Two-Photon Imaging of Neural Responses

Animals were prepared for two-photon imaging in the same manner as described for the awake head fixed epifluorescence imaging experiments, except that animal were habituated to run on a free floating Styrofoam ball. Imaging was carried out with a two-photon microscope (MOM; Sutter Instruments) coupled to a pulsed Ti:Sapphire laser (Mai Tai HP; Spectra-Physics) and controlled by Scanimage 3.9 (Pologruto et al., 2003 (link)). In all experiments, imaging was performed through a 16 × 0.8 numerical aperture objective (Nikon) and emitted light was collected by multialkali photomultiplier tubes (Hamamatsu R6357). Images were acquired at 3.7 Hz in most experiments. Fluorescence time series were extracted and analyzed with custom Matlab scripts, and ΔF/F was calculated as in in vitro experiments. Pseudocolor activation maps reflect an average of 8 trials in which movies were spatially filtered using a Gaussian window with a sigma of 0.75 pixels and temporally filtered using a fourth-order Butterworth filter with a cutoff frequency of 0.25 Hz. Correlation coefficients of odor evoked activity maps were calculated from pairwise correlations of averaged (8 odor presentations) ΔF maps.