Coucheron David André, Helle Øystein Ivar, Øie Cristina Ionica, Tinguely Jean-Claude, Ahluwalia Balpreet Singh
Department of Physics and Technology, UiT The Arctic University of Norway.
Vascular Biology Research Group, Department of Medical Biology, UiT The Arctic University of Norway.
J Vis Exp. 2019 Nov 16(153). doi: 10.3791/60378.
Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 µm x 500 µm region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.
全内反射荧光（TIRF）常用于基于单分子定位的超分辨率显微镜，因为它通过光学切片提供增强的对比度。传统方法是使用高数值孔径显微镜TIRF物镜进行激发和收集，这严重限制了视野和通量。我们提出了一种用于光波导成像产生TIRF激发的新方法，称为基于芯片的纳米显微镜技术。本方案的目的是演示如何在已构建的装置中进行基于芯片的成像。基于芯片的纳米显微镜技术的主要优点是激发和收集路径是解耦的。然后可以用低倍放大镜头进行成像，从而得到大视野的TIRF图像，代价是分辨率略有降低。使用直接随机光学重建显微镜（dSTORM）对肝窦内皮细胞（LSEC）进行成像，其分辨率与传统超分辨率显微镜相当。此外，我们通过用低倍放大镜头对500 µm x 500 µm的区域进行成像，展示了高通量能力，提供了76 nm的分辨率。基于芯片成像具有紧凑的特点，可以改装到大多数普通显微镜中，并可与其他片上光学技术相结合，如片上传感、光谱学、光镊等。因此，该技术非常适合高通量二维超分辨率成像，同时也为多模态分析提供了巨大的机会。
