Multiple-color Fluorescence Imaging of Chromosomes and Microtubules in Living Cells

Cell Struct Funct. 1999 Oct;24(5):291-8. doi: 10.1247/csf.24.291.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Benzimidazoles / chemistry
  • Cell Cycle / physiology
  • Cell Division / physiology
  • Chromosomes / chemistry
  • Chromosomes / metabolism*
  • Chromosomes, Fungal / chemistry
  • Chromosomes, Fungal / metabolism
  • Chromosomes, Human / chemistry
  • Chromosomes, Human / metabolism
  • DNA / chemistry
  • Fluorescent Dyes / chemistry
  • Green Fluorescent Proteins
  • HeLa Cells
  • Histones / chemistry
  • Histones / genetics
  • Humans
  • Indoles / chemistry
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics
  • Microscopy, Fluorescence / instrumentation
  • Microscopy, Fluorescence / methods
  • Microtubules / chemistry
  • Microtubules / metabolism*
  • Mitosis / physiology
  • Rhodamines / chemistry
  • Schizosaccharomyces / genetics
  • Schizosaccharomyces / metabolism
  • Schizosaccharomyces pombe Proteins / genetics
  • Thiamine / pharmacology
  • Tubulin / chemistry
  • Tubulin / genetics

Substances

  • Benzimidazoles
  • Fluorescent Dyes
  • Histones
  • Indoles
  • Luminescent Proteins
  • NMT1 protein, S pombe
  • Rhodamines
  • Schizosaccharomyces pombe Proteins
  • Tubulin
  • hoechst 32258
  • Green Fluorescent Proteins
  • DAPI
  • DNA
  • bisbenzimide ethoxide trihydrochloride
  • Thiamine