A mercury arc lamp-based multi-color confocal real time imaging system for cellular structure and function.
Multi-point scanning confocal microscopy using a Nipkow disk enables the acquisition of fluorescent images with high spatial and temporal resolutions. Like other single-point scanning confocal systems that use Galvano meter mirrors, a commercially available Nipkow spinning disk confocal unit, Yokogawa CSU10, requires lasers as the excitation light source. The choice of fluorescent dyes is strongly restricted, however, because only a limited number of laser lines can be introduced into a single confocal system. To overcome this problem, we developed an illumination system in which light from a mercury arc lamp is scrambled to make homogeneous light by passing it through a multi-mode optical fiber. This illumination system provides incoherent light with continuous wavelengths, enabling the observation of a wide range of fluorophores. Using this optical system, we demonstrate both the high-speed imaging (up to 100 Hz) of intracellular Ca(2+) propagation, and the multi-color imaging of Ca(2+) and PKC-gamma dynamics in living cells.
Saito
Kenta
K
Nikon imaging center, Research Institute for Electronic Science, Hokkaido University, Sapporo, Hokkaido, Japan.
Kobayashi
Kentaro
K
Tani
Tomomi
T
Nagai
Takeharu
T
D002118:Calcium / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D022082:Cellular Structures / Q000502:physiology / Q000648:ultrastructure*; D003199:Computer Systems; D003593:Cytoplasm / Q000378:metabolism; D005336:Fiber Optic Technology; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007834:Lasers; D008027:Light; D008164:Luminescent Proteins / Q000032:analysis; D008628:Mercury / Q000737:chemistry*; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism; D055100:Optical Fibers
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D008628:Mercury; D002118:Calcium