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*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)252-257
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume434
Issue number2
DOIs
Publication statusPublished - 2013 May 3
Externally publishedYes

Keywords

  • Ca oscillation
  • Genetically encoded Ca indicators
  • Illumination power
  • Multipoint scanning confocal system
  • Single-point scanning confocal system
  • Wide-field fluorescent microscope

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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