Orca er digital

The fluorescent indicator, 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolylcarbocyanine iodide (JC‐1; 200 nmol/L, excitation 480 nm, emission 580/535 nm; Molecular Probes), was used to measure Ψ_m at 37°C, as previously described.^{10 (link)} Fluorescence at 480‐nm excitation and 580/535‐nm emission signal were measured at 2‐minute intervals with an exposure of 50 ms on a Hamamatsu Orca ER digital camera (Hamamatsu Photonics, K.K.) attached to an inverted Nikon TE2000‐U microscope (Nikon Corporation). Ratiometric 580/535‐nm signal of individual myocytes were quantified using MetaMorph 6.3 (Molecular Devices) to measure signal intensity of manually traced myocytes. An equivalent region not containing cells was used as background and was subtracted. Fluorescent ratios recorded over 3 minutes before and 7 minutes after addition of drugs were averaged and alterations in fluorescent ratios reported as a percentage increase from the basal average. To confirm that the JC‐1 signal was indicative of Ψ_m, 4 mmol/L of sodium cyanide (NaCN) was added at the end of each experiment to collapse Ψ_m. In addition, individual 580‐ and 535‐nm signals were assessed in each experiment to confirm that the fluorescent indicator was accurately measuring Ψ_m.