Hamamatsu-photonics K.k.

Other

Shizuoka University

Atsuo Miyakawa

Masahiko Hirano

Kiyoshi Kamiya

Yoshimasa Kawata

An optical microscope for optically measuring a sample () includes: a fluorescent thin membrane () which at least partly contains fluorescent substance and on which the sample () is placed; an electron source () for generating an electron beam; an electron lens () for focusing the electron beam generated by the electron source () in such a manner as to excite a minute light source having a wavelength shorter than a visible light wavelength from the fluorescent thin membrane () so as to irradiate the fluorescent thin membrane () with the electron beam, and further, scanning the focused electron beam; and an optical detector () for detecting a measurement light beam which is generated in the minute light source and acts on the sample ().

Optical microscope

A device for measuring an intracellular ion concentration in a living cell into which a fluorescent probe dye has been introduced. A concentration of an ion in the cell is measured based on intensities of fluorescence generated by irradiating the cell with excitation beams, and comparing the intensities of fluorescence emitted by the cell with fluorescence intensities generated from each of 3 reference solutions irradiated with excitation beams. The device is composed of a container means for holding an object to be measured, an excitation beam irradiating means, a fluorescence intensity detecting means, a first processing means for receiving output signals of the fluorescence intensity detecting means and a second processing means for solving simultaneous equations to determine a concentration of the ion to be measured within the cell.

Device for measuring an intracellular ion concentration

In a method for measuring an intracellular ion concentration of the present invention, 4 or more solutions of a fluorescence probe dye and the ions to be measured, 3 or more solutions of an interfering biosubstance and the fluorescent probe dye, and 3 or more solutions of the interfering biosubstance, the fluorescent probe dye and the ions to be measured are prepared. Each solution is irradiated with excitation beams at three different wavelengths, and a fluorescence intensity generated from each solution is measured. Three kinds of equilibrium constants in equilibrium constant equations and 12 kinds of fluorescence coefficients in fluorescence intensity calculating equations are optimized in accordance with a method of successive approximation. With the optimized 15 constants, the equilibrium constant equations and the fluorescence intensity calculation equations are solved to obtain the concentration of ions to be measured. Accordingly, labor for preparing samples is decreased, and the constants are given by a method of successive approximation, which means that the constants are highly reliable, and accordingly the intracellular ion concentration measured based on such highly reliable constants can be accurate.

Method of optimizing equilibrium constants and fluorescence coefficients

A fluorescence probe dye is introduced into a cell, and excitation beams at three defferent wavelengths are irradiated to the cell to measure intensities of the fluorescence generated by the excitation beams, corresponding to the three wavelengths. Then, an equilibrium reaction equation for concentrations of the fluorescence probe dye, protein, free ions and their complexes in the cell, and a relationship equation between the fluorescence probe dye, protein, free ions and their complexes are used as simultaneous equations to give concentrations of the respective components. This process can correct interactions among various components of the cell due to bonding among them and a correct ion concentration can be determined.

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Hamamatsu, Japan

Atsuo Miyakawa