Ken-ichi Hirano

Single-molecule detection by laser-induced fluorescence technique with a position-sensitive photon-counting apparatus

A new method of detecting single-molecule fluorescence has been implemented using a conventional optical microscope and a position-sensitive photon-counting apparatus. The samples were solvent-free fluorescent dyes adsorbed on a solid substrate in air at room temperature by spraying microdroplets of a dye solution followed by solvent evaporation. At the concentration used, each droplet contained a very small number of dye molecules. Quantized fluorescence intensity was determined and the number of occurrences of the quantized intensity agreed well with that calculated from Poissons formula, thus confirming success of the position-sensitive single-molecule fluorescence detection.

Hydrogen peroxide-induced cellular injury is associated with increase in endogenous fluorescence from rat gastric mucosal epithelial cell culture: A new method for detecting oxidative cellular injury by fluorescence measurement

Abstract: To develop a new method of detecting cellular injury caused by oxygen radicals, we studied endogenous fluorescence from the cultured cells of a rat gastric mucosal epithelial cell line. Measurement with an ultra-high sensitivity camera-image processor system under an inverted epifluorescence microscope showed that the fluorescence intensity of the cells increased time- and dose-dependently after the addition of hydrogen peroxide (H2O2), an oxygen radical precursor, to the medium. This increase was inhibited by the presence of catalase. Phase-contrast and fluorescence microscopy revealed that the fluorescence was emitted from granular substances in the cytoplasm of the injured cells. The spectral pattern of excitation and emission indicated that the fluorescent substances were flavins. In cell-free experiments, glutathione reductase which has flavin adenine dinucleotide (FAD) at the active site, increased in fluorescence after incubation with H2O2 in the presence of reduced glutathione and glutathione peroxidase. These findings indicate that FAD in the cytoplasm of cells injured by H2O2 increased in endogenous fluorescence according to the extent of injury, and suggest that fluorescence measurement may be a simple method in cellular toxicology to detect oxygen radical-induced injuries.

Autofluorescence in onset of gastric mucosal injury induced by hemorrhagic shock in rats

In an attempt to clarify whether gastric mucosal autofluorescence can help us to recognize gastric lesions at the onset of their formation, we investigated the fluorescence generated from the gastric mucosa of rats under ischemia-reperfusion stress. Redcolored fluorescence appeared and began to increase within 5 min after reperfusion. Such an increase in fluorescence did not occur in the gastric mucosa under prolonged ischemia without reperfusion. The epifluorescence microscopy of mucosal cryosections revealed that fluorescence was present even when only superficial mucosal damage occurred. Spectrofluorometric and high-performance liquid chromatographic analysis of the fluorescent mucosa extract identified the fluorescent substances as porphyrins. These findings suggested that fluorescent porphyrins are generated in the mucosal layer during the introductory phase of mucosal lesion formation induced by ischemia-reperfusion stress.

Change in Membrane Fluidity of Sand Dollar Egg Cortices Caused by Ca2+‐Induced Exocytosis: Microscopic Analysis with Fluorescence Anisotropy

The surface membrane fluidity of sand dollar eggs after exocytosis was investigated by using a specially designed video‐microscope system to measure the fluorescence depolarization of isolated cortices stained with hexadecanoylaminofluorescein. When unfertilized eggs were stained before isolation, the plasma membranes became labeled with fluorescent dye, but cortical vesicles did not. The fluorescence anisotropy of the isolated cortices increased from 0.256 to 0.285 during exocytosis induced by Ca2+. The increased anisotropy was not changed by lowering the Ca2+concentration after exocytosis. Dislodging of cortical vesicles by shearing with a stream of solution had no affect on the anisotropy. These results suggest that the fluidity of the plasma membrane decreases after exocytosis. When cortices were stained after isolation, both plasma membranes and cortical membrane organelles became labeled. These cortices possessed an anisotropy of 0.215. After dislogding the cortical organelles the anisotropy increased to 0.232. These results indicate that the fluidity of the cytoplasmic membrane leaflets of cortical organelles is higher than that of the plasma membrane. Therefore, it was suggested that only the outer leaflet of the plasma membrane becomes less fluid after exocytosis.

Autofluorescence in indomethacin-induced gastric mucosal lesions in rats.

Abstract: Autofluorescence observations enable scientists to sensitively identify various lesions. Non-steroidal anti-inflammatory drugs such as aspirin and indomethacin are well known to induce gastric mucosal injuries. Our purpose was to clarify whether the observation of mucosal autofluorescence could help us to recognize indomethacin-induced gastric lesion formation. Gastric mucosal fluorescence intensity and gastric lesion scores were time-sequentially measured after indomethacin treatment in rats. To identify the localization of autofluorescent substances, stomach cryosections were observed with an epifluorescence microscope. Fluorescent substances from damaged tissue were also analyzed by high-performance liquid chromatography. In addition, to elucidate whether oxidative stress directly generates fluorescent substances from heme, we investigated the reaction between hydrogen peroxide and hemoglobin in a cell-free system. Treatment with indomethacin induced gastric lesions by tissue peroxidation, with mucosal fluorescence intensity increasing time-dependently. The fluorescence products were mesoporphyrin and protoporphyrin, and they were localized in disrupted mucosal tissue. In the cell-free system, porphyrins were directly generated by hydrogen peroxide from hemoglobin. These findings indicate that indomethacin treatment increased the intensity of porphyrin fluorescence. Gastric mucosal lesion formation can be sensitively detected with fluorescence observations.

A fluorescence dequenching method for monitoring exocytotic membrane fusion in fertilization of single sea urchin eggs

A method for monitoring exocytotic membrane fusion by using a fluorescent membrane probe is presented. The method is based on the relief from concentration‐dependent self‐quenching (dequenching) of fluorescence of 5‐N‐(octadecanoyl)aminofluorescein (AF18), an amphiphilic derivative of fluorescein. The validity and usefulness of this method were shown by the following results: 1) self‐quenching of AF18 fluorescence occurred in the plasma membrane of unfertilized eggs of a sea urchin, Pseudocentrotus depressus, which were heavily stained with the fluorescent dye; 2) dequenching of AF18 fluorescence occurred upon fertilization in normal eggs but not in EGTA‐injected eggs; 3) Ca2+ induced both AF18 fluorescence dequenching and cortical granule disappearance in the isolated sea urchin egg cortex; and 4) simultaneous measurements of the intracellular Ca2+ concentration ([Ca2+]i) and dequenching of AF18 fluorescence by using a simple one‐excitation and two‐emission wavelength system.