The measurement of intracellular Ca²⁺ concentration ([Ca²⁺]_i) is one of the major experimental procedures because of its important role in various cellular events such as contraction, secretion, metabolism, excitability and proliferation, etc. To obtain the exact concentration of intracellular Ca²⁺, the ratiometric fluorescent dye, fura-2, was used. 340 nm and 380 nm are usually selected for excitation because the emission peaks in Ca²⁺-saturated and Ca²⁺-free conditions, respectively. The ratio (F₃₄₀/F₃₈₀) of the emission intensities at around 500 nm by each excitation wavelength is measured. To convert ratio to [Ca²⁺], Grynkiewicz et al. (1985) developed an equation that needs four parameters such as the maximum ratio (R_max), the minimum ratio (R_min), dissociation constant (K_d) and the ratio of maximum and minimum (F_380,max/F_380,min) at 380 nm excitation. Theoretically, if we could obtain the exact values of these four parameters, we could convert the ratio to the exact Ca²⁺ concentration. Practically, it is very difficult to obtain exact values of four parameters in dye loading conditions in cells. Worse, because of the change of Ca²⁺ binding affinities by pH changes, if there are pH changes, it is almost impossible to obtain those parameters in situ. Fortunately fura-2 has very peculiar characteristics: the Ca²⁺-binding form of fura-2 has virtually no emission at 400 nm excitation while the Ca²⁺-free form produces considerable emission (Ogden et al. 1995). Based on this fact, we developed the following equation:

[Ca²⁺] = K_d X F_400,max/F_340,max X (R – R_min).

K_d is a dissociation constant, R is F₃₄₀/F₄₀₀, R_min is a minimum ratio and F_400,max/F_340,max is the ratio of maximum value at 400 nm excitation and maximum value of 340 nm excitation. The new equation has several benefits over old the equation. It needs only three parameters and all the values can be easily obtained from the experiments. Also, the error in parameter measurement can be decreased considerably. Another important benefit is that the ratio is linear to Ca²⁺ concentration, which means the rate of ratio change can be directly correlated to the rate of Ca²⁺ change. If we select the emission at isobestic excitation wavelength for the numerator, the equation can be changed into a much simpler form, as follows: [Ca²⁺] = K_d X 1/R_min X (R – R_min) at constant pH conditions. We verified that this new equation can be applied within reasonable Ca²⁺ concentration ranges. We obtained the changes of each parameter by pH changes which must be system specific except K_d. Finally we obtained strong evidence that the K_d values in free conditions and in situ are virtually the same. Based on these facts, the resting [Ca²⁺] may be measured more accurately. At present, several ion-sensitive fluoroprobes such as fura-2, bis-fura-2, fura-2-FF, pyranine, etc., can be applied to the new equation. The new development of dyes in this direction may improve its applicability.

This work was supported by grant no. R01-2000-00172 from the Korea Science & Engineering Foundation.

All procedures accord with current local guidelines.