Super Resolution Imaging of Thick Samples by Means of Selective Plane Illumination Microscopy

Abstract

Recently, super-resolution techniques based on single molecule localization became a popular tool to understand biological processes and image structures below the diffraction limit. In the last few years a fast development in the field allowed multicolor and 3D super-resolution imaging of biological samples. However, super-resolution techniques are currently well established at the cellular level but their application to more complicate samples, such as tissues and embryos, still remain a challenging task. within this context, recently, the coupling of localization based techniques and selective plane illumination microscopy [1], allowed to extend the application range to thicker tissues ( 50 μm), by coupling far-field individual molecule localization (IML) and selective plane illumination microscopy (SPIM) both in the linear and non linear regime. Particular attention is addressed to the advantages provided by two photon photo-activation in scattering environments.IML-SPIM allows to image cellular spheroids with sub-diffraction resolution in depth and can be applied to image cellular substructures in Zebrafish early developmental stages as well.(1) Cella Zanacchi F. et al., “Live-cell 3D super-resolution imaging in thick biological samples.,” Nat Methods, 8, no. 12, pp. 1047-1049, (2011)(2) Truong T. V. et al. “Deep and fast live imaging with two-photon scanned light-sheet microscopy.,” Nat Methods, 8, no. 9, pp. 757-760, (2011). Huisken, J., et al. Science 305, 1007-1009 (2004).