3D Multicolor STED Nanoscope a Super-Resolution Approach to Mammalian Photoreceptor

Abstract

Photoreceptors in mammalian retina present two distinct subcellular regions: the Inner Segment (IS) and the Outer Segment (OS). The OS shows an unique architecture and is responsible for the photo-transduction, despite the metabolic activity of photo-transduction remains controversial and still unclear(1), recent studies suggest that the presence of OXidative PHOSphorylation proteins (OXPHOS) in rods OS support the energetic requirements of photo-transduction(2).We propose a STimulated Emission Depletion (STED) superresolution approach to investigate the structural proteins and the OXPHOS proteins distribution in photoreceptor cells. STED nanoscopy allows overcoming the diffraction limit, enabling to distinguish details of cellular and molecular structures at the nanoscale(3). Our 3D multicolor STED nanoscope is based on a supercontinuum pulsed laser with 20MHz repetition frequency and three synchronized outputs: a supercontinuum laser beam in the 450-2000nm spectral range, used for excitation, and two laser beams at 710 and 745nm respectively, which have a bandwidth of about 10nm, used for depletion(4). In order to obtain 3D super-resolution we divide the STED beam and shape the two beam in donut and bottle profiles. Such beam profiles are obtained by two phase plates, a vortex (0-2π) and a homemade 0-π one respectively(5). Since we can adjust the power ratio in the two depletion pathways we are flexible in the choice of the final three-dimensional resolution.Nevertheless we have optimized the system and the fluorophores choice in order to separate three channels spectrally. Thus we show three colors image with two colors super-resolved.