Error correction due to background subtraction in ratiometric calcium measurements with CCD camera

摘要

Ca2+ plays an important role in many physiological processes and an accurate study of these signals is important. In modern fluorescence microscopy, a charge-coupled device (CCD) camera is widely deployed for calcium imaging. The ratiometric method is used for the fluorescence dye Fura-2 and Grynkiewitz's formula (Grynkiewicz et al., 1985) is commonly used to convert fluorescence to free Ca2+ concentration ([Ca2+]). But the need to subtract the background signal can lead to a big error in ratiometric calcium measurements. When the error due to background subtraction occurs, the fluorescence ratio of 340 nm divided by 380 nm lights may be twice as large as the actual value. Under conditions when the excitation intensity is not adjusted to ensure the same throughput of the objective lens for ultraviolet dye illumination, the indicator does not gradually bleach out for channels with a wavelength of 340 nm and 380 nm light, which lead to an additional error in determining the concentration of Ca2+.