Orca flash 4

Minicultures were imaged in pairs at DIV9–DIV13, a developmental stage in which the number and position of the clusters was stable and spontaneous activity high. Neuronal activity was monitored through fluorescence calcium imaging using Fluo-4-AM as Ca²⁺ probe (Teller et al., 2014 (link), 2015 (link)). Prior recording, cultures were incubated for 25 min in a transparent, pH-stable medium (recording solution, RS) that contained 2 μg of Fluo-4 per milliliter of solution. At the end of incubation and after washing off residual Fluo-4, the cultures were transferred to an observation chamber that contained 2 ml of RS. The chamber was sealed with a glass coverslip to prevent evaporation and left 5 min in darkness for stabilization.
The observation chamber was mounted on a multimodal microscope attached to a high-speed sCMOS camera (Hamamatsu Orca Flash 4, USB3 mode) that allowed for the simultaneous imaging of two minicultures. The multimodal microscope is a modified commercial confocal microscope (Nikon C1) that integrates a femtosecond-pulsed laser source for two-photon fluorescence microscopy (Mathew et al., 2009 (link)). This pulsed laser input was optimized for accurate multiphoton microsurgery and optical manipulation/stimulation of biological samples (Santos et al., 2013 (link)).

iPSCs-CMs were superfused with IMDM supplemented with NaCl (final concentrations in mmol/L: 140 NaCl, 3.6 KCL, 1.2 CaCl2, 1 MgCl2. 10 HEPES and 5.5 D-glucose) and loaded with 15 μM of the potentiometric voltage sensitive dye RH-237 (Molecular Probes, Eugene, OR). Blebbistatin, a myosin ATPase inhibitor (Sigma-Aldrich), was employed to avoid motion artifact. The iPSC-CMs were kept in a 37 °C plate and were excited by 532 nm LEDs. Voltage sensitive dye, RH-237 emission was monitored using >710 nm long-pass and signal was captured with a single Hamamatsu ORCA Flash 4 digital CMOS camera. Doxorubicin was diluted in DMSO and added to the culture dish in sequentially increasing concentrations (1.0, and 10.0 μM) at 20-minute intervals. Electrical field stimulation was applied using stainless steel electrodes. The electrodes were ~1 cm apart and were placed in the imaging chamber. Signal processing of the optically mapped data was performed using a custom IDL software program^{23 ,24 (link)}. APD₅₀ data represent independent, biological replicates with n = 3 cases and n = 3 controls.

To test whether unc-49 is expressed in AVA interneurons, we coinjected four plasmids, wp1427 (Punc-49::GFP), wp1339 (Pgpa-14::Cre), wp1392 (Pflp-18::loxP::LacZ::STOP::loxP::mStrawberry), and lin-15(+) into the lin-15(n765) strain. The expression of wp1339 and wp1392 results in mStrawberry labeling of the two AVA interneurons^{34 (link),81 (link)}. The expression patterns of GFP and mStrawberry were imaged with an inverted microscope (TE-2000U, Nikon) enhanced GFP/fluorescein isothiocyanate and mCherry/Texas Red filter sets (49002 and 49008, Chroma Technology Corporation, Rockingham, VT, USA) and a Hamamatsu ORCA-Flash4.0 digital camera (Model C11440-22CU). Pflp-18 and Pgpa-14 were gifts from Dr Alexander Gottschalk^{81 (link)} whereas Punc-49 was cloned from genomic DNA of the Bristol N2 strain by PCR using primers listed in Supplementary Table 1.

Laser ablation of collagen was performed with a custom-made nano-dissection setup based on the one described previously^{50 (link)}. Our setup includes a spinning-disc unit (CSU-X1, Yokogawa) equipped with an Andor NEO sCmos camera, a 638 nm and a 488 nm laser (Cobolt) for fluorescence confocal imaging, as well as a HAMAMATSU Orca Flash 4 for bright field imaging along a separate optical path. Cuts were done by shining a pulsed UV-laser with emission of 355 nm, 400 psec pulse duration, 72 kW peak power and 25 mW average power through a HC PL APO 40x/1.10 water objective (Leica microsystems) with a working distance of 0.65 mm. Movies were taken at frame rates between 120 fps and 200 fps. For analysis movies were processed with Fiji. The tip cell was tracked manually to measure the relaxation after the cut.

To assess SLOW-1 expression in F₁ progeny from reciprocal crosses between mScarlet::SLOW-1 NIL and EG6180 strains, we conducted 2 sets of crosses: (1) SLOW-1::mScarlet dpy (INK461) hermaphrodites to EG6180 males for maternal inheritance; and (2) EG6180 dpy (QX2355) hermaphrodites to mScarlet::SLOW-1 NIL males (INK459) for paternal inheritance. Wild-type young adult F₁ progeny were immobilized in NemaGel on a glass slide and imaged using an Axio Imager.Z2 (Carl Zeiss) widefield microscope with a Hamamatsu Orca Flash 4 camera, (excitation 545/30 nm filter). The analysis was performed in FIJI, by tracing the germline in the DIC channel and measuring mean fluorescence, including gut autofluorescence.

Images were acquired using a Zeiss AxioObserver Z1 widefield microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY) equipped with 10x plan-apochromat (N.A. 0.45) objective lens, an Axioscam MRc5 color CCD camera for brightfield imaging, a Hamamatsu ORCA Flash 4 v2 sCMOS camera for fluorescence imaging, a CoolLED pE-4000 multi-LED fluorescence excitation light source, and Zen Blue (v2.3) image acquisition and processing software. Brightness and contrast in fluorescence images was adjusted by linear histogram stretching; the same adjustments were made for each image in the dataset. Images were exported as TIFF files and arranged into figures using Adobe Photoshop CC 2017.