To acquire the contractile parameters of adult flies, similar protocols were adopted.13,19 Briefly, in each experiment, two-dimensional (2-D) OCT images were first obtained in the longitudinal direction to identify the dorsal midline of the A1–A3 abdominal segments of the Drosophila. Then, the OCT image orientation was rotated by 90 deg, such that the conical chamber (CC) of the heart was acquired in the transverse plane. Measurements were always made in the same location in the abdominal segment. A group of 20 flies was examined with an SS-OCT system (OCM1300SS, Thorlabs Inc., Newton, New Jersey). The median wavelength of the SS-OCT system was 1310 nm with an axial resolution of in tissue, a total power of 10 mW, and an A-scan rate of 16 kHz. Since heart function of Drosophila decelerates greatly between 5 and 7 weeks of age, to assess the age-dependent heartbeat parameters, 1-, 3-, and 7-week old flies were used. It has been previously reported that anesthetic treatment induces cardiac arrhythmias in mice, and that carbon dioxide or ether affects heart performances in Drosophila.22–24 To sidestep the aforementioned issues, during the image assessment, the Drosophila were first anesthetized with triethyl amine, and then gently immobilized on wax gel with the dorsal side facing the OCT probe. The flies were subsequently allowed to return to a fully awake state on the bench for 20 min before being subjected to OCT scanning.