Fibrinogen irradiated by X-ray (X-fibrinogen) resulted in a delay in the polymerization by the addition of thrombin in an X-ray-dose dependent manner (Koh et al., 1997). The aim of the present study was to visualize and analyze this fibrin with confocal laser-scanning fluorescence microscopy (CLSM). Using CLSM, we analyzed the structures of the networks formed from RITC-labeled X-fibrinogen, FITC-labeled unirradiated normal fibrinogen (N-fibrinogen), and mixtures of the two. The X-network demonstrated an irregular, poorly-organized mass. Composite networks formed by a combination of X- and N-fibrinogen in various amounts were also abnormal in shape, although they were not as irregular as those formed by X-fibrinogen alone, and they contained fibrils with homogeneously mixed colors. The dissolution of the fibrin by plasmin showed similar fluorescence localization in agglomerates at the periphery of the network and in the unraveled network inside the lysing area. Dissolution by urokinase-type plasminogen activator resulted in loosely connecting fibers. Because no changes occurred in the chemical properties of the X- and N-fibrinogens—electrophoretic cross-linking patterns by Factor XIIIa, and quantities of fibrinopeptide A by thrombin activation—those results suggest that X-rays might induce molecular lesions at the sites necessary for polymerization, thus facilitating delayed association of the fibrin monomers.
The nucleus at interphase can be characterized by distinct morphological features and the distribution pattern of certain chromosomal regions such as nuclear chromocenters (CCs). Parameters of CCs within a nucleus are used to determine the type of resting chromosomes, which is a useful cytological characteristic that can serve as an indicator of cross-compatibility among orchid species. However, it is difficult using conventional imaging methods to identify the CCs within the interphase nucleus with the same level of acuity as human visual perception. This is because numerous variations on density and size are observed among CCs. An unsharp mask filter enables us to simulate human visual perception and allows us to identify CCs within the nucleus regardless of their density and size. As a result, CCs of Spiranthes sinensis (Pers.) Ames. were identified to the same standard as human visual recognition. Moreover, not only the quantitative parameters indicating morphological features but also the distribution pattern of CCs were successfully determined using this method. The numerical parameters of CCs can provide useful information not only for phylogenetic studies of Orchidaeceae but also for the future breeding of new hybrids of orchids.
The disorganization of actin polymerization associated with lamelipodia formation in neutrophils of patient with leukocyte adhesion dysfunction syndrome (LAD), which lack Mac-1 on their membranes due to point mutation, was analyzed with a bioimaging system using a polarized microscopic system LC-Pol Scope. Neutrophils of the patient showed depression in spreading, random migration, and chemotaxis to fMet-Leu-Phe (FMLP) and IL-8, and expression of integrin adhesion molecules CD11a, CD11c, and CD18 on cell surfaces of peripheral leukocytes. However, CD11b expression on the surface, the expression of CD18 mRNA, and adherence to a cover glass were close to the normal levels or slightly decreased. In addition, O-2 production, myeloperoxidase release, and sensitivity to FMLP of neutrophils of the patient were also within normal levels. The disorganization of the fibrous cytoskeleton was observed with a scanning electronic microscope after Triton X-100 treatment followed by actin polymerization with rhodamin-phalloidin staining. Moreover, the disorganization of actin polymerization of neutrophils stimulated with FMLP on non-specific adherence of neutrophils to the cover glass was analyzed with a newly developed bioimaging technique using a polarized light microscope LC-Pol Scope for a kinetic analysis of polarized material in living cell under real-time observation. In addition, films processed under the LC-Pol Scope system showed that neutrophil cells of the patient still were round-shaped with disorganization of actin polymerization different from that in control cells.