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.
Authors
Keseroglu, Kemal OguzCollections
- Theses [4116]