R6357

In vivo 2P-LSM was performed using a custom-built microscope equipped with a resonant scanner (RESSCAN-MOM, Sutter instrument) and a 20× water-immersion objective (W Plan-Apochromat 20x/1.0 DIC D=0.17 M27; Zeiss). Images were acquired with a frame rate of 30 Hz and a frame-averaging factor of 10, resulting in an effective acquisition rate of 3 Hz. Recorded fields of view (FOVs) had a size of 256 × 256 μm, sampled with 512 × 512 pixels (0.5 μm/pixel). To minimize photo-damage, incident laser power was kept between 30 and 40 mW for a sufficient signal-to-noise ratio. Laser wavelength was set to 890 nm (Chameleon Ultra II, Ti:Sapphire Laser, Coherent). The emitted light was detected by photomultiplier tubes (R6357, Hamamatsu) [10 ] and pre-amplified (DHPCA-100, Femto). Digitizer (NI-5734) and control hardware (NI-6341) were housed in a NI PXIe (1082) chassis, connected to a control PC via a high bandwidth PXIe-PCIe8398 interface. Scanning and image acquisition were controlled by ScanImage (SI 5.6R1) [28 (link)].

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.

Confocal images of fluorescence labeled sections were acquired with a Leica AOBS SP2 laser scanning confocal microscope (Leica, Heidelberg, Germany), using a high resolution Leica 63×/1.4 plan apochromatic oil immersion objective. The acquisition software was Leica Confocal Software TCS SP2. The laser lines used were 405 nm (for DAPI), 488 nm (for Alexa Fluor 488) and 594 nm (for Alexa Fluor 594), produced by ultraviolet diode, argon and helium-neon lasers (Leica AOBS SP2 module), respectively. The respective emission signals were collected sequentially to avoid cross excitation, as well as the crosstalk using AOBS tunable filters as follow; 410–480 nm for DAPI, 504–571 nm for Alexa Fluor 488, and 597–751 nm for Alexa Fluor 594. While collecting the images, appropriate PMT offset level was used to minimize the auto-fluorescence and images were framed an average of 3–4 times to minimize the noise. All images and spectral data were captured using the PMT detectors (R6357, Hamamatsu) located inside the scan head. Spectral scanning was performed throughout the experiments to confirm the specificity of the Alexa Fluor signal emission profiles.