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

Background: Ca 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 Ca concentration ([Ca]). 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 Ca.

New Method: Here we present a new approach for calculating [Ca] from the ratiometric fluorescence of Fura-2 dye imaged by a CCD camera. It is designed to optimize [Ca] measurements with photobleaching correction without background subtraction error. A mathematical method is also provided for removing the existing underestimated value of fluorescence at an excitation wavelength of 340 nm and compensating for the bleaching rate for both channels with wavelengths of 340 nm and 380 nm using a power function.

Results: In cultured neurons, the calculations of the free Ca concentration during Ca transients estimated by the old and new methods, determine it to the same extent. This comparison was made under conditions without errors through background subtraction. If there is this error, the old method calculates [Ca] with a much higher, rather than the actual value.

Conclusions: We present a modified Grynkiewitz's formula for calculation [Ca] for ratiometric dye, such as Fura-2 imaged by a CCD camera, with photobleaching correction without background subtraction error.