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All imaging was performed on a custom 2-photon microscope built by the authors. Coherent light was provided by a Chameleon Ti-Sapphire laser (Chameleon XR, Coherent Inc.) tuned to 920 nm. All images were acquired with an Olympus water immersion objective (XLUMPlanFLNW 20x, NA 1.0). Collected green light passed through a 750 nm high-pass filter (Semrock, FF01-750/SP-50), a FF552-Di02-50 dichroic (Semrock), and finally through a “green” band-pass filter (Semrock, FF01-525/50). Signals were detected with an R10699 photomultiplier tube (Hamamatsu) and amplified with an SR570 amplifier (Stanford Research Systems). Scanning was conducted using 3 mm Cambridge Technologies galvanometric scan mirrors (part number 8315 KB) and laser power regulated by a Pockels cell (Conoptics). The microscope was controlled by an unmodified version of ScanImage 3.8 [9] . Data acquisition and galvo control were performed using National Instruments PCI-6110 and PCIe-6343 boards.
The test slide was made by painting a green patch on a standard slide using a Sharpie accent highlighter. We used the darker, rather than paler green, pen. A cover slip was placed over the patch and it was sealed with clear nail polish. Pollen grains were imaged from a “mixed pollen grain slide” (carolina.com).

A custom-built two-photon microscope was used for in vivo imaging. Fluorophores were excited and imaged with a water immersion objective (20×, 0.95 NA, Olympus) at 920 nm using a Ti:Sapphire laser (Mai Tai HP, Spectra-Physics). Images were acquired at 16-bit resolution and 4–8 frames/s. The pixel size was 0.6 μm OSN somata imaging and 1.2–2.4 μm for imaging glomeruli. Fields of view ranged from 180 × 180 μm in the epithelium to 720 × 720 μm in the glomerular layer. The point-spread function of the microscope was measured to be 0.51 × 0.48 × 2.12 μm. Image acquisition and scanning were controlled by custom-written software in LabView (National Instruments). Emitted light was routed through two dichroic mirrors (680dcxr, Chroma and FF555- Di02, Semrock) and collected by a photomultiplier tube (R3896, Hamamatsu) using filters in the 500–550 nm range (FF01–525/50, Semrock).

A custom-built two-photon microscope104 was used for in vivo imaging. Fluorophores were excited and imaged with a water immersion objective (20×, 0.95 NA, Olympus) at 950 nm using a Ti:Sapphire laser (Mai Tai HP, Spectra-Physics). Frame rates were typically 4 Hz. Image acquisition and scanning were controlled by custom-written software in Labview. Emitted light was routed through two dichroic mirrors (680dcxr, Chroma and FF555-Di02, Semrock) and collected by 2 photomultiplier tubes (R3896, Hamamatsu) using filters in the 500–550 nm range (green channel, FF01-525/50, Semrock) and 572–642 nm range (red channel, FF01-607/70, Semrock).
To identify sparsely labeled granule cells that allow imaging of both their soma and dendrites the laser was tuned to 1020 nm and the red channel was used to record a detailed z-stack with 1 μm step size. The entire dendritic tree of a given GC was then examined to ensure that it could be clearly separated from processes of neighboring cells and faithfully traced to its corresponding soma. After choosing one imaging plane containing the soma and one imaging plane containing several branches of the same dendrite the laser was tuned back to 950 nm for imaging odor-evoked activity in either plane.