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Analytical Methods Using Light for Trace-amount Biomolecules
Functioning in Living Organisms
MITSUO HIRAMATSU
Central Research Laboratory, Hamamatsu Photonics K. K.
Hirakuchi, Hamakita, 434-8601, Japan
1. Introduction
We will present you outcome obtained through test research in a biological field by making use of reagents to support light measurement as well as photonic technologies. We performed research and development centering on ultra-sensitive fluorescence analysis. Elemental technologies developed optics and light detection components to eliminate background light interference from the test materials and photo-reagents such as fluorescent reagents and light-sensitive reagents. Key technologies for systems to detect and analyze functional trace substances in living organisms developed the methods for non-destructive and destructive measurement.
All the results of this presentation are the outcome of the investment project by Japan Key Technology Center, a special corporation, which is under the control of MITY and the ministry of Postal Services, from March 1994 to March 2000 (six years).
2. Direct Observation of Specific mRNA in a Single Living Cell
Localization, movements, and degradation of mRNA within cells are important factors in genetic control. Specific messenger RNA was observed in a single living cell under a fluorescence microscope by detecting hybrid formed with two fluorescently labeled oligodeoxynucleotides (oligoDNAs) and mRNA in the cytoplasm. When two oligoDNAs hybridized to an adjacent sequence on the target mRNA, the distance between the two fluorophores became very close and fluorescence resonance energy transfer (FRET) occurred, resulting in changes in fluorescence spectra. Hybridization of the pair of oligoDNAs to mRNA in the cytoplasm was detected in fluorescence images indicating FRET.1)
Mainly the intensity of background light, such as cell autofluorescence and fluorescence from medium, coverslips, and other optical elements limit the number of hybrids that can be detected. Measurement methods on a longer wavelength would reduce background light.
3. Restricted Photorelease of Biologically Active Molecules from Caged Compounds
A variety of photo-activatable or caged molecules exist, including caged ATP and Ca2+. The caged molecules have conventionally been used in studies of intracellular or intercellular dynamics by photo-irradiation.
An evanescent wave of ultraviolet light was successfully used to release biologically active molecules from caged compounds in living cells. The evanescent wave was generated by the total internal reflection in a limited region near the plasma membrane attached to the illuminated interface. The method provides a useful tool for studying signal transduction near the plasma membrane in a living cell.2)
4. Highly Sensitive Detection Using Light for Biomolecules
We have carried out simultaneous measurement of fluorescence and chemiluminescence using neutrophil relating to defense response.3)
We selected chemiluminescent method for superoxide ion and fluorescent method for calcium ion. Argon laser was used as an excitation light source. The key point of our set-up is the chopper, 1kHz. Fluorescence was passed through dichroic mirror and monochrometer and was detected by a photomultiplier. Chemiluminescence was reflected by the dichroic mirror and passed through band pass filter and detected by the other photomultiplier.
5.References
