Na water immersion objective

Imaging used the red-shifted Ca²⁺ indicator Cal-590 AM (AAT Bioquest, Sunnyvale, CA), which was prepared and injected as previously described (Tischbirek et al., 2015 (link)). Briefly, the dura was removed and dye (1 mM) was pressure-injected into the PCx at a depth of ~200 µm using a glass pipette (tip diameter ~10 µm). A coverslip was glued over the PCx and imaging commenced >1 hr after injection. Imaging frames were acquired at 30 Hz using a custom-modified B-scope two-photon microscope (Thorlabs, Newton, NJ) with a 16×/0.8 NA water immersion objective (Nikon, Tokyo, Japan), resonance-galvanometer scanners and a Chameleon Ultra Ti:Sapphire laser (Coherent, Santa Clara, CA) tuned to 800–820 nm. Cells were included in the dataset if they unambiguously satisfied the fluorescence and soma morphology criteria given in Results, and if they exhibited clear odor responses according to the criteria under Data analysis, below.

Mice were head-fixed and placed under a two-photon microscope (Denk et al., 1990 (link)). Anesthetized mice were maintained on a heating pad to keep the body temperature at 36°C. The microscope (Scientifica Multiphoton VivoScope or custom designed by Independent NeuroScience Services, UK) was coupled with a MaiTai DeepSee laser (Spectra-Physics, Santa Clara, CA, USA) tuned to 940 nm (<30 mW average power on the sample) for imaging. Images (512 × 512 pixels) were acquired with a resonant scanner at a frame rate of 30 Hz using a 16× 0.8 NA water-immersion objective (Nikon).

Two-photon imaging was performed with a resonant scanning microscope (Ultima II, Bruker Corporation) using a Chameleon Ultra II laser (Coherent) driven by PrairieView. A 16×/0.8-NA water-immersion objective (Nikon) was used for all experiments. An ETL (Optotune EL-10-30-TC, Gardasoft driver) was used to perform volumetric imaging, spanning a 100 μm range with 33.3 μm spacing between 4 planes. The FOV size was 710 × 710 μm at a resolution of 512 × 512 pixels. The number of cells recorded (ROIs after curation) per experiment ranged from 1266 to 4891 (mean = 2765 ± 995). The per-plane frame rate was 7 Hz (total acquisition rate 30 Hz). GCaMP6s was imaged at 920 nm and mRuby (conjugated to C1V1-Kv2.1) was imaged at 765 nm. Power on the sample was 50 mW at the shallowest plane (~150–200 μm below pia) and increased to ~85 mW at the deepest plane (~250–300 μm below pia), interpolating for intermediate planes, to equalize imaging quality across planes. To maximize imaging quality^{127 (link)} we calculated the tilt of the sample relative to the microscope and then rotated the objective along two axes to be perpendicular to the implanted coverslip window.

EGFP-labeled microglia were imaged by two-photon microscope through a small craniotomy as described above. By raster scanning a femtosecond-pulsed laser beam (Chameleon Ultra II, Coherent) via standard galvanometer raster scanning with a moving in vivo microscope (Bruker Corporation), two-photon imaging was performed. A 16×/0.8-NA water-immersion objective (Nikon) was used in all the experiments. The Ti-sapphire laser was set at the excitation wavelength of 920 nm for both EGFP-expressing microglia and Texas red-labeling blood vessels imaging. A stack of image planes (1064 × 1064 pixels) with a step size of 2 μm was acquired using the water-immersion objective at a zoom of 2.0. The maximum imaging depth was ~350 μm from the pial surface. Images were acquired with low laser power (<50 mW at the sample).
To visualize the vasculature and the motion of red blood cells (RBCs) with TPLSM, we injected 7 mg/kg Texas Red dextran (70,000 MW, neutral; Thermo) in 0.9% NaCl intravenously. Line scans were used along single vessels with a maximum scan rate of 5 kHz to quantify RBCs velocity. RBCs movement resulted in dark diagonal streaks in the image with a slope that was inversely proportional to the RBCs velocity.

Four weeks after the surgery, mice were imaged under an Ultima IV two-photon microscope from Bruker/Prairie Technologies^{56 (link)}, with a Nikon 16 × 0.8 NA water-immersion objective (model CFI75 LWD 16XW). The fluorescence of GCaMP6f and jRGECO1a was excited at 999 nm with a Spectra-Physics InSight DeepSee laser. All optical filters mentioned in the following description of the two-photon microscope are by Chroma Technology Corporation: after the collected light is reflected towards the detection unit by the main dichroic filter (ZT473-488/594/NIRtpc), the signal light enters the detector house (four channels), passing a ZET473-488/594/NIRm filter, that is shielding the photomultiplier tubes from reflective light. Inside the detector house, the light is then split into two fractions separated at a wavelength of 560 nm by a dichroic filter (T560lpxr). The green light (GCaMP6f) is further guided by a secondary dichroic beam splitter at 495 nm (T495lpxr) and filtered by a ET525/50m-2p bandpass filter, whereas the red light (jRGECO1a) is similarly guided by a secondary beam splitter at 640 nm (T640lpxr) and subsequently filtered by a ET595/50m-2p bandpass filter. The emitted photons were detected with Peltier cooled photomultiplier tubes (model 7422PA-40 by Hamamatsu Photonics K.K.). Images (512 × 512 pixels) were acquired at 30 Hz in layer 2/3 of barrel cortex.

In vivo imaging was performed with an Ultima IV two-photon laser-scanning microscope system (Bruker), using a 16 × 0.8 NA water immersion objective (Nikon) with a femtosecond laser (MaiTai DeepSee, Spectra Physics, Mountain View, CA, USA) tuned to 950 nm for imaging of GCaMP6f expressing cells. Time-series movies of neuronal populations expressing GCaMP6f were acquired at 7 Hz (182 × 182 microns). Each focal plane movie duration was 3.6 minutes (1500 frames) to track spontaneous neuronal activity. Care was taken to use less than 10 mW of laser power at the surface of the tissue. For in vivo two-photon imaging of microglia cells of the CX3CR1 mice (also injected with the AAV1.CAG.tdTomato vector), the femtosecond laser tuned to 960 nm and laser power was kept below 5 mW to avoid phototoxic effects. Time series were acquired (1024x1024 pixels) at a 10-second interval for a total of 10 min (60 iterations).

Data were collected using a custom-built two-photon microscope. A Ti:Sapphire laser (Coherent Chameleon Vision II) was used to deliver 950 nm excitation light for calcium imaging through a Nikon 16 × 0.8 NA water immersion objective, with an average power of ~60–70 mW at the sample. The scan head consisted of a resonant-galvonometric scanning mirror pair separated by a scan lens-based relay. Collection optics were housed in a light-tight aluminum box to prevent contamination from visual stimuli. Emitted light was filtered (525/50, Semrock) and collected by a GaAsP photomultiplier tube (Hamamatsu). Microscope hardware was controlled by ScanImage 2018 (Vidrio Technologies). Rotation of the spherical treadmill along three axes was monitored by a pair of optical sensors (ADNS-9800) embedded into the treadmill support communicating with a microcontroller (Teensy, 3.1). The treadmill was mounted on an XYZ translation stage (Dover Motion) to position the mouse under the objective.