In vivo two-photon brain microscopy.

An Ultima IV 2P microscope (Prairie Technologies/Bruker) equipped with a Mai Tai eHP DeepSee and an Insight X3 Ti:sapphire femtosecond laser (pulse width <120 fs, tuning range 690–1040 nm [Mai Tai] or 680–1300 nm [Insight X3], repetition rate 80 MHz; Spectra-Physics/Newport) was used for all in vivo imaging studies. PrairieView software (v5.4; Bruker) was used for microscope and laser control and image acquisition. The laser excitation wavelength was 910–1100 nm depending on the fluorophore(s). Imaging was performed ~170–200 μm below the dura mater in the whisker barrel cortex for microglial process extension and neuronal activity imaging in awake mice. Imaging was performed ~100–120 μm below the dura mater for dendritic spine imaging or 40–100 μm below the dura mater for microglial surveillance/motility imaging in anesthetized mice. A Nikon 40 × 0.8 NA or an Olympus 25 × 1.05 NA water-immersion lens was used depending on the experiment as specified below. The maximum laser power exiting the objective was ≤ 40 mW during all imaging experiments, except during point scan laser ablations. An IR-blocking filter and 560-nm dichroic were placed in the primary emission beam path before the non-descanned photomultiplier tubes (PMTs). A 660-nm dichroic and a 692/24-nm + 607/45-nm bandpass filter set were used to separate far red (e.g., SiR-actin probe) and red fluorescence emission (e.g., jRCaMP1b), respectively; a 520-nm dichroic and a 542/27-nm + 494/41-nm bandpass filter set separated YFP and GFP fluorescence emission, respectively. A GaAsP PMT (Hamamatsu H7422PA-40) was installed after the 607/45-nm bandpass filter; all other PMTs were of the multi-alkali type (Hamamatsu R3896).