title: Wide field intravital imaging by two-photon-excitation digital-scanned light-sheet microscopy (2p-DSLM) with a high-pulse energy laser.
abstract: Digital-scanned light-sheet microscopy (DSLM) illuminates a sample in a plane and captures single-photon-excitation fluorescence images with a camera from a direction perpendicular to the light sheet. This method is potentially useful for observing biological specimens, because image acquisition is relatively fast, resulting in reduction of phototoxicity. However, DSLM cannot be effectively applied to high-scattering materials due to the image blur resulting from thickening of the light sheet by scattered photons. However, two-photon-excitation DSLM (2p-DSLM) enables collection of high-contrast image with near infrared (NIR) excitation. In conventional 2p-DSLM, the minimal excitation volume for two-photon excitation restricts the field of view. In this study, we achieved wide-field 2p-DSLM by using a high-pulse energy fiber laser, and then used this technique to perform intravital imaging of a small model fish species, medaka (Oryzias latipes). Wide fields of view (>700 μm) were achieved by using a low-numerical aperture (NA) objective lens and high-peak energy NIR excitation at 1040 nm. We also performed high-speed imaging at near-video rate and successfully captured the heartbeat movements of a living medaka fish at 20 frames/sec.
authors.affiliation: Laboratory for Spatiotemporal Regulations, National Institute for Basic Biology, 38 Nishigonaka, Myodaijicho, Okazaki, Aichi, 444-8585, Japan ; These authors contributed equally to this work.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Maruyama
authors.affiliation: Molecular Medicine for Pathogenesis, Graduate School of Medicine, Ehime University, Shitukawa Toon city, Ehime, 791-0295, Japan ; Division of Bio-imaging, Proteo-Science Center, Ehime University, Shitukawa Toon city, Ehime, 791-0295, Japan ; Translational Research Center, Ehime University Hospital, Shitukawa Toon city, Ehime, 791-0295, Japan ; These authors contributed equally to this work.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Oshima
authors.affiliation: Laboratory for Spatiotemporal Regulations, National Institute for Basic Biology, 38 Nishigonaka, Myodaijicho, Okazaki, Aichi, 444-8585, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Kajiura-Kobayashi
authors.affiliation: Laboratory for Spatiotemporal Regulations, National Institute for Basic Biology, 38 Nishigonaka, Myodaijicho, Okazaki, Aichi, 444-8585, Japan.
authors.forename: Shigenori
authors.initials: S
authors.lastname: Nonaka
authors.affiliation: Molecular Medicine for Pathogenesis, Graduate School of Medicine, Ehime University, Shitukawa Toon city, Ehime, 791-0295, Japan ; Division of Bio-imaging, Proteo-Science Center, Ehime University, Shitukawa Toon city, Ehime, 791-0295, Japan ; Translational Research Center, Ehime University Hospital, Shitukawa Toon city, Ehime, 791-0295, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Imamura
authors.affiliation: Laboratory of Bioresources, National Institute for Basic Biology, 38 Nishigonaka, Myodaijicho, Okazaki, Aichi, 444-8585, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Naruse
keywords: (000.1430) Biology and medicine; (110.6880) Three-dimensional image acquisition; (180.0180) Microscopy; (180.2520) Fluorescence microscopy; (180.4315) Nonlinear microscopy