Fluorescence Nanoscopy with Stochastic Switching and 4pi Detection

Lecturer: Mark Bates, Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

Abstract

The stochastic on-off switching of single fluorophores enables high-resolution fluorescence microscopy such as STORM and PALM [1]. These methods routinely achieve a 3D image resolution of ~20nm in X-Y and ~50nm in Z, and have recently been applied to studies of actin organization in neurons and the molecular architecture of the protein complexes [2]. When combined with interferometric fluorescence detection, image resolution in the third dimension may be improved dramatically. Building on the concepts of 4Pi and I5M microscopy [3], this approach employs two high-angle objective lenses to coherently detect light from the specimen. Single molecule switching events are recorded on a CCD camera in a multi-phase measurement, enabling fluorophore localization with a precision as high as 3nm in the Z dimension [4]. In this presentation I discuss the design and characterization of a 4Pi-STORM fluorescence microscope, and present initial measurements of 3D protein localization within the Nuclear Pore Complex.

[1] M. Bates, B. Huang, and X. Zhuang, Curr. Op. Chem. Biol., 12, 505-514 (2008)
[2] A. Szymborska et al., Science, 341, 655-658 (2013)
[3] S. Hell and E.H.K. Stelzer, J. Opt. Soc. Am. A, 9, 2159–2166 (1992)
[4] G. Shtengel et al., PNAS, 106, 3125-3130 (2009)