The classical approach for calibrating non-ratiometric fluorescent Ca²⁺ dyes entails the measurement of the fluorescence maximum (F_max) and minimum (F_min), as well as the dissociation constant (Kd) of the Ca²⁺—Dye reaction (model 1). An alternative equation does not need the F_min but requires the rate constants k_on and k_off (model 2). However, both approaches are experimentally time consuming and the rate constants for several dyes are unknown. Here, we propose a set of equations (model 3) that simplify the calibration of fluorescent Ca²⁺ transients obtained with non-ratiometric dyes. This equation allows the calibration of signals without using the F_min: [Ca²⁺] = Kd(F − F_rest/F_max − F) + [Ca²⁺]_IR(F_max − F_rest/F_max − F), where [Ca²⁺]_IR is the resting [Ca²⁺]. If the classical calibration approach is followed, the F_min can be estimated from: F_min = F_rest − ([Ca²⁺]_IR(F_max − F_rest)/Kd). We tested the models’ performance using signals obtained from enzymatically dissociated flexor digitorum brevis fibers of C57BL/6 mice loaded with Fluo-4, AM. Model 3 performed the same as model 2, and both gave peak [Ca²⁺] values 15 ± 0.3% (n = 3) lower than model 1, when we used our experimental F_min (1.24 ± 0.11 A.U., n = 4). However, when we used the mathematically estimated F_min (6.78 ± 0.2 A.U) for model 1, the peak [Ca²⁺] were similar for all three models. This suggests that the dye leakage makes a correct determination of the F_min unlikely and induces errors in the estimation of [Ca²⁺]. In conclusion, we propose simpler and time-saving equations that help to reliably calibrate cytosolic Ca²⁺ transients obtained with non-ratiometric fluorescent dyes. The use of the estimated F_min avoids the uncertainties associated with its experimental measurement.

校准非比例荧光 Ca ²⁺染料的经典方法需要测量荧光最大值 ( F _max ) 和最小值 ( F _min )，以及Ca ²⁺ -Dye 反应的解离常数 ( Kd )（模型1）。另一个方程不需要F _min但需要速率常数k _on和k _off（模型 2）。然而，这两种方法在实验上都是耗时的，并且几种染料的速率常数是未知的。在这里，我们提出了一组简化荧光 Ca ^{2+校准的方程（模型 3）}用非比例染料获得的瞬变。该方程允许在不使用F _min的情况下校准信号： [Ca ²⁺ ] =  Kd ( F - F _rest / F _max - F ) + [Ca ²⁺ ] _IR ( F _max - F _rest /F _max - F ) ，其中[Ca ²⁺ ] _IR是静止的[Ca ²⁺ ]。如果遵循经典校准方法，则F _min可以通过以下公式估算：F_min  =  F _rest - ([Ca ²⁺ ] _IR ( F _max - F _rest )/Kd )。我们使用从加载 Fluo-4、AM 的 C57BL/6 小鼠的酶解离的趾短屈肌纤维获得的信号测试了模型的性能。模型 3 的性能与模型 2 相同，当我们使用我们的实验F _min (1.24 ± 0.11 AU, n = 4)，两者的峰值 [Ca ²⁺ ] 值都比模型 1 低 15 ± 0.3% (n = 3. 然而，当我们对模型 1 使用数学估计的F _min (6.78 ± 0.2 AU) 时，峰值 [Ca ²⁺] 对于所有三个模型都是相似的。这表明染料泄漏使得F _min^{的正确确定不太可能并且在[Ca 2+} ]的估计中引起错误。总之，我们提出了更简单且省时的方程，有助于可靠地校准用非比例荧光染料获得的细胞溶质 Ca ^2+瞬变。使用估计的F _min避免了与其实验测量相关的不确定性。