title: Two-Photon Excitation STED Microscopy with Time-Gated Detection.
abstract: We report on a novel two-photon excitation stimulated emission depletion (2PE-STED) microscope based on time-gated detection. The time-gated detection allows for the effective silencing of the fluorophores using moderate stimulated emission beam intensity. This opens the possibility of implementing an efficient 2PE-STED microscope with a stimulated emission beam running in a continuous-wave. The continuous-wave stimulated emission beam tempers the laser architecture's complexity and cost, but the time-gated detection degrades the signal-to-noise ratio (SNR) and signal-to-background ratio (SBR) of the image. We recover the SNR and the SBR through a multi-image deconvolution algorithm. Indeed, the algorithm simultaneously reassigns early-photons (normally discarded by the time-gated detection) to their original positions and removes the background induced by the stimulated emission beam. We exemplify the benefits of this implementation by imaging sub-cellular structures. Finally, we discuss of the extension of this algorithm to future all-pulsed 2PE-STED implementationd based on time-gated detection and a nanosecond laser source.
authors.affiliation: Nanoscopy, Nanophysics, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy.|Department of Physics, University of Genoa, Via Dodecaneso 33, 16146, Genoa, Italy.
authors.forename: Iván
authors.initials: I
authors.lastname: Coto Hernández
authors.affiliation: Nanoscopy, Nanophysics, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy.|Department of Informatics, Bioengineering, Robotics and Systems Engineering, University of Genoa, Via Opera Pia 13, 16145, Genoa, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Castello
authors.affiliation: Nanoscopy, Nanophysics, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Lanzanò
authors.affiliation: Nanoscopy, Nanophysics, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: d'Amora
authors.affiliation: Nanoscopy, Nanophysics, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Bianchini
authors.affiliation: Nanoscopy, Nanophysics, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy.|Department of Physics, University of Genoa, Via Dodecaneso 33, 16146, Genoa, Italy.|Nikon Imaging Center, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Diaspro
authors.affiliation: Nanoscopy, Nanophysics, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Vicidomini
mesh_terms: D002460:Cell Line; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods* / Q000592:standards; D008870:Microtubules / Q000378:metabolism; D017785:Photons; D059629:Signal-To-Noise Ratio