Protein unfolding on single molecule level - relating the radius of gyration to the hydrodynamic radius
Zusammenfassung der Projektergebnisse
Fluorescence microscopy permits the three-dimensional imaging of living cells, tissues and even small organisms. However, features smaller than approximately half the emission wavelength (~200 – 300 nm) cannot be resolved in conventional far-field microscopy due to the optical diffraction limit. During the last decade, the optical diffraction limit has been overcome with the introduction of several new concepts. All these superresolution methods are capable of enhancing the resolution in 3D, but often at the expense of major technical demands or modifications to the microscope. We have developed a new 3D superresolution method which not only overcomes the diffraction limit, but also generates virtually background-free, contrast-enhanced images with a few seconds of acquisition time. It is based on the analysis of temporal fluorescence fluctuations of emitters (such as the blinking of quantum dots or the triplet state blinking of conventional fluorescent dyes), which we termed superresolution optical fluctuation imaging or SOFI. The core idea of SOFI is that the blinking of different emitters in a sample such as a labeled cell is statistically independent from each other, enabling to spatially disentangle the different fluorescence contributions from different emitters in a given image. The only hardware prerequisite of this method is the ability to record with high sensitivity and speed consecutive fluorescence images of a sample. Resolution improvement is achieved by evaluating the temporal fluorescence fluctuations in the recorded movie using a software algorithm. This makes SOFI exceptionally simple from a hardware point of view, because virtually any widefield microscope equipped with a fast and sensitive camera can be converted into a superresolution microscope. The SOFI concept has attracted attention beyond the scientific community and several articles in the popular media have been published. 23.3.2010 im Göttinger Tageblatt in der print als auch in der online Ausgabe: http://www.goettinger-tageblatt.de/Nachrichten/Wissen/Regionale-Wissenschaft/Goettinger-Forscher-bringen-Licht-in-lebende-Zellen 25.3.2010 microscopy news.com: http://www.microscopy-news.com/news/sofi--turning-fluorescence-fluctuations-into-improved-optical-resolution.html
Projektbezogene Publikationen (Auswahl)
- ‘Measuring diffusion with polarization-modulation dual-focus fluorescence correlation spectroscopy’, Optics Express 16, 19, 14609-14616, 2008
Korlann, Y; Dertinger, T; Michalet, X; et al.
- ‘Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI), Proceedings of the National Academy of Sciences of the United States of America 106, 52 22287-22292, 2009
Dertinger, T; Colyer, R; Iyer, G; et al.
- ‘Achieving increased resolution and more pixels with Superresolution Optical Fluctuation Imaging (SOFI)’, Optics Express 18, 18, 18875-18885 2010
Dertinger, T; Colyer, R; Vogel, R; et al.
- ’Superresolution Optical Fluctuation Imaging with Organic Dyes’, Angewandte Chemie-International Edition 49, 49, 9441-9443, 2010
Dertinger, T; Heilemann, M; Vogel, R; et al.
