title: Confocal imaging of subcellular Ca2+ concentrations using a dual-excitation ratiometric indicator based on green fluorescent protein.
abstract: Dual-excitation ratiometric dyes are excited alternately at two different wavelengths, but the emission is collected at a single fixed wavelength. Therefore, the pair of intensity measurements must be collected sequentially. Ratiometric-pericam is a fluorescent Ca(2+) indicator based on a chimeric fusion protein of circularly permuted green fluorescent protein and calmodulin. Upon binding to calcium, its excitation peak shifts from 415 nm to 494 nm. Ca(2+) imaging using ratiometric-pericam was thought to be inadequate to follow very fast Ca(2+) dynamics or Ca(2+) changes in highly motile cell samples; however, we describe a technique that allows high spatial and time resolution of images acquired with ratiometric-pericam. To obtain confocal images of Ca(2+) using ratiometric-pericam, we established a system in which two laser beams (excitation 408 nm and 488 nm) are alternated on every scanning line under the control of two acousto-optic tunable filters. This system increases the rate at which ratio measurements are done to 200 Hz, and provides confocal images at 1 to 10 Hz depending on the image size. The ratio images are free from noise caused by the fluctuation of laser power, because the system is equipped with a violet laser diode (408 nm) and a diode-pumped solid-state laser (488 nm), both of which are stable. We visualized the dynamic propagation of Ca(2+) waves from the cytosol to the nucleus and changes in Ca(2+) concentrations in motile mitochondria of HeLa cells. We demonstrate that this new confocal imaging system expands the range of potential applications of ratiometric-pericam and other dual-excitation ratiometric indicators.
authors.affiliation: Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, RIKEN, Wako-city, Saitama, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Shimozono
authors.forename: Takashi
authors.initials: T
authors.lastname: Fukano
authors.forename: Takeharu
authors.initials: T
authors.lastname: Nagai
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kirino
authors.forename: Hideaki
authors.initials: H
authors.lastname: Mizuno
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004058:Diffusion; D005456:Fluorescent Dyes / Q000032:analysis; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents / Q000032:analysis; D008164:Luminescent Proteins / Q000032:analysis*; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D002118:Calcium