Lokalisierungsmikroskopie unter Ausnutzung überkritischer Fluoreszenz
Zusammenfassung der Projektergebnisse
Advances in light microscopy have allowed circumventing the diffraction barrier, once thought to be the ultimate resolution limit in optical microscopy, and gave rise to various superresolution microscopy techniques. Among them, single-molecule localization microscopy (SMLM) exploits the blinking of fluorescent molecules to precisely pinpoint the positions of many emitters individually, and subsequently to reconstruct a superresolved image from these positions. While SMLM enables the study of cellular structures and protein complexes with unprecedented details, severe technical bottlenecks still reduce the scope of possible applications. In particular, 3D SMLM generally suffers from a limited z resolution. While isotropic resolution is possible with extremely complex instrumentation, most common 3D methods are limited to a resolution that is several times worse in the z direction than in the x and y direction. In this project, we established a conceptually new approach to obtain threedimensional superresolution images. Supercritical-angle localization microscopy (SALM) exploits the phenomenon of surface-generated fluorescence arising from fluorophores close to the coverslip. Close to the interface between water and glass this extra fluorescence can account for a large portion of the total fluorescence, but it decays rapidly with the distance to the interface. Because the surface-generated fluorescence depends so strongly on the z-position of the fluorophore, it is an excellent read-out for it. Interestingly enough, surface-generated fluorescence is propagating in the glass above the critical angle, as opposed to light refracted at the coverslip. By selecting only light propagating above the critical angle, one is able to measure the amount of surface-generated fluorescence for single-emitters. In SMLM, the brightness of single fluorophores is not the same for all molecules. Therefore, the supercritical intensity alone is not enough to resolve the z position of the emitters. However, by using a dual-channel microscope in which we additionally detect the normal, undercritical fluorescence for normalization, we can determine precise z-positions independent of the absolute brightness. In a first proof-of-principle study, we used SALM to superresolve objects smaller than the diffraction limit (DNA origamis, clathrin-coated pits). While SALM has the prospect of an isotropic spatial resolution with simple instrumentation, our first implementation suffered from limited depth, inaccurate intensity estimation and lower theoretical precision due to aberrations. In order to optimize SALM and reach the expected resolution, we implemented a new microscope incorporating a very high NA objective (NA 1.7) and adaptive optics. In addition, we developed a new algorithm that allows experimental PSF fitting and accurate brightness estimation at very high speed. Together, the new analysis pipeline and advanced optics should enable high precision 3D localization microscopy with SALM.
Projektbezogene Publikationen (Auswahl)
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2014. 3D superresolution microscopy by supercritical angle detection. Optics express, 22(23), pp.29081-29091
Deschamps, J., Mund, M. and Ries, J.
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2016. Efficient homogeneous illumination and optical sectioning for quantitative single-molecule localization microscopy. Optics express, 24(24), pp.28080-28090
Deschamps, J., Rowald, A. and Ries, J.
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2017. Fast, robust and precise 3D localization for arbitrary point spread functions
Li, Y., Mund, M., Hoess, P., Matti, U., Nijmeijer, B., Sabinina, V.J., Ellenberg, J., Schoen, I. and Ries, J.
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2017. Systematic analysis of the molecular architecture of endocytosis reveals a nanoscale actin nucleation template that drives efficient vesicle formation. bioRxiv, p.217836
Mund, M., van der Beek, J.A., Deschamps, J., Dmitrieff, S., Monster, J.L., Picco, A., Nedelec, F., Kaksonen, M. and Ries, J.
