title: Optimizing and extending light-sculpting microscopy for fast functional imaging in neuroscience.
abstract: A number of questions in system biology such as understanding how dynamics of neuronal networks are related to brain function require the ability to capture the functional dynamics of large cellular populations at high speed. Recently, this has driven the development of a number of parallel and high speed imaging techniques such as light-sculpting microscopy, which has been used to capture neuronal dynamics at the whole brain and single cell level in small model organisms. However, the broader applicability of light-sculpting microcopy is limited by the size of volumes for which high speed imaging can be obtained and scattering in brain tissue. Here, we present strategies for optimizing the present tradeoffs in light-sculpting microscopy. Various scanning modalities in light-sculpting microscopy are theoretically and experimentally evaluated, and strategies to maximize the obtainable volume speeds, and depth penetration in brain tissue using different laser systems are provided. Design-choices, important parameters and their trade-offs are experimentally demonstrated by performing calcium-imaging in acute mouse-brain slices. We further show that synchronization of line-scanning techniques with rolling-shutter read-out of the camera can reduce scattering effects and enhance image contrast at depth.
authors.affiliation: Research Institute of Molecular Pathology, Vienna, Austria ; Max F. Perutz Laboratories, University of Vienna, Vienna, Austria ; Research Platform Quantum Phenomena & Nanoscale Biological Systems (QuNaBioS), University of Vienna, Vienna, Austria ; Current address: Friedrich Miescher Institute, Basel, Switzerland.
authors.forename: Peter
authors.initials: P
authors.lastname: Rupprecht
authors.affiliation: Research Institute of Molecular Pathology, Vienna, Austria ; Max F. Perutz Laboratories, University of Vienna, Vienna, Austria ; Research Platform Quantum Phenomena & Nanoscale Biological Systems (QuNaBioS), University of Vienna, Vienna, Austria.
authors.forename: Robert
authors.initials: R
authors.lastname: Prevedel
authors.affiliation: Research Institute of Molecular Pathology, Vienna, Austria.
authors.forename: Florian
authors.initials: F
authors.lastname: Groessl
authors.affiliation: Research Institute of Molecular Pathology, Vienna, Austria.
authors.forename: Wulf E
authors.initials: WE
authors.lastname: Haubensak
authors.affiliation: Research Institute of Molecular Pathology, Vienna, Austria ; Max F. Perutz Laboratories, University of Vienna, Vienna, Austria ; Research Platform Quantum Phenomena & Nanoscale Biological Systems (QuNaBioS), University of Vienna, Vienna, Austria.
authors.forename: Alipasha
authors.initials: A
authors.lastname: Vaziri
keywords: (110.0110) Imaging systems; (110.0180) Microscopy; (170.1420) Biology; (170.2655) Functional monitoring and imaging; (170.3880) Medical and biological imaging; (170.5810) Scanning microscopy; (180.2520) Fluorescence microscopy; (180.6900) Three-dimensional microscopy; (320.0320) Ultrafast optics; (320.5540) Pulse shaping