FIGURE 3.
In vivo calibration of the two-photon SBFI fluorescence signal. (A) Two-photon fluorescence intensity from the entire cell during a calibration experiment. After an equilibration period in normal Tyrode's solution, the myocyte was perfused with divalent-free solutions with various [Na]o in the presence of 10 μM gramicidin D and 100 μM strophanthidin. (B) Two-photon fluorescence images of the cell at the points indicated by arrowheads in A. (C) Two-photon fluorescence calibration curve in rat ventricular myocytes (—) and in buffer solutions (− − −). The in vivo data represent the mean ΔF/F0 from 26 myocytes. The in vitro measurements were done in the same solutions as used for in vivo calibration. Data were fitted with a one-site binding equation (ΔF/F0 = (ΔF/F0)max × [Na]i/(Kd+[Na]i)) to derive the Kd and (ΔF/F0)max.