0.8 numerical aperture objective

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.

All Ca²⁺ imaging recordings were performed in the dark using a custom-made two-photon microscope (Janelia MIMMS design). GCaMP6f and, if expressed, tdTomato were excited at 920 nm (typically 40–70 mW) by a Ti:sapphire laser (Chameleon Ultra II, Coherent) and imaged through a Nikon 16×, 0.8-numerical-aperture objective. Emission light passed through a 565 DCXR dichroic filter (Chroma) and either a 531/46 nm (GCaMP channel, Semrock) or a 612/69 nm (tdTomato channel, Semrock) bandpass filter. It was detected by two GaAsP photomultiplier tubes (11706P-40SEL, Hamamatsu). Images (512 × 512 pixels) were acquired at about 30 Hz using the ScanImage software (R2015 and R2018, Vidrio).
For CA1 pyramidal neuron Ca²⁺ imaging, imaging fields (size varied from 280 × 280 to 380 × 380 μm) were selected on the basis of the presence of Ca²⁺ transients in the somata. One field of view was imaged per day. If possible, the same field of view was imaged on days 0 and 1 (n = 14/18 animals).
For EC3 axonal Ca²⁺ imaging, imaging fields (size of 230 × 230 μm) were selected on the basis of the presence of the fibre morphology in the tdTomato channel and the occasional Ca²⁺ transient in the field of view. No attempt was made to locate the same imaging field from day to day.