title: Single-cell resolution fluorescence imaging of circadian rhythms detected with a Nipkow spinning disk confocal system.
abstract: Single-point laser scanning confocal imaging produces signals with high spatial resolution in living organisms. However, photo-induced toxicity, bleaching, and focus drift remain challenges, especially when recording over several days for monitoring circadian rhythms. Bioluminescence imaging is a tool widely used for this purpose, and does not cause photo-induced difficulties. However, bioluminescence signals are dimmer than fluorescence signals, and are potentially affected by levels of cofactors, including ATP, O(2), and the substrate, luciferin. Here we describe a novel time-lapse confocal imaging technique to monitor circadian rhythms in living tissues. The imaging system comprises a multipoint scanning Nipkow spinning disk confocal unit and a high-sensitivity EM-CCD camera mounted on an inverted microscope with auto-focusing function. Brain slices of the suprachiasmatic nucleus (SCN), the central circadian clock, were prepared from transgenic mice expressing a clock gene, Period 1 (Per1), and fluorescence reporter protein (Per1::d2EGFP). The SCN slices were cut out together with membrane, flipped over, and transferred to the collagen-coated glass dishes to obtain signals with a high signal-to-noise ratio and to minimize focus drift. The imaging technique and improved culture method enabled us to monitor the circadian rhythm of Per1::d2EGFP from optically confirmed single SCN neurons without noticeable photo-induced effects or focus drift. Using recombinant adeno-associated virus carrying a genetically encoded calcium indicator, we also monitored calcium circadian rhythms at a single-cell level in a large population of SCN neurons. Thus, the Nipkow spinning disk confocal imaging system developed here facilitates long-term visualization of circadian rhythms in living cells.
authors.affiliation: Photonic Bioimaging Section, Research Center for Cooperative Projects, Hokkaido University Graduate School of Medicine, N15 W7 Kita-ku, Sapporo 060-8638, Japan. enoki@pop.med.hokudai.ac.jp
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Enoki
authors.forename: Daisuke
authors.initials: D
authors.lastname: Ono
authors.forename: Mazahir T
authors.initials: MT
authors.lastname: Hasan
authors.forename: Sato
authors.initials: S
authors.lastname: Honma
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Honma
mesh_terms: D000818:Animals; D002940:Circadian Rhythm / Q000502:physiology*; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal / Q000379:methods*; D009474:Neurons / Q000502:physiology*; D013493:Suprachiasmatic Nucleus / Q000502:physiology*
chemical_list: D049452:Green Fluorescent Proteins