A study of the precision of confocal, ratiometric, Fura-2-based [Ca2+] measurements

The precision with which quantitative confocal ratiometric Fura-2-based calcium measurements can be performed has been investigated. A standard confocal scanning laser microscope system has been modified so as to enable excitation with the 351 nm and 364 nm lines of the UV argon laser and simultaneous separation of the fluorescence emanating from the two different excitation wavelengths. Experiments have been performed on living cells and mainly on Fura-2 containing calibrated Ca2+ buffer solutions in order to study the variation of the precision with various experimental parameters in a controlled manner. Furthermore, expressions relating the precision of the calcium determination to various measured parameters are derived. These expressions have been used in conjunction with published excitation intensity spectra for Fura-2 in order to verify the experimental results and to analyse the variation of parameters that could not be experimentally tested. The results show that a photon number of the order of 10,000 per pixel, at maximum Ca2+ concentration and 351 nm excitation, is required in order to reduce the relative error at all concentrations to less than 30%. The commercially available laser wavelength combinations, 325 nm/380 nm and 334 nm/380 nm, were found to be the best as concerns the precision of the method.