Optimal microscopic systems for long-term imaging of intracellular calcium using a ratiometric genetically-encoded calcium indicator

Akitoshi Miyamoto, Hiroko Bannai, Takayuki Michikawa, Katsuhiko Mikoshiba*

*この研究の対応する著者

研究成果: Article査読

4 被引用数 (Scopus)

抄録

Monitoring the pattern of intracellular Ca2+ signals that control many diverse cellular processes is essential for understanding regulatory mechanisms of cellular functions. Various genetically encoded Ca2+ indicators (GECIs) are used for monitoring intracellular Ca2+ changes under several types of microscope systems. However, it has not yet been explored which microscopic system is ideal for long-term imaging of the spatiotemporal patterns of Ca2+ signals using GECIs. We here compared the Ca2+ signals reported by a fluorescence resonance energy transfer (FRET)-based ratiometric GECI, yellow cameleon 3.60 (YC3.60), stably expressed in DT40 B lymphocytes, using three different imaging systems. These systems included a wide-field fluorescent microscope, a multipoint scanning confocal system, and a single-point scanning confocal system. The degree of photobleaching and the signal-to-noise ratio of YC3.60 in DT40 cells were highly dependent on the fluorescence excitation method, although the total illumination energy was maintained at a constant level within each of the imaging systems. More strikingly, the Ca2+ responses evoked by B-cell antigen receptor stimulation in YC3.60-expressing DT40 cells were different among the imaging systems, and markedly affected by the illumination power used. Our results suggest that optimization of the imaging system, including illumination and acquisition conditions, is crucial for accurate visualization of intracellular Ca2+ signals.

本文言語English
ページ(範囲)252-257
ページ数6
ジャーナルBiochemical and Biophysical Research Communications
434
2
DOI
出版ステータスPublished - 2013 5月 3
外部発表はい

ASJC Scopus subject areas

  • 生物理学
  • 生化学
  • 分子生物学
  • 細胞生物学

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