2D/3D Super-Resolution Microscopy by Means of Individual Molecule Localization (IML) and Selective Plane Illumination Microscopy

Abstract

In the last few years localization based techniques, which exploit photoactivation, photoconversion or ground state depletion of fluorescent molecules, became a popular tool for super-resolution imaging of biological samples. Recently, approaches based on two photon excitation have been implemented in order to get axial confinement of the photoactivation process allowing for 3D super-resolution imaging of biological samples [1]. However, a topic of interest is still represented by the widening of super-resolution applications to thick samples (>15 μm). Within this scenario, light sheet based fluorescence microscopy techniques provide optical sectioning since illumination is confined to a thin planar region perpendicular to the detection axis and represent a suitable tool to confine the photoactivation process along the optical axis. In particular, single plane illumination microscopy (SPIM), has been proved to be a useful tool for biological investigations of thick living samples [2].Here we demonstrate three-dimensional super-resolution live cell imaging through thick biological specimen (>50 μm), by coupling far-field individual molecule localization (IML) and selective plane illumination microscopy (SPIM).The confined excitation provided by SPIM and the improved signal-to-noise ratio allows for nanometric localization of single molecules in thick scattering samples. A PALM approach [3] and elliptical stretching of the point spread function allow to perform 3D super-resolution imaging of biological live samples in depth (up to 100 μm). IML-SPIM allowed to image cellular spheroids with < 35 nm lateral precision and sub-diffraction resolution in depth [4].(1) York, A.G. et al. Nat Methods, 8, 327–333 (2011).(2) Huisken, J., et al. Science305, 1007–1009 (2004).(3) Hess, S.T., Girirajan,T.P.K. and Mason, M.D. Biophys J., 91(11), 4258–4272 (2006).(4) Cella Zanacchi et al. Nature Methods (accepted).