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Fluorescence Nanoscopy   
[See also Nobel Prize in Chemistry 2014]

With a conventional objective lens and visible light, the maximum resolution of optical microscopy is fundamentally limited by diffraction, about 200 nm. Stimulated emission depletion (STED) microscopy is one of the most powerful approaches that overcome the diffraction barrier. In STED microscopy, an excitation beam is overlapped with a doughnut-shaped depletion beam where the STED light turns off fluorescence of molecules in the periphery region via stimulated emission depletion; as a result, it produces sub-diffraction sized focal spot. As the intensity of STED light increases, the effective spot size can shrink down below 10 nm.

To overcome current fluorescence nanoscopy, we aim to develop 1) high-speed 3D nanoscope with low photo-damage, and 2) wide field-of view nanoscope with high throughput imaging for biomedical applications.

Related papers

STED microscopy

  • Rittweger* & Han* et al., Nature Photon. 3,144-147 (2009)

  • Han et al., Nano Lett. 9, 3323-3329 (2009)

  • Vicidomini*, Moneron*, Han* et al., Nature Methods 8, 571-573 (2011)

  • Han & Ha, Opt. Lett. 40, 2653 (2015)

RESOLFT microscopy

  • Han et al., Nano Lett. 10, 3199-3203 (2010)

  • Kwon et al., Sci. Reports, 5, 17804 (2015)

Fluorescent Probes

Fluorescence microscopy requires fluorescent probes to visualize targets of interest. Organic dyes, nanoparticles and fluorescent proteins are good examples. Understanding their optical properties is as important as developing new optical microscopes. We aim to use novel fluorescent probes for applying in fluorescence nanoscopy as well as reporters to sense pH, temperature, voltage, electromagnetic fields or biochemicals.

Nitrogen-vacancy (NV) center: NV center is a defect made of nitrogen and adjacent vacancy in diamond. It has several unique optical properties: 1) its ground state is triplet; 2) it neither shows photo-blinking nor photo-bleaching; 3) it is possible to readout spin state using fluorescence intensity. These advantages have allowed NV center to be used in quantum computation, nanoscale magnetometry and bioimaging.

Spinach RNA mimic of green fluorescent protein (GFP): RNA plays essential roles in biology; it transmits genetic information from DNA to proteins and also controls gene expression in various ways. Particularly, its location and copy number strongly affect the development of organisms. However, visualizing RNA in living cells has been challenging due to the lack of genetically modifiable fluorophores like GFP. We use RNA aptamer system (called Spinach) that can specifically bind to a small (non-fluorescent) molecule, eliciting bright fluorescence, in order to study gene expression of mRNA and non-coding RNA in bacteria and mammalian cells.

Related papers

NV centers

  • Han et al., Nano Lett. 10, 3199-3203 (2010)

  • Han et al., New J. Phys. 14, 123002 (2012)

  • Lee et al., Molecular BioSystems 9, 1004-1011 (2013)

RNA imaging probes

  • Han et al., J. Am. Chem. Soc. 135, 19033-19038 (2013)

  • Zhang et al., Sci. Reports 5, 17295 (2015)

  • Masuda et al. Nucleic Acids Res. doi: 10.1093/nar/gkw1229 (2016)

Single-molecule pull-down assay (SiMPull)

Traditional biochemical assays usually require large amount of samples. However, SiMPull enables to (i) directly count immunoprecipitated proteins from crude cell lysates with single-molecule sensitivity, and (ii) determine an stoichiometry of macromolecular complexes using photobleaching analysis. We are studying important biomarkers of neurodegenerative diseases using human brain samples from Parkinson's and Altzheimer's diseases patients.

Advanced bioimaging techniques

Coming soon.

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