Fv mpe rs multiphoton microscope

Mice were habituated to the head-fixation setup for 3 days beginning 8–10 weeks after surgery. Ca²⁺ imaging data was acquired using an Olympus FV-MPE-RS Multiphoton microscope with Spectra Physics MaiTai HPDS laser, tuned to 920 nm with 100 fs pulse width at 80 MHz. Each 128×128 pixel scan was acquired with a 20x air objective (LCPLN20XIR, Olympus), using a Galvo-Galvo scanner at 5Hz. Stimulus delivery and behavioral measurements were controlled through a custom software written in LabVIEW (National Instruments) and operated through a DAQ (USB-6008, National Instruments). Each imaging session lasted between 30–45 minutes and was synchronized with the stimulus delivery software through a TTL pulse. The imaging depth was manually adjusted to closely match that of the first imaging day such that we recorded from overlapping populations across days of imaging. Animals were excluded from analysis if a) histology showed that either the GRIN lens or the jGCaMP7s virus was mistargeted or b) the motion during imaging was too severe for successful motion-correction. 2 animals were excluded due to mistargeting and 2 animals were excluded due to excessive motion.

AAVs expressing the GRAB5-HT3.5 biosensor (AAV9-hSyn-5-HT3.5) were stereotaxically injected into the DRN of C57BL/6J mice, as described above. Four weeks later, ex vivo two-photon fluorescent imaging was conducted, using an Olympus FVMPE RS multiphoton microscope paired with Mai Tai Si:Sapphire laser (Spectra-Physics, Mountain View, CA, USA). First, acute brain slices containing the DRN were prepared. Then, they were transferred to a recording chamber filled with aCSF saturated with 95% O₂/5% CO₂, flowing at the rate of 2 ml/min. The excitation wavelength was 920 nm, and fluorescence was detected with a 495–540-nm filter. Data were analyzed using ImageJ and MATLAB software.