Multiple-color fluorescence imaging of chromosomes and microtubules in living cells.
Microscopic observation of fluorescently-stained intracellular molecules within a living cell provides a straightforward approach to understanding their temporal and spatial relationships. However, exposure to the excitation light used to visualize these fluorescently-stained molecules can be toxic to the cells. Here we describe several important considerations in microscope instrumentation and experimental conditions for avoiding the toxicity associated with observing living fluorescently-stained cells. Using a computer-controlled fluorescence microscope system designed for live observation, we recorded time-lapse, multi-color images of chromosomes and microtubules in living human and fission yeast cells. In HeLa cells, a human cell line, microtubules were stained with rhodamine-conjugated tubulin, and chromosomes were stained with a DNA-specific fluorescent dye, Hoechst33342, or with rhodamine-conjugated histone. In fission yeast cells, microtubules were stained with alpha-tubulin fused with the jellyfish green fluorescent protein (GFP), and chromosomes were stained with Hoechst33342.
Haraguchi
T
T
Kansai Advanced Research Center, Communications Research Laboratory, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe 651-2401, Japan.
Ding
D Q
DQ
Yamamoto
A
A
Kaneda
T
T
Koujin
T
T
Hiraoka
Y
Y
D001562:Benzimidazoles / Q000737:chemistry; D002453:Cell Cycle / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002875:Chromosomes / Q000737:chemistry / Q000378:metabolism*; D015825:Chromosomes, Fungal / Q000737:chemistry / Q000378:metabolism; D002877:Chromosomes, Human / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007211:Indoles / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology; D012235:Rhodamines / Q000737:chemistry; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics; D013831:Thiamine / Q000494:pharmacology; D014404:Tubulin / Q000737:chemistry / Q000235:genetics
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D006657:Histones; D007211:Indoles; D008164:Luminescent Proteins; C093953:NMT1 protein, S pombe; D012235:Rhodamines; D029702:Schizosaccharomyces pombe Proteins; D014404:Tubulin; C447680:hoechst 32258; D049452:Green Fluorescent Proteins; C007293:DAPI; D004247:DNA; C017807:bisbenzimide ethoxide trihydrochloride; D013831:Thiamine