构建多模式全光谱暗场显微镜用于纳米单颗粒局域表面等离子共振实时动力学研究

金属纳米颗粒由于其局域表面等离子共振(LSPR),能显示出独特的光吸收和散射特性,常被应用于物理、化学和生物领域的分析检测。这类探针具有高强度、高稳定性,以及可以长时间成像观察等优势。对于单个金属纳米颗粒的LSPR光谱研究通常采用暗场显微镜(DFM)与光谱仪来观察。但是,现有的暗场显微镜-光谱仪联用装置受限于自带照明光源的强度与光谱范围等原因,造成对散射信号较弱的样品光谱采集时间长、采集范围窄,例如,无法做到对粒径在30 nm以下的小颗粒纳米金进行实时观察。本文针对这一问题使用超连续激光器作为光源,使对单个金属纳米颗粒的光谱采集时间可以缩短至1 ms。此外,针对细胞功能成像的需求,增加了光片成像模式,通过切换滤块,能够实现荧光成像与暗场成像的共定位。

Multi-Mode Full Spectrum Dark Field Microscope for Single Nanoparticle Localized Surface Plasmon Resonance Dynamics Study

In the last few decades, noble metal nanoparticles (MNP) have been widely used as imaging probes, in the field of bio-imaging, due to their localized surface plasmon resonance (LSPR) phenomenon. Compared to fluorescent probes, MNP imaging exhibits high sensitivity and outstanding signal-to-noise ratio, while the particle itself has good photostability; this makes the MNP probe the perfect candidate for long-term imaging. Currently the most popular MNP imaging and analysis method employs a dark-field microscope with a spectroscope. Since most dark-field microscopes use halogen lamp or mercury lamp as their illumination source, the illumination intensity and wavelength spectrum are limited. Both camera and spectroscopy require longer exposure time to collect sufficient scattering signal to generate a reasonable quality image and scattering spectrum. The narrow illumination spectrum also limits the size of the MNP that can be used (larger-diameter MNP tend to scatter in the near-infrared region). Therefore, a high-intensity and wide-spectrum illumination source is urgently needed in MNP imaging. In this study, we custom-designed a multi-mode dark field microscope by using a supercontinuum laser, comprising of a lightsheet illumination mode for wide-field imaging and a back focus mode for live spectrum analysis, as its illumination source. The total output of the supercontinuum laser was 2 W. Since it was a coherent illumination source it could be focused by the microscope objective to a near diffraction limit area for sufficient intensity. Moreover, since its wavelength spectrum was between 450 nm and 2200 nm, which covered most of the visible and near infrared region, it made the detection of the large-diameter MNP single particle possible. In the back-focus mode, the supercontinuum laser first passed through an annular filter and then entered the objective from the microscope back port. In the lightsheet illumination mode, the laser was focused by a 400-mm cylindrical concave mirror to create a "sheet" and illuminate the sample from its side. In both the illumination modes, the illumination radiation was blocked from the camera to obtain the dark field illumination effect. By using a multi-mode dark field microscope, we could observe a 30-nm-diameter MNP single particle with a color CCD camera in its lightsheet illumination mode and a spectrum time resolution of 1 ms in its back-focus illumination mode. This custom-designed microscope could not only be used to study the MNP single particle in living cells, but more importantly, its application could also be potentially extended to all the MNP-probe-based cell imaging.