Scmos camera

Cells were incubated for 30 min at 37 °C with 3 × 10⁻⁶m Fluo3 AM calcium indicator (ThermoFisher Scientific) in cell culture medium containing 0.02% Pluronic F‐127 (Sigma Aldrich). Cells were then rinsed in cell culture media and incubated for 30 min at 37 °C before imaging.
Calcium imaging of neurons containing the MAP2‐GCamP3 reporter and the cells loaded with Fluo3AM was performed in the same way, as follows. Cell culture media was replaced with 100 µL baseline buffer containing 1.2 × 10⁻³m MgCl₂, 2 × 10⁻³m CaCl₂, 150 × 10⁻³m NaCl, 5 × 10⁻³m KCl, 5 × 10⁻³m glucose, and 10 × 10⁻³m HEPES. Imaging was performed on an inverted Ti2 microscope (Nikon) equipped with a CSU‐W1 spinning disc system (Yokogawa), a sCMOS camera (Teledyne Photometrics), and a 20× objective. Environment control chamber was used to maintain the temperature at 37 °C and CO₂ level at 5% during imaging. Exposure time was set to 30 and 100 ms depending on the dynamics of calcium transients. Firstly, spontaneous activity was recorded. Finally, stimulated calcium influx was recorded by inducing cell membrane depolarization through the injection of 50 µL of stimulation buffer containing 1.2 × 10⁻³m MgCl₂, 2 × 10⁻³m CaCl₂, 5 × 10⁻³m NaCl, 450 × 10⁻³m KCl, 5 × 10⁻³m glucose, and 10 × 10⁻³m HEPES buffer. Images were analyzed in ImageJ (NIH) and plotted in QtiPlot.

L4 or 1-day adult worms were immobilized using 30 mM sodium azide and mounted on 2–5% agarose pads. Images were acquired on an Olympus IX73 Epifluorescence microscope with an Andor EMCCD camera or the Olympus Fluoview FV1000 confocal laser scanning microscope or Olympus IX83 with Perkin Elmer Ultraview Spinning Disc confocal microscope fitted with a Hamamatsu EMCCD camera. Since AP-3 localization is sensitive to levels of ATP [73 (link)], static imaging of APB-3::GFP was performed using 5 mM Tetramisole. APB-3::GFP was imaged on Olympus Spin SR10 (SoRA, 50 μm disk) fitted with Teledyne Photometrics sCMOS camera.

Fluorescence imaging of both live cells and immunolabeled printed constructs was performed on an inverted Ti2 microscope (Nikon) equipped with a CSU‐ W1 spinning disc system (Yokogawa) and a sCMOS camera (Teledyne Photometrics). 4x and 20x objectives were used. Z‐stacks were acquired and tile‐scans for larger samples. Reflectance microscopy was performed as follows. Samples were scanned at 647 nm excitation on a Nikon A1RHD confocal with a 100x Apo TIRF NA 1.49 objective, a nonpolarizing 80:20 beam splitter was used to collect the reflected light from the sample. Images were processed in ImageJ (NIH).