We applied image processing technology for selecting monoclonal antibodies recognizing surface antigens of the rat pheochromocytoma PC12 cell. BALB/c mice preinjected with water emulsified in Freund's complete adjuvant were immunized by three subcutaneous injections of intact cell suspensions in the hind foot pads, and lymphnode cells from the immunized mice were fused with myeloma cells by electrofusion. For screening, culture supernatants from 450 hybridoma wells were incubated with PC12 cells cultured on collagen-coated 96-well plates, and reacted antibodies were visualized by FITC-conjugated anti-mouse lgG. Immunofluorescence images were taken into an image processor through an SIT camera, and positive clones were selected on the basis of two different imaging analyses. First, antibodies reacting with PC12 cells were screened by quantifying fluorescence intensities of the images. Second, those preferentially recognizing surface antigens were characterized by contrast-enhancement of the images. As a result, we identified 22 clones as positive ones. From reactivity with live cells, we found that 12 of them recognized the native conformation of surface antigens. By quantitative imaging using these monoclonal antibodies, we identified one membrane protein which decreased its expression level during neurite outgrowth in the PC12 cell. These results indicate that image processing is a useful method for screening and characterizing monoclonal antibodies.

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The three-dimensional sensing method for a local surface of the human body is introduced and the possibility of three dimensional shape reconstruction of the entire surface is investigated. For 3-D local surface sensing, a photometric stereo method using 3 point light sources is introduced. The idea of photometric stereo is to sense the 3-D position of surface points by interpreting the differences among shading information on surface points which are obtained by serially varying the direction of incident illumination on the state of the viewing direction held. It can provide 3-D sensed results of the local surface. In order to reconstruct the 3-D structure of the entire surface, plural viewing directions were used in this paper because the photometric stereo can sense only the surface exposed to a CCD camera. Each 3-D sensed result on local surfaces by photometric stereo in plural viewing directions is modified and combined using its silhouette information. In one experiment, a chicken bone is used for reconstructing the 3-D surface structure.

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To elucidate cytoskeletal changes induced by an elevation of intracellular calcium ion concentration ([Ca²⁺]_i) during the degranulation in mast cells, we observed changes in three-dimensional (3-D) arrangements of actin filaments and microtubules with confocal laser scanning microscopy. Rat peritoneal mast cells, obtained from peritoneal lavage in rats, were degranulated with compound 48/80, a secretagogue. Serial optical tomograms, subsequently reconstructed into stereoscopic images, were obtained from single mast cells stained with fluorescein-conjugated phalloidin and anti-tubulin antibodies. In addition, expressions of caldesmon, calmodulin-regulated actin-binding protein, were immunohistochemically analyzed to illuminate the relationship between [Ca²⁺]_i and actin filament. 3-D observations revealed compound 48/80-evoked changes in actin filament distribution. At the resting state, a mesh of thick actin filaments wrapped round the cell cortex. It was remarkable that the mesh of subplasmalemmal filamentous actin was solated and streamed to the cytoplasmic space as thinner filaments surrounding the granules after the stimulation. Present observations suggest that the cortical actin filaments may function as a “net” to hold and rapidly release the secretory granules in cell cortex rather than as a complete barrier to prevent interactions between granules and cell membrane. A novel mechanism that is compatible with these observations is proposed.

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An application of Computer Image Analysis (CIA) to beef cattle science serves as a valuable tool. In this study, the CIA technique was used to investigate the procedure and reliability of extracting specific information from the 6th and 7th cross-sections of beef carcass, and to determine the biological relationship between the CIA information extracted and the weight of M. longissimus thoracic (M11). The results indicate that the CIA technique is reliable, and is highly precise in extracting information from the carcass cross-section. It was also observed that there is a biological relationship between the CIA information, such as data on long and short radius axes, direction, and eccentricity, and weight of M11. The coefficient of determination (R²) for estimating the weight of M11 using the information extracted from the carcass cross-section was .824. This result suggests that the CIA technique has greater potential for extracting various types of information from the carcass cross-section that can be used to estimate muscle weight.

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The lymphatic system plays a major role in material and fluid transport in tissues and organs to maintain, like blood-microcirculation, homeostasis. The rhythmical contraction of lymph vessels has been observed frequently in mammalian species; however, the mechanisms of lymphatic contraction and lymph transport are not well known. In order to analyze the rhythmical contraction of lymph vessels in rat mesentery in vivo, microscopic image processing technique and effective algorithm for image analysis were developed in this study. They served to automatically extract indistinct edge lines of lymph vessel walls and to measure the vessel diameter from video images. Continuous measurement of diameter changes at equi-distant positions along the lymph vessel was carried out frame by frame. The results of diameter measurement showed high correlation with those of direct visual measurement on the video display. Graphic description of the lymphatic diameter changes related to time and position was obtained for further analysis with a contractile mode of lymph vessel. Lymph volume change was also calculated using a cylindrical model of lymph vessel. Periodic contraction of collecting lymphatics mostly occurred every 4 to 8 sec in rat mesentery, and was independent of heartbeat, respiration or skeletal muscle contraction. The results indicated that the rhythmical contraction of lymph vessel must be regulated by an active control system.

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A crystallographic workbench or workstation installed with software packages and database systems is described, which enables a through processing from X-ray measurement to protein structure analysis. As workstations have been installed in many crystallography laboratories, a through X-ray analysis system can be now constructed via a local area network. We describe the construction of an integrated system for measuring the X-ray intensities from any crystal specimen, transferring structure factor data via the network, carrying out the structure analysis, and displaying the molecular structure.

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Neutrophil-adhered lung carcinoma A549 cells labeled with acridine orange (AO) caused a rapid and transient increase in fluorescence in nuclei upon addition of a novel diketopiperadine compound, N-TAF1/SF2771. Image analysis successfully revealed that such a fluorescence increase was dependent on the duration of prior neutrophil adhesion to A549 cells; the peak time obtained from intranuclear changes of fluorescence intensity shifted closer to the time of addition of the compound the longer the cells had been adherd by neutrophils. AO fluorescence which was localized in a few spots in each A549 cell nucleus was diffused with concurrent increase of fluorescence in the whole nuclear region. Similar AO diffusion and increase of fluorescence were also observed in A549 cells treated with etoposide, a topoisomerase II inhibitor as well as an apoptosis stimulator, but only in the absence of neutrophils. These results suggest that N-TAF1/SF2771 may exhibit a neutrophil-dependent killing activity with respect to A549 cells through change in association of DNA and/or RNA in the nuclear region in an action similar to that of etoposide.

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