Fluoview confocal scan head

A custom two-photon laser-scanning microscope was used for in vivo imaging (Ti: Sapphire, Mai-Tai, Spectraphysics; modified Fluoview confocal scan head, 20x water immersion objective lens, 0.95 numerical aperture, Olympus, Center Valley, PA). Excitation for fluorescent imaging was achieved with 100-fs laser pulses (80MHz) tuned to 920 nm for GFP with a power of ~30–40 mW measured at the sample. Fluorescence was detected using a photomultiplier tube in whole-field detection mode using a 580/180 filter. Images were collected from 20 μm to 300 μm into the brain. For repeated imaging, blood vessels were used as gross landmarks and stable microglia were also used as fine landmarks to re-identify the correct region for imaging. Image analysis was done offline using ImageJ and Matlab with custom algorithms as described in Stowell et al., 2019a (link) and available at https://github.com/majewska-lab (Stowell et al., 2019b ).

A custom two-photon laser-scanning microscope was used for intravital imaging (Ti:Sapphire, Mai-Tai, Spectraphysics; modified Fluoview confocal scan head, × 20 lens, 0.95 numerical aperture, Olympus). Two-photon excitation was achieved using 100-fsec laser pulses (80 MHz) tuned to 840 nm with a power of ∼ 50 mW measured at the sample after the objective lens. For all experiments, a 565-nm dichroic mirror was used in conjunction with 580/180 (GFP) and 605/55 (rhodamine B) filters. Rhodamine B dextran (2% w/v, 75 μL total) was injected retro-orbitally after ketamine (100 mg/kg)/xylazine (10 mg/kg) anesthesia and 5 min prior to intravital imaging. Mice were maintained at 37 °C during intravital imaging and recovery. Importantly, intravital imaging sessions were limited to 40 min per mouse to avoid re-exposure to ketamine/xylazine anesthesia. Intravital imaging was performed using × 4 digital zoom and at a 512 × 512-pixel resolution. Stacks containing 41 slices (1 μm step size) were imaged every 90 s for 15 min to obtain the XYZT stacks used for analysis.

Immediately after surgery and while still anesthetized, the L7^cre/Ai9^+/−/Cx3cr1^G/+ mice were affixed to an imaging stage. A custom two-photon laser-scanning microscope was used for in vivo imaging of microglia dynamics, morphology, and Purkinje cell interactions (Ti: Sapphire, Mai-Tai, Spectra Physics; modified Fluoview confocal scan head, 20X water-immersion objective, 0.95 numerical aperture, Olympus). Excitation was achieved with 100-fs laser pulses (80 MHz) at 920 nm with a power of ~30 mW measured at the sample. A 565 dichroic with 520/40 (GFP) and 598/30 (Ai9) filters was used to visualize microglia (GFP) and Purkinje cells (Ai9) in different channels simultaneously. Imaging of 101 μm z-stacks at a 1 μm z-step size with a 4x digital zoom with an 800 × 600 pixel frame size occurred with time-lapse imaging at 5-min intervals for 1 h per imaging session. All image analysis was run offline using Ilastik (Berg et al., 2019 (link); RRID:SCR_015246), NIH ImageJ¹ (RRID:SCR_003070) or FIJI (see footnote 1; RRID:SCR_002285), and MATLAB (MathWorks, version R2020a; RRID:SCR_001622). Some bleed-through of the Ai9 Purkinje cell fluorescence into the background of the GFP microglia channel occurred due to spectral overlap and bleed-through correction was performed in ImageJ/FIJI as described below.