title: Combining fluorescence and bioluminescence microscopy.
abstract: Bioluminescence microscopy has revealed that gene expression in individual cells can respond differently to the same stimulus. To understand this phenomenon, it is important to sequentially observe the series of events from cellular signal transduction to gene expression regulated by specific transcription factors derived from signaling cascades in individual cells. However, these processes have been separately analyzed with fluorescence and bioluminescence microscopy. Furthermore, in culture medium, the background fluorescence of luciferin-a substrate of luciferase in promoter assays of gene expression in cultured cells-confounds the simultaneous observation of fluorescence and bioluminescence. Therefore, we optimized conditions for optical filter sets based on spectral properties and the luciferin concentration based on cell permeability for fluorescence observation combined with bioluminescence microscopy. An excitation and emission filter set (492-506 nm and 524-578 nm) was suitable for green fluorescent protein and yellow fluorescent protein imaging of cells, and >100 μM luciferin was acceptable in culture medium based on kinetic constants and the estimated intracellular concentration. Using these parameters, we present an example of sequential fluorescence and bioluminescence microscopic observation of signal transduction (translocation of protein kinase C alpha from the cytoplasm to the plasma membrane) coupled with activation of gene expression by nuclear factor of kappa light polypeptide B in individual cells and show that the gene expression response is not completely concordant with upstream signaling following stimulation with phorbol-12-myristate-13-acetate. Our technique is a powerful imaging tool for analysis of heterogeneous gene expression together with upstream signaling in live single cells.
authors.affiliation: Corporate Research and Development Center, Olympus Corporation, Hachioji, Tokyo, 192-8512, Japan.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Goda
authors.affiliation: Corporate Research and Development Center, Olympus Corporation, Hachioji, Tokyo, 192-8512, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Hatta-Ohashi
authors.affiliation: Corporate Research and Development Center, Olympus Corporation, Hachioji, Tokyo, 192-8512, Japan.
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Akiyoshi
authors.affiliation: Corporate Research and Development Center, Olympus Corporation, Hachioji, Tokyo, 192-8512, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sugiyama
authors.affiliation: Corporate Research and Development Center, Olympus Corporation, Hachioji, Tokyo, 192-8512, Japan.
authors.forename: Ikuko
authors.initials: I
authors.lastname: Sakai
authors.affiliation: Corporate Research and Development Center, Olympus Corporation, Hachioji, Tokyo, 192-8512, Japan.
authors.forename: Takeo
authors.initials: T
authors.lastname: Takahashi
authors.affiliation: Corporate Research and Development Center, Olympus Corporation, Hachioji, Tokyo, 192-8512, Japan.
authors.forename: Hirobumi
authors.initials: H
authors.lastname: Suzuki
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D042541:Intracellular Space / Q000737:chemistry / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D008164:Luminescent Proteins
keywords: NF-κB; PKC; luciferin; single-cell analysis