| Autor: |
Coucheron DA; Department of Physics and Technology, UiT The Arctic University of Norway., Helle ØI; Department of Physics and Technology, UiT The Arctic University of Norway., Øie CI; Vascular Biology Research Group, Department of Medical Biology, UiT The Arctic University of Norway., Tinguely JC; Department of Physics and Technology, UiT The Arctic University of Norway; jean-claude.tinguely@uit.no., Ahluwalia BS; Department of Physics and Technology, UiT The Arctic University of Norway. |
| Jazyk: |
angličtina |
| Zdroj: |
Journal of visualized experiments : JoVE [J Vis Exp] 2019 Nov 16 (153). Date of Electronic Publication: 2019 Nov 16. |
| DOI: |
10.3791/60378 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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