Extended Data Figure 1. Volumetric population imaging of PCx L2 and L3 during wakefulness using rationally designed odor sets.

a, (Left), Cartoon of volumetric multiphoton imaging approach to characterize odor responses in PCx in wakeful, semi-paralyzed mice (see Methods). (Right), Approximate position of an imaging volume (green dotted line) in a typical experiment superimposed on a Nissl-stained coronal section through PCx. Scanning volumes were oriented to acquire similarly-sized cortical populations in L2 and L3 (red dotted lines), despite decreased neuron density in L3 (see Methods). Imaging was performed in the most anterior portion of the posterior PCx.

b, Sample fields of view for a single imaging session. PCx L2 is depicted on top; PCx L3 on bottom. Segmentation masks associated with each layer are shown on the right.

c, Global, clustered, and tiled odor sets superimposed on the collection of odors constituting odor space as defined by principal components analysis (see Methods). Global odors are indicated by black dots; tiled and clustered odor sets via the indicated color code

d, Plot of the amount of molecular variance contributed by each additional principal component for each odor set in descriptor space; this analysis reveals that each odor set tiles odor space at a distinct level of resolution.

e, Molecular structures and associated PID signals of the odors comprising the global, clustered, and tiled odor sets. These PID traces are shown to illustrate the controlled kinetics of the olfactometer only; because detector reports depend upon ability of an odor to be photo-ionized, the relative amplitudes of the traces between odors are not meaningful. For example, heavy aliphatics elicit a minimal PID response because their photo-ionization energies lie outside the range of the detector; however, odors with low/absent PID traces still induced cortical activity in 5–20% of the imaged population, consistent with effective odor delivery. Five odors are shared between the global and clustered odor sets. These are indicated by bold lettering (and in c, as black circles with colored edges). Color code as in c.