The muzzle pattern is very useful as a means to identify each individual animal in cattle. An image processing system for muzzle prints, including inputting, binarizing the digital picture with shade, extracting of feature values and identification of the muzzle prints has been investigated. The results suggest that the system is applicable to the identification of cattle muzzle prints.

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Magnetic resonance (MR) imaging was performed in 15 normal healthy individuals and 6 patients, 4 with complete central type idiopathic diabetes insipidus (DI) and 2 with partial central idiopathic DI. Two cases of complete idiopathic DI were associated with empty sella. Characteristic high intensity of the posterior pituitary lobe on T1-weighted image was seen in all 15 normal pituitary glands; however, it was undetectable in all 6 cases of DI. In dynamic study, the posterior lobe was well enhanced within 30 seconds after injection of gadopentetate dimeglumine in healthy subjects and in one of mild partial DI. In contrast, no or faint early enhancement of the posterior pituitary lobe was observed in 4 cases of complete and 1 case of partial DI. Regardless of whether sella were empty or not, the early enhanced area of posterior pituitary was not seen in complete central idiopathic DI. The posterior pituitary high intensity on MR T1-weighted images probably reflects the stored antidiuretic hormone (ADH) granules in the posterior lobe, and the posterior pituitary vascularity may be decreased in neurohypophysial hypofunctioning states such as central idiopathic DI. These findings were clearly demonstrable by MR dynamic imaging of pituitary glands after intravenous injection of contrast materials, using an anteroposterior orientation of frequency encoding gradient.

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Confocal laser scanning microscopy and conventional digital-imaging microscopy were used to record changes in intracellular free Ca induced by electric stimuli, or by the action of caffeine, in giant neuronal cells of circumaesophageal ganglia dissected from a land slug, Incilaria bilineata. Recording of fluorescence changes of a Ca probe, indo-1, with a confocal laser scanning microscope (CLSM) equipped with a UV laser (325 nm) demonstrated clear distinction in the mode of Ca changes in cytoplasm and nucleoplasm. Injection of outward current caused repetitive action potentials and Ca influx, resulting in a rapid Ca rise in cytoplasm followed by a slower rise in nucleoplasm. The nuclear envelope appeared to serve as a barrier to Ca diffusion from cytosol to nucleoplasm. When the cell was tetanically stimulated in the presence of 10 mM caffeine, the rate of the Ca rise in the nucleoplasm became more rapid, thereby suggesting participation of a Ca-induced Ca release from caffeine-sensitive intracellular Ca stores. Bath application of caffeine to a resting cell caused a calcium transient in most cells. Caffeine also induced in some cells repeated bursts of spontaneous action potentials and corresponding Ca oscillation. Cytoplasmic phasic rise in Ca corresponding to each action potential was clearly visualized when fluo-3 as a Ca indicator dye was used for CLSM. Calcium oscillation in this type of cell may be caused by a membranous oscillator involving voltage-dependent Ca and K channels and Ca-induced Ca release from caffeine-sensitive intracellular Ca stores.

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A method of reconstructing a three-dimensional structure from live serial section images taken with a light or an electron microscope is described. The method frees researchers from the cumbersome task of tracing the contour lines of objects. Two methods of achieving automatic alignment were tested in order to obtain some technical bases. One used cross-correlation functions of neighbouring images; the other, local image features. In the former, rotational difference and relative displacement between images were measured at high speed with the aid of hardware exclusively devoted to the fast Fourier transform and affine transformations. In the latter case, techniques used in digital stereophotogrammetry were employed for identifying corresponding points in the image pairs. The problem of image distortion due to local deformations of sections was solved by using an elastic model where the image plane was modelled on a material such as a rubber sheet. With the model a distorted resampling geometry for the correction was obtained by numerical calculations under boundary conditions given by a set of corresponding points in the image pair. This reconstruction method was applied to a light micrograph series of minute colonic adenomas in human familial adenomatosis coli. Some voxel processings for noise removal and smoothing are demonstrated. Volumetric presentations and cutting of the reconstructed data are also demonstrated in a final three-dimensional rendering.

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To elucidate the anticonvulsant mechanism of peony root extract, effects of peony root extract on intracellular calcium concentration changes induced by pentylenetetrazol (PTZ) and caffeine were investigated in the primary cultured cerebral cortical neurons of mice. Peony root extract showed inhibitory effect on PTZ- and caffeine-induced elevation of intracellular calcium concentration. Peony root extract also inhibited bursting activity induced by elevation of intracellular calcium concentration with forced intracellular diffusion of calcium chloride and inositol 1,4,5-trisphosphate. Our previous results demonstrated the clear inhibitory effect of peony root extract on PTZ-induced bursting activity in snail neurons and also the clear inhibitory effect of peony root extract on PTZ-induced EEG power spectrum changes as well as extracellular calcium concentration changes in cerebral cortex of rats. The present findings together with the previous results suggest that peony root extract manifests anticonvulsant action derived from inhibition of intracellular calcium release, inhibition of intracellular pathological phenomena induced by elevation of intracellular calcium concentration, and also inhibition of calcium inward current.

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By carboxymethylation at the γ-carboxyl group of glutamic acid-58, ribonuclease T₁ (RNase T₁) loses its enzymatic activity entirely while retaining substrate-binding ability almost completely. To elucidate the mechanism of this phenomenon as based on the three-dimensional structure, molecular dynamics simulation and energy minimization calculation were carried out for the complex of guanosine 2'-monophosphate (2'-GMP) with carboxymethylated RNase T₁. The conformation thus obtained was compared with that of the complex of 2'-GMP and intact RNase T₁. The results indicated that upon carboxymethylation the guanine-binding loop (histidine-40 to phenylalanine-48) is displaced to open the active site cleft so that 2'-GMP binds to the active site in such a way that the phosphate group of the inhibitor moves away from the active site residues, glutamic acid-58 and arginine-77.

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Nuclear envelope breakdown (NEBD) in the living sea urchin egg was investigated by video microscopy. Small and deep dimples at the apex of the ovoid nucleus appeared, elongated, and increased in number during prophase just before NEBD. These dimples grew with time, and projected deeply into the nucleus to more than 5 μm in length, but their thickness was uniform at 0.3 μm in diameter. Therefore, we called them nuclear envelope projections (NEPs). NEPs were not stationary but moved slowly in the nucleus, suggesting that NEPs are rotationally movable around the centrosome. NEPs were also observed at each division just before NEBD as well as at the first cleavage. Some microtubules were recognized in the nucleus at late prophase, by immunofluorescence, using anti-tubulin antibody. No NEPs were observed in the nucleus during NEBD when the fertilized eggs were treated with 50 μM colcemid. Therefore, it is concluded that when microtubules grew from the centrosome near the nucleus and increased in number as the mitotic stage proceeded, microtubules also began to elongate toward the nucleus and pushed the nuclear envelope. At the same time as the nuclear lamina was presumed to dissolve, the growing microtubules protruded into the nucleus with the nuclear envelope around them, and finally they projected deeply into the nucleus. Subsequently, the nuclear envelope disappeared gradually, but NEBD did not occur instantaneously or obviously.

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An optical system suitable for ultraviolet (UV) laser-scanning confocal microscopy was designed and custom-made. The optical system consisted of a hybrid objective of reflective and refractive elements (achromatic for 200—500 nm), a relay lens (200—500 nm) and a conventional confocal laser-scanning head. The system yielded lateral and axial resolutions of 0.6 μm and 3.5 μm, respectively, which were measured at a half decay length of the fluorescence of a bead (0.5 μm), and demonstrated the sliced image of cultured bullfrog sympathetic ganglion cells loaded with a Ca²⁺-sensitive fluorescent dye, lndo-1.

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The growth of balloon and laser angioplasty technology, and the challenging surgical treatment for vascular diseases have created a need for three dimensional (3-D) imaging modalities, for both precise diagnosis and assessment of therapeutic results. As an intravascular pathological arterial disease such as dissecting aneurysm of the aorta (DAA) has a 3-D morphology, 2-D imaging equipment is not able to visualize its complicated intravascular volumic character. A data set of multiple consecutive 2-D echograms has the possibility of reconstructing a 3-D image. We have developed a 3-D imaging system for intravascular surface characterization with short processing time and high fidelity. In this study, we present the development of this system for 3-D intravascular surface characterization using consecutive echographic cross-section images which were collected transesophageally or percutaneously. In the case of DAA, the intimal flap and both true and false lumens were clearly visualized, as well as the rough surface of the atherosclerotic aorta. Approximately 10 minutes were required to input echographic data and then generate one 3-D image on the CRT display. In conclusion, the reconstructed images provided superior resolution of the endovascular surface characteristics with short processing time.

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To elucidate the function of the microcirculation system, which is essential for material transport in tissues and organs, it is very important to know blood flow velocity and its distribution in the microvessels. Although many methods of measuring microcirculatory blood flow velocity have been developed, all of them have both advantages and disadvantages, and cannot satisfy our requirements completely. In this situation, we tried to measure microcirculatory blood flow velocity vector by processing images obtained in situ under microscopic observation. The unit used for our measurement contained a supersensitive high-speed video system and a real-time velocity vector measurement system based on two-dimensional correlation. In this study, we determined red blood cell (RBC) velocity vector in the rat mesenteric microcirculation using the unit, and obtained data on blood flow velocity, its change over time and its velocity distribution, which were very interesting from the viewpoint of microcirculatory rheology.
 The results indicate that using this system might be useful in analyzing RBC velocity with a high time resolution. The prominent advantage of this system is the possibility of measuring the two-dimensional velocity distribution of blood flow at many windows, and the velocity profile at any cross-section can be easily investigated.
 This system offers real-time and simultaneous multi-window measurement of red blood cell velocity, giving information on the following: velocity distribution of branched vessels; pulse waves; and analysis of velocity profiles of curved vessels. Therefore, this system promises to greatly contribute to further development of hemorheology.

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The heterogeneous distribution of functional glutamate receptor subtypes was visualized by calcium fluorometry in the fura-2-loaded organotypic slice culture of the rat hippocampus. The organotypic culture prepared according to the method described by Gähwiler (1984) was loaded with fura-2, a fluorescent Ca²⁺ indicator, and subjected to image analysis. In the culture the cells grew in the mono- or bi-layer manner on the glass coverslip preserving their original arrangement. Monitoring the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i), we detected the synaptic activities as spontaneous and electrically evoked [Ca²⁺]_i changes. Each of the agonists for glutamate receptor subtypes (at a concentration of 10 μM) showed characteristic two-dimensional patterns of [Ca²⁺]_i change. The responses to N-methyl-D-aspartic acid (NMDA) and DL-α-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) were always the largest in the CA1 region, but that to kainic acid was the largest in the CA3 region. Trans-1-amino-cyclopentyl-1,3-dicarboxylic acid (t-ACPD) produced a clear elevation of [Ca²⁺]_i in the cells that migrated out of the margin of the slice, which were negative in the immunohistochemical staining with a neuron-specific marker, MAP-2 antibody. Present results confirmed the heterogeneous distribution of functional glutamate receptor subtypes in the hippocampus which we previously described in a fresh slice preparation.

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A confocal laser scanning microscope was successfully used to visualize the T-system in skeletal muscle fibers of the frog skeletal muscle stained with the Golgi silver method. The muscle tissues were selectively stained for the T-system and prepared for electron microscopic observations. The staining pattern varied among muscle fibers, but the fibers selectively stained for the T-system could be readily identified in reflected light by their yellowish color among black-colored unstained fibers on the trimmed face of an Araldite-embedded block under a dissecting microscope. Under a confocal laser scanning microscope with a reflection filter, the stained fibers showed characteristic networks of the T-system at the level of the Z-disc, providing sharp and high-contrast images in optical sections of thick slices. Such images resembled those obtained with a transmission electron microscope in thick sections cut from the same blocks. Sharp optical section images were obtained at depths up to ～10 μm from the specimen surface. The three-dimensional distribution of the T-tubules was examined in stereo-pair images reconstructed from serial optical sections. In such stereo images, one could follow the plane of the network of the T-system, often recognizing a helicoidal pattern of the plane of the T-system.

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We have studied the effects of a tyrosine kinase inhibitor, herbimycin A, on the nuclear calcium signals in antigen-specific B cells (TP67.21). After antigen stimulation (trinitrophenol conjugated ovalbumin, TNP₂₁-OVA), we measured first the intracellular free calcium ion concentration, [Ca²⁺]_i, in fura-2-loaded B cells with a fluorescence spectrophotometer. Herbimycin A decreased the [Ca²⁺]_i in antigen-stimulated B cells in a dose-dependent manner. Then we measured the effects of herbimycin A on the calcium signals in individual B cells with a confocal fluorescence microscope. We found that antigen-induced calcium signals in the cytoplasm and the nucleus of B cells were completely inhibited by preincubation of the cells with 5—10 μM herbimycin A for 14 hr. On the other hand pretreatment of the cells with a lower concentration of herbimycin A (0.5—1 μM) partly inhibited calcium signals where the intracellular free calcium ion concentrations ([Ca²⁺]_i) showed rapid rises and falls over much shorter periods during the time course of the ([Ca²⁺]_i) in the drug-untreated cells. This indicated that at low concentrations (0.5—1 μM), herbimycin A inhibited mainly the calcium entry from the external medium. In all the experiments it seemed that nuclear calcium signals were controlled by the elevated level of the [Ca²⁺]_i in the cytoplasm of B cells.

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Computer-assisted image processing based on the confocal microscopic observation of the retina stained with rhodamine phalloidin made it possible to reconstruct the three-dimensional architecture of cytoskeletal actin filaments in rod photoreceptor cells. Fluorescent strands corresponding to bundles of actin filaments were tapered at both proximities and ran parallel to the long axis of the cell from the tips of the calycal processes to the myoid portion, in such a way that the inner segment was surrounded by actin bundles. These longitudinal actin bundles were intermingled with the actin ring associated with the adherens junction. Another actin-rich domain in the cell was the synaptic terminal, where fluorescence appeared to show strands or blobs. The prominent fluorescent strand found at the neck of the synaptic terminal was presumed to represent a circumferential ring, suggesting that the typically constricted synaptic shape may be formed by its contraction.

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