The system for two-photon imaging in behaving mice is shown in Figure 1B. In vivo calcium imaging was conducted with a homebuilt two-photon microscope. For optimal detection sensitivity, we used a two-inch optical path (2′ lenses and hardware for Thorlabs, 2″ filters and dichroics from Semrock and Chroma), and gallium arsenide photomultiplier tubes (PMTs, 2× increased sensitivity; Hamamatsu #H7422P-40MOD) coupled to electronic shutters (Uniblitz) to prevent PMT damage. The microscope permitted rapid alternation between blue/green and green/red dichroic cubes, in order to isolate signals from CFP or GFP (blue), YFP or OGB1-AM (green), or sulforhodamine (red; FF01-457_50, FF01-542_50, and FF01-629_53, respectively; Semrock). We achieved high excitation efficiency using an Olympus 25× (NA 1.05) or Nikon 16× (NA 0.8) objective together with a Mai Tai laser (830 nm for YC3.6, 800 nm or 920 nm for OGB 1-AM) with group delay dispersion compensation (Deep See ‘pre-chirp’ module, Newport). Scanning galvanometers (Cambridge Technology) provided a frame rate up to 64 Hz (32 × 32 pixels/frame, pixel dwell time: 16 μs), which required passive cooling of mirrors and active cooling of mirror drivers (using a chiller, WAtronix).
To achieve complete three-dimensional imaging of small volumes, we scanned the objective in a trapezoidal pattern using a piezo-scanner (Physik Instrumente, P-721.LLQ; 48 μm displacement, 3.5 μm/plane; Figure 1B) controlled by a PC computer running custom-written Labview software, synchronized to output triggers sent from the two-photon acquisition computer. Image acquisition was controlled by a modified version of ScanImage (Pologruto et al., 2003 (link)) and MATLAB (The MathWorks). To maintain constant fluorescence excitation across multiple depths and to avoid passing laser power during the two ‘flyback’ frames, we often modulated laser power with a Pockels cell (e.g., between 16 and 13 mW in Figure 6), which was controlled by the MATLAB program.