title: Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging.
abstract: A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to "pinhole cross-talk," which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging.
authors: Optical Image Analysis Unit, RIKEN Center for Developmental Biology, Kobe650-0047, Japan.
authors: Togo
authors: T
authors: Shimozawa
authors: Kazuo
authors: K
authors: Yamagata
authors: Takefumi
authors: T
authors: Kondo
authors: Shigeo
authors: S
authors: Hayashi
authors: Atsunori
authors: A
authors: Shitamukai
authors: Daijiro
authors: D
authors: Konno
authors: Fumio
authors: F
authors: Matsuzaki
authors: Jun
authors: J
authors: Takayama
authors: Shuichi
authors: S
authors: Onami
authors: Hiroshi
authors: H
authors: Nakayama
authors: Yasuhito
authors: Y
authors: Kosugi
authors: Tomonobu M
authors: TM
authors: Watanabe
authors: Katsumasa
authors: K
authors: Fujita
authors: Yuko
authors: Y
authors: Mimori-Kiyosue
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000166:cytology / Q000196:embryology; D002470:Cell Survival; D004331:Drosophila melanogaster / Q000166:cytology / Q000196:embryology; D004622:Embryo, Mammalian / Q000166:cytology; D004625:Embryo, Nonmammalian / Q000166:cytology; D049452:Green Fluorescent Proteins; D021621:Imaging, Three-Dimensional / Q000379:methods*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D017785:Photons; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
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