title: Simultaneous imaging of multiple cellular events using high-accuracy fluorescence polarization microscopy.
abstract: Förster resonance energy transfer (FRET) has been widely used to design indicators for biomolecules. Conventional FRET-based indicators enable quantitative measurements of analyzes by calculating the ratio between donor and acceptor fluorophores. However, such 'hetero-FRET'-based indicators, which use multiple differently colored fluorophores, restrict the simultaneous use of other colors of fluorescent molecules. To overcome this problem, we developed a 'homo-FRET'-based Ca2+ indicator, W-Cameleon, composed of two identical yellow fluorescent proteins. The binding of Ca2+ to the indicator induces a change in FRET efficiency, which in turn transforms into changes in fluorescence anisotropy. Given that the fluorescence polarization is depolarized by light passing through a high numerical aperture lens and reflecting on a dichroic mirror, we also developed a microscopy technique that reliably detects fluorescence anisotropy with high precision. Our design is aided by photonic-crystal technology, to compensate for the fluorescence depolarization. We thereby succeeded in the simultaneous visualization of three individual intracellular events by using three different fluorescent indicators. Our system may contribute to an expansion of the number of events that can be observed, which will enable a more quantitative understanding of biological phenomena.
authors.affiliation: Laboratory for Nanosystems Physiology, Research Institute for Electronic Science, Hokkaido University, Kita 20, Nishi 10, Kita-ku, Sapporo, Hokkaido 001-0020, Japan.
authors.forename: Sang-Yeob
authors.initials: SY
authors.lastname: Kim
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Arai
authors.affiliation: Laboratory for Nanosystems Physiology, Research Institute for Electronic Science, Hokkaido University, Kita 20, Nishi 10, Kita-ku, Sapporo, Hokkaido 001-0020, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Tani
authors.affiliation: Scientific Solutions Product Development Division, Product Development Department, Olympus Corporation, 2951 Ishikawa-cho, Hachijoji-shi, Tokyo 192-8507, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Takatsuka
authors.affiliation: Scientific Solutions Product Development Division, Product Development Department, Olympus Corporation, 2951 Ishikawa-cho, Hachijoji-shi, Tokyo 192-8507, Japan.
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Saito
authors.affiliation: Photonic Lattice, Inc., ICR 2F, 6-6-3 Minami-Yoshinari, Aoba, Sendai, Miyagi 989-3204, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Kawashima
authors.affiliation: Photonic Lattice, Inc., ICR 2F, 6-6-3 Minami-Yoshinari, Aoba, Sendai, Miyagi 989-3204, Japan.
authors.forename: Shojiro
authors.initials: S
authors.lastname: Kawakami
authors.affiliation: Laboratory for Cell Function and Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan.
authors.forename: Takeharu
authors.initials: T
authors.lastname: Nagai
mesh_terms: D002468:Cell Physiological Phenomena; D002477:Cells / Q000648:ultrastructure*; D005454:Fluorescence Polarization / Q000295:instrumentation / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins
keywords: FRET; fluorescence anisotropy; photonic crystals; polarization microscopy