Possibility of simultaneously measuring low and high calcium concentrations using Fura-2 and lifetime-based sensing.
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 microM 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.
Szmacinski
H
H
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, USA.
Lakowicz
J R
JR
D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D005069:Evaluation Studies as Topic; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D016257:Fura-2; D007424:Intracellular Fluid / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence; D015335:Molecular Probes; D010777:Photochemistry; D012996:Solutions; D013053:Spectrophotometry
D005456:Fluorescent Dyes; D015335:Molecular Probes; D012996:Solutions; D002118:Calcium; D016257:Fura-2