基于光热效应的显微光谱技术在单粒子检测中应用和发展

高灵敏度的单粒子检测技术是纳米粒子在生物医学、化学、光电子等领域应用的前提条件。常见的单粒子检测技术主要包括基于粒子的荧光、拉曼、散射和吸收等信号而发展起来的光学显微成像及光谱技术。其中,拉曼光谱和荧光光谱技术主要适用于一些具有拉曼活性的分子/粒子或可发光的荧光分子或粒子,然而即使对于荧光效率高的有机染料分子和半导体纳米粒子,固有的光漂白和blinking现象也对单粒子探测形成了挑战。散射光谱测量是应用于单粒子检测的另外一种方法,从理论上讲,由于瑞利散射随着尺寸的减小而呈六次方减弱的趋势,在细胞或生物组织内,小尺寸粒子的散射信号很难从背景散射噪声中分离出来。众所周知,介质吸收激发光后会引起介质内的折射率变化,进而在光加热区附近出现折射率的梯度分布,称为光热效应(photothermal effect)。基于粒子光热效应的光学显微成像和光谱测量技术具有信号灵敏度高、无背景散射、原位和免标记等优点,在单粒子检测领域展现了良好的应用潜力。综述了近年来基于光热效应的显微光谱技术在单粒子检测中应用和研究发展,首先介绍了光热效应的测量原理;接着分别讨论了光热透镜测量技术、微分干涉相差测量技术和光热外差测量技术的实验装置,比较了各种测量技术的信噪比、灵敏度、分辨率等特点,并且介绍这些测量技术在单粒子检测中的应用研究进展;接着,论述了近年来研究人员在提高光热显微测量的信噪比、改善动态测量性能以及在红外波段拓展等方面的最新研究成果;最后,简单总结了光热测量技术在单粒子检测领域所面临的挑战。

Application and Development of Photothermal Based Microscopy in Single Particle Detection

High sensitive single particle/molecule detection technique is an important prerequisite for nanoparticle application in biomedicine, chemistry and optoelectronics. Common single particle detection techniques include optical microscopy and spectroscopy based on fluorescence, Raman scattering, Rayleigh scattering, and absorption signals from single particle or molecule. Raman and fluorescence spectra analysis are applicable to fluorescence molecules or molecules with SERS activity. However, even for organic dye and semiconductor nanoparticles with high fluorescence efficiency, inherent photobleaching and blinking is a challenge for single particle detection. Scattering based technique is another solution for single particle detection, but as the scattering signal decreases with the particles size with the sixth power, it is difficult to separate the scattering signal of small size particle from background scattering noise. As we known, the light absorption of medium will induce refractive index change, and a refractive index gradient distribution will be present in the light heating zone. This phenomenon is known as the photothermal effect. Microscopy based on particle or molecule light absorption induced thermal effect has the merit of high sensitivity, freedom from background scattering, in-situ and label-free. It has been demonstrated potential application in single-particle/molecule detection field. In this paper, the application and development of photothermal based microscopy and spectroscopy was summarized. Firstly, the measurement principle was introduced. Then experimental set-ups of several different techniques including photothermal lens, differential interference contrast and photothermal heterodyne were discussed. The signal-noise ratio, sensitivity and resolution of these techniques were compared, and the recent investigations on applications of these techniques to single particle detection were also introduced. And then the recent research progresses in improve photothermal microscopy was elaborated, including signal/noise ratio increasing, dynamic measurement improvement and infrared spectrum expanding. Finally, the challenge for the photothermal technique used in single particle detection was summarized briefly.