La1951

Experiments were performed in the late morning or early afternoon Zeitgeber time (Z.T.), inside a dark imaging chamber. An adult female animal 2–3 days-post-eclosion (dpe), was mounted onto a custom stage (Chen et al., 2018 (link)) and allowed to acclimate for 5 min on an air-supported spherical treadmill (Chen et al., 2018 (link)). Optogenetic stimulation was performed using a 617 nm LED (Thorlabs, Newton, NJ) pointed at the dorsal thorax through a hole in the stage, and focused with a lens (LA1951, 01" f = 25.4 mm, Thorlabs, Newton, NJ). Tethered flies were otherwise allowed to behave spontaneously. Data were acquired in 9 s epochs: 2 s baseline, 5 s with optogenetic illumination, and 2 s without stimulation. Individual flies were recorded for five trials each, with one-minute intervals. Data were excluded from analysis if flies pushed their abdomens onto the spherical treadmill—interfering with limb movements—or if flies struggled during optogenetic stimulation, pushing their forelimbs onto the stage for prolonged periods of time.

In order to excite rhod-2, cell cultures were illuminated with two filtered green LED light sources (CBT-90-G; peak output 58W; peak wavelength 524 nm; Luminus Devices, Billerica, MA, United States), with a plano-convex lens (LA1951; focal length = 25.4 mm; Thorlabs, Newton, NJ, United States) and a green excitation filter (D540/25X; Chroma Technology, Bellows Falls, VT, United States) (Climent et al., 2015 (link)). Fluorescence was recorded using an electron-multiplying charge-coupled device (EMCCD; Evolve-128: 128 × 128, 24 μm × 24 μm-square pixels, 16 bit; Photometrics, Tucson, AZ, United States) with a custom emission filter (ET585/50-800/200M; Chroma Technology) suitable for rhod-2 emission placed in front of a high-speed camera lens (DO-2595; Navitar Inc., Rochester, NY, United States). Fifteen-second movies of fluorescence were recorded at 100 frames/s throughout the protocol duration. The area of the field of view (FOV) was ∼2 cm × 2 cm (95 pixels × 95 pixels) and included the entire flexible well, both in control and stretch situations (Climent et al., 2015 (link)).

As shown in Fig. 5, this system uses four identical CMOS cameras CAM 1 – CAM 4 (part #: UI-3220CP-M-GL; IDS Imaging Development Systems GmbH) to acquire images from the anterior and posterior surface of the heart. Each camera is configured the same way as in Optical Mapping System 1.
Four green LEDs (part #: CBT-90 Green; Luminus Devices Inc., Woburn, MA) are used to excite RH237 and rhod-2AM dye-loaded tissue (Fig. 5a). The excitation light from each LED passes through a plano-convex lens L1 (part #: LA1951; Thorlabs Inc.) and excitation filter F1 (part #: FF01-534/20-25; Semrock Inc., Rochester, NY). Fluorescence emission from both dyes are first separated by a dichroic mirror D1 (part #: T685LPXR; Chroma Technology Corp). Separated emission light from rhod-2AM and RH237 then pass through emission filter F2 (part #: ET590/50 M; Chroma Technology Corp) and custom emission filter F3 (part #: ET700LP; Chroma Technology Corp), respectively. Both filtered lights are then collected by camera lenses, both labeled L2 (part #: DO-1795; Navitar Inc.). And because of the reflections at the dichroic mirrors, images from CAM 2 and CAM 4 are flipped horizontally during image processing.