Scmos orca flash4 v3.0 camera

HEK 293T cells were imaged at 30 °C in extracellular solution containing (in mM): 135 NaCl, 5.4 KCl, 10 HEPES, 2 CaCl₂, 1 MgCl₂, pH = 7.4 with continuous perfusion on a Zeiss LSM880 scanning confocal microscope using ZEN Black software with a 63x objective, a 488 nm and/or 561 nm laser for imaging and a 405 nm laser for photoactivation. For photoactivation experiments, a brief 10–100 μM glutamate application was used to identify healthy cells, based on calcium responses, which would be suitable for photoactivation experiments. Photoactivation was performed by scanning a 405 nm laser at 100% power in a defined region for 40 iterations between each imaging frame. For drug application experiments, imaging was performed at room temperature on an Olympus IX-73 microscope using a 60× 1.49 NA APO N TIRFM objective (Olympus) and snapshots were taken with a sCMOS ORCA-Flash4 v3.0 camera (Hamamatsu). GFP was excited with a 488 nm laser diode and glutamate was added by a gravity-driven perfusion system. Astrocytes were imaged at 37°C with the same protocol described above in an extracellular solution containing (in mM): 138 NaCl, 1.5 KCl, 1.2 MgCl₂, 2.5 CaCl₂, 10 glucose and 5 HEPES, pH 7.4. DHPG was purchased from Tocris and applied using a gravity-driven perfusion system. For calcium-free experiments, calcium was exchanged for magnesium to maintain osmolarity and ionic strength.

Imaging was performed on an IX-73 microscope (Olympus) with a cellTIRF laser illumination system and an sCMOS ORCA-Flash4 v3.0 camera (Hamamatsu). GFP was excited using a 488 nm laser diode and a 561 nm DPSS laser was used for Alexa-546. Cellular imaging was performed in wide field mode using a 60× objective (NA = 1.49). Data was acquired using cellSens software (Olympus) and analyzed using ImageJ. Prior to imaging, cells were incubated with purified, Alexa-546 labeled NBs (50 nM) for 45 min at 37 °C. For genetically encoded ss-NB-SNAP experiments, BG-Alexa-546 was applied at 1 μM for 45 min at 37 °C. Single molecule pulldown of mGluR2 was performed as previously described.16 (link) Briefly, following immobilization using an anti-mGluR2 antibody, images were acquired with a 100× objective (NA = 1.49) at 20 Hz in TIRF mode, and bleaching steps for individual molecules were manually analyzed using a previously described custom software.35 (link)