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

荧光法是最灵敏的化学物种检测工具之一，但测定生物细胞中功能物质的量用于诊断一直面临着许多实际问题：荧光探针和有机物质的非特异性吸附造成的大背景使高灵敏度检测不可能；来自细胞壁的光散射干扰了再生性；等等......。本研究旨在解决这些问题，并为基于细胞的荧光诊断方法提供实用指南。1)组成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)利用荧光显微光谱仪对单个微粒进行了鉴别。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。混合后作为测试样品。研究发现，即使每个微粒的荧光发射光谱形状相同，但它们是可区分的，其峰值波长仅相差5-10 nm。并证明了在高背景条件下的粒子识别。3)吸附染料在单个微颗粒上的检出限低至10^<-18> mol/颗粒。4)根据实验结果，提出了一种基于荧光强度、光谱和背景水平评价的微量物质中化学种类的荧光检测指南。

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.