POSSIBILITY OF SIMULTANEOUSLY MEASURING LOW AND HIGH-CALCIUM CONCENTRATIONS USING FURA-2 AND LIFETIME-BASED SENSING

被引:32
作者
SZMACINSKI, H [1 ]
LAKOWICZ, JR [1 ]
机构
[1] UNIV MARYLAND, SCH MED, CTR FLUORESCENCE SPECT, DEPT BIOL CHEM, BALTIMORE, MD 21201 USA
关键词
D O I
10.1016/0143-4160(95)90046-2
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
We characterized the fluorescence probe Fura-2 for calcium measurements using frequency-domain phase-modulation fluorometry. By the use of different excitation wavelengths from 345 to 380 nm, the apparent calcium dissociation constants can be altered from 41 nM to 1.92 mu M Ca2+. This change in apparent Kd results from changes in the relative extent of excitation of the calcium-bound and calcium-free forms, and the excitation wavelength-dependent contribution of each form to the intensity decay. These results indicate that lifetime-based measurements with Fura-2 can be used for imaging of calcium over a wide range of concentrations. An additional favorable feature of Fura-2 is that the calcium-free form can be almost exclusively excited at wavelength of 390 nm or longer, and can thus be used as a reference providing the lifetime in the absence of calcium, without removing the calcium. Additionally, exposure of Fura-2 to intense illumination shifts but does not distort the frequency response. For cellular imaging, these favorable properties of Fura-2 may allow calibration of the calcium concentrations without the use of ionophores.
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页码:64 / 75
页数:12
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