Ratiometric Analysis of Voltage Dynamics

CC2-DMPE and DiBAC₄(3) voltage reporter dyes were obtained from Invitrogen and used as per the standard protocol, including dark-field and flat-field correction (Adams and Levin, 2012 (link)). Briefly, the use of two dyes with opposite emission profiles simultaneously provides an internal control and allows ratiometric normalization. CC2-DMPE stock (5 mM) was dissolved 1:1000 in 0.1× MMR and the embryos were incubated in dark in this solution for at least 1 h followed by washes with 0.1× MMR. DiBAC₄(3) stock (1.9 mM) was dissolved 1:4000 in 0.1× MMR and the CC2-DMPE-stained embryos were then incubated in dark in this solution for at least 30 min followed by visualization under the microscope. An Olympus BX-61 microscope equipped with a Hamamatsu ORCA AG CCD camera, and controlled by Metamorph software (Molecular Devices), was used to collect signal. NIH Image J software was used to quantify the fluorescence intensities of the CC2-DMPE:DiBAC signal.