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Highly-Selective Determination of Chemical Sepecies in Cells with 3D Fluorescence Microscopy

使用 3D 荧光显微镜高度选择性地测定细胞中的化学物质

基本信息

批准号：
10555294
负责人：
HARATA Akira
金额：
$ 8.38万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

A fluorescence method is the one of the most sensitive detection tools for chemical species but determination of amounts of functional substances in a biological cell for diagnostics always faces many practical problems : Large backgrounds caused by nonspecifically adsorbed-fluorescence probe and organic substances makes high sensitive detection impossible ; Light scattering from the cell walls disturbs reproducibility ; and so on. This study aims to solve these problems and get practical guidelines for a fluorescence method of cell-based diagnostics.1) A three-dimensional fluorescence micro-spectrometer is composed. A laser source or monochromated halogen-lamp light source, a microscope and an imaging spectrometer are combined to observe spectral images of microscopic substances.2) Discrimination of individual microparticles is demonstrated with the fluorescence micro-spectrometer. Different types of fluorescence dyes are adsorbed on different microparticles, respectively. They are made of polystyrene or ion-exchange resin, and their diameters are from 3 to 11 μm. After mixing, they are used as test samples. It was found that each microparticle was distinguishable even if their fluorescence emission spectra have the same shapes with a difference in their peak wavelengths of only 5-10 nm. Particle discrimination under a high background condition was demonstrated as well.3) Detection limit for adsorbed dyes on one microparticle was determined to be as low as 10^<-18> mol/particle.4) Based on the experimental results, a guideline for fluorescence determination of chemical species in micro-substances was proposed where fluorescence intensity, spectra and background levels are evaluated for the diagnostics.

荧光方法是最灵敏的化学物种检测工具之一，但用于诊断的生物细胞中功能物质含量的测定始终面临许多实际问题：非特异性吸附荧光探针和有机物质造成的大背景使高灵敏度检测变得不可能;细胞壁的光散射干扰重现性;本研究旨在解决这些问题，为荧光细胞诊断方法的研究提供实用指导。1）研制了三维荧光显微光谱仪。将激光光源或单色卤素灯光源、显微镜和成像光谱仪相结合，观察微观物质的光谱图像。2）用荧光显微光谱仪对单个微粒进行区分。不同类型的荧光染料分别吸附在不同的微粒上。它们由聚苯乙烯或离子交换树脂制成，直径为3 - 11 μm。混合后，将其用作试验样品。发现即使它们的荧光发射光谱具有相同的形状，其峰值波长的差异仅为5-10 nm，每个微粒也是可区分的。在高背景条件下也表现出颗粒的歧视。3）被确定为一个微粒上吸附的染料的检测限低至10 μ <-18>mol/particle。4）基于实验结果，提出了一个指导方针，荧光测定的化学物种的微量物质，其中荧光强度，光谱和背景水平的诊断进行评估。