Theoretical analysis on ratiometric fluorescent indicators caused biased estimates of intracellular free calcium concentrations

Ratiometric fluorescent calcium indicator dyes have been widely used for the study of the role of Ca2+ in cell physiopathology. Although these ratiometric dyes offer several advantages over others, they suffer some drawbacks which cause serious errors in measurement of intracellular Ca2+ concentration, [Ca2+]i. The present study systematically analyzes theoretical reasons and technical sources of discrepancies occurring in the measurement of the characteristics of the agonists-induced cells [Ca2+]i. In order to avoid the errors and achieve the accurate determination of [Ca2+]i, this study proposes solutions and suggests some critical measures in both theoretical and technical aspects. Therefore, this analysis can be a valuable tool in clarifying proper usages of fluorescent dyes for [Ca2+]i measurements.