme, 1,2-propanediol oxidoreductase. The immobilized antibody--enzyme complex that formed was active after the addition of substrate (1,2-propanediol and NAD) and this activity could be detected by visual inspection of NADH fluorescence under ultraviolet illumination.

Rapid estimation of biochemical oxygen demand.

The feasibility of using columnar reactors containing immobilized microorganisms for the rapid estimation of BOD was demonstrated in this study. Dilutions of three types of industrial effluents were tested by the BOD5 test and by this experimental system. A high degree of correlation (r = 0.98) was observed between results of the two tests. The mean standard error of estimation of the experimental system was 11%.

Enzymatic detection of Neisseria gonorrhoeae.

In a study using a non-serological enzymatic approach for the detection of Neisseria gonorrhoeae in cervical and urethral swabs, the technique was shown to be technically feasible. The enzyme, 1, 2-propanediol oxidoreductase, was used as a presumptive diagnostic marker for N gonorrhoeae. Enzymatic activity was measured with a fluorometer. Two assay procedures were performed: (a) enzyme detection (two-tube and three-tube assays) requiring 60 minutes; and (b) enzyme inhibition (EI) (90-minute and modified 20-minute assays). Sensitivities of the two-tube, three-tube, and the 90-minute EI assays with male urethral specimens from a high-prevalence population were 80%, 84%, and 91% respectively. The specificities of these assays in a low-prevalence male population were not determined. Sensitivity of the 90-minute EI assay in a high-prevalence female group was 77% and specificity in a low-prevalence female group was 75%. The modified EI assay was tested only in a low-prevalence female group and had 87% specificity. Although the specificity of the assays needs improvement, several advantages--including early case detection, rapid availability of results, detection of current active infections, and the possibility of automation--are intrinsic in this enzymatic approach.

The isolation and characterization of a 1,2-propanediol oxidoreductase from Neisseria gonorrhoeae.

An enzyme which oxidizes 1,2-propanediol in the presence of NAD+ has been purified from lysates of Neisseria gonorrhoeae. The enzyme was activated by monovalent cations, had a pH optimum between 9 and 10, and showed a substrate specificity unlike any known alcohol or glycerol dehydrogenase. The enzyme had an apparent Km of 17 mM for 1,2-propanediol and 0 . 37 mM for NAD+. When chromatographed on a Sephadex G-150 column, the enzyme eluted as a single peak in the molecular weight region of a bovine serum albumin marker. An antibody to the purified enzyme was prepared in goats. When antiserum was reacted with the enzyme in immunodiffusion experiments, a single precipitin band was detected. When the enzyme was mixed with an excess of antibody and then reacted with substrate, enzyme activity was completely inhibited.

The occurrence of 1,2-propanediol oxidoreductase in micro-organisms and its use as a possible diagnostic marker for Neisseria gonorrhoeae.

The cervical microbial flora of 25 females and stock cultures of various micro-organisms which may be present in the human female cervix were examined using a fluorimetric assay for 1,2-propanediol oxidoreductase. Results indicated that only members of the genera Neisseria and Acinetobacter possess appreciable activities of the enzyme, whose physiological function is not yet known. The activity of this enzyme in N. gonorrhoeae appeared to be significantly higher than the activities observed in mot of the other Neisseria species and in the Acinetobacter species. These results indicated that it may be possible to utilize this enzyme as a presumptive diagnostic marker for N. gonorrhoeae in cervical secretions. 1,2-Propanediol oxidoreductase may also be of taxonomic significance for the classification of various bacterial species.

Applications of fluorophore-containing microbial growth media.

Media containing the fluorogenic compound 8-anilino-1-naphthalene sulfonic acid may be used to discriminate between gram-positive and gram-negative bacteria and to differentiate between various species of bacteria. Fluorescent light emitted from colonies of gram-negative bacteria on 8-anilino-1-naphthalene sulfonic acid-containing agar was visually more intense than that on gram-positive bacteria. The emitted light from the gram-negative bacteria differed in wave-lengths from that of light emitted by colonies of gram-positive bacteria. The fluorescent intensity of colonies on complete 8-anilino-1-napthalene sulfonic acid agar supplemented with 1% of single substrates varied depending on the bacterial species, thus allowing the development of profiles used to identify 12 different species.

Constricted tube system for presumptive identification and differentiation of group D streptococci.

A single, constricted tube containing two differential media to identify and differentiate group D streptococci was developed. Test results with a limited number of group D streptococcal isolates were in complete agreement with results of conventional procedures.