Super-Resolution Imaging via Spectral Separation of Quantum Dots
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There has been significant progress in the optical resolution of microscopes over the last two decades. However, the majority of currently used methods (e.g. STED, PALM, STORM) have a number of drawbacks, including high intensities of light that result in damage to living specimens in STED, and long data acquisition time leading to limitations on live-cell imaging. Therefore, there is a niche for faster image acquisition at lower intensities while maintaining resolution beyond the diffraction limit. Here, we have developed a new methodology – Quantum Dot-based Optical Spectral Separation (QDOSS) – which relies on using Quantum Dots (QDs) as fluorophores, and on their separation and localisation based on their spectral signatures. We utilise the key advantages of QDs over the usual organic fluorophores: broad excitation, narrow emission spectra and high resistance to photobleaching. Besides, since QDOSS is based on spectral differences for separation, QDs can be deterministically localised in a relatively short time – milliseconds and, potentially, microseconds. Last but not least, QDOSS is suitable for obtaining super-resolution images using a standard confocal fluorescence microscope equipped with a single laser excitation wavelength and capable of spectral signal separation (e.g. Leica TCS SP series or Zeiss LSM series). First, we demonstrated resolution down to 60 nm using triangular DNA origami as a reference. Furthermore, we labelled and imaged the alpha-tubulin structure in HEK293T cells. We showed that using a mixture of standard off-the-shelf QDs of different sizes, resolution down to 40 nm could be achieved via spectroscopic separation of QDs. Finally, we demonstrated that QDOSS could also be used for multicolour imaging of synaptic proteins distributed around synapsis in neurons within diffraction limit. All in all, we believe that these features of QDOSS make it a potential method for long-term live super-resolution imaging, which is going to have a high impact in biological sciences.
AuthorsKeseroglu, Kemal Oguz
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