TY - JOUR
T1 - Optimal microscopic systems for long-term imaging of intracellular calcium using a ratiometric genetically-encoded calcium indicator
AU - Miyamoto, Akitoshi
AU - Bannai, Hiroko
AU - Michikawa, Takayuki
AU - Mikoshiba, Katsuhiko
N1 - Funding Information:
We thank Dr. T. Nagai and A. Miyawaki for YC3.60. We thank Nikon Instruments Inc. and T. Tajima and staffs of RIKEN BSI-Olympus Collaboration Center (BOCC) for technical support. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan ( 20220007 to K.M. and 24500476 to T.M.).
PY - 2013/5/3
Y1 - 2013/5/3
N2 - 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.
AB - 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.
KW - Ca oscillation
KW - Genetically encoded Ca indicators
KW - Illumination power
KW - Multipoint scanning confocal system
KW - Single-point scanning confocal system
KW - Wide-field fluorescent microscope
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U2 - 10.1016/j.bbrc.2013.02.112
DO - 10.1016/j.bbrc.2013.02.112
M3 - Article
C2 - 23535376
AN - SCOPUS:84877060461
VL - 434
SP - 252
EP - 257
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 2
ER -