W plan apochromat 20 1.0 dic

Two-photon calcium imaging was performed on a modified upright microscope (Axio Examiner, Zeiss), exciting the specimens with a Ti:Sapphire laser (Chameleon Vision, Coherent) tuned to 920 nm (power at the back aperture ~25 mW). We imaged with a ×20 water-immersion objective (W Plan-Apochromat ×20/1.0 DIC, Zeiss). Data acquisition was controlled by Slidebook (version 6, 3i). Single-plane images were taken at ~10 Hz with a spatial resolution of approximately 285 × 142 pixels (100 × 50 μm, pixel size ≅ 0.35 μm, dwell time ≅ 2.5 μs). GCaMP responses were recorded from the presynaptic terminals of T3 neurons. For each preparation, we identified the most caudal presynaptic terminals and then shifted the region of interest (ROI) downward ~30 μm. Images and external stimulations were synchronized a posteriori using frame capture markers (TTL pulses output from Slidebook) and stimulus events (analog outputs from the LED display controller) sampled with a data acquisition device (DAQ) (PXI-6259, NI) at 10 kHz. The DAQ interfaced with MATLAB (R2020a, MathWorks) via rack-mount terminal block (BNC-2090, NI).

At 3 dpf, Tg(myl7:GAL4FF)^hu6531;Tg(UAS:Kaede)^rk8 embryos were mounted in 1.5% low melting point agarose and 30 ​mM BDM to stop the heart for the duration of imaging. Prior to photoconversion, hearts were imaged using a dual 488/543 excitation and a Z-stack collected. Immediately thereafter, a defined region in each embryo that included all heart Kaede-green-marked cells and their vicinity was photoconverted to Kaede-red by manual focusing throughout the Z-stack under continuous laser scanning using a 405 ​nm laser (Hatta et al., 2006 (link)) while assessing visually for residual green fluorescence. Embryos were re-scanned under dual excitation 488/543 ​nm post-photoconversion, then released from agarose and recovered in system water in the dark until 5 dpf when they were embedded and imaged again. Only larvae that were healthy after recovering were used for analysis. Photoconversion and imaging was undertaken on a Zeiss LSM Exciter confocal with a Zeiss W Plan-Apochromat 20×/1.0 DIC (UV) VIS-IR objective.