Two-Photon Imaging of Microglial Dynamics

Live imaging of microglial processes was performed on 250 µm coronal brain slices from Nf1 ± or Nf1flox/wt mice and their WT littermates using a custom-built two-photon laser-scanning microscope (Till Photonics, Gräfelfing, Germany). EGFP or eYFP was excited by a Chameleon Ultra II laser (Coherent, Dieburg, Germany) at a wavelength of 940 nm. A 40X water-immersion objective (NA 0.8, Olympus, Hamburg, Germany) was used, with scanned 60 µm thick z-stacks and a step size of 3 µm covering a field of 320 × 320 µm. Laser lesions were set to 40 µm under the slice surface in the cortex by focusing the laser beam, set to a wavelength of 810 nm and to maximum power in the selected imaging volume, and scanned until autofluorescence of the injured tissue was visible. This procedure resulted in lesions of ~ 20 µm in diameter in the middle of the observed region. For the recording of microglia surveillance, no laser lesion was performed. IGOR Pro 6.37 (Lake Oswego, USA) was used for data analysis as in Davalos et al. [13 (link)] and Madry et al. [42 (link)]. The sequences of 3D image stacks were converted into sequences of 2D images by a maximum intensity projection algorithm. Grayscale images were first converted into binary form using a threshold. For quantification of laser lesion-induced movements, microglial response to focal lesion was defined as EGFP + pixel count in a proximal circular region 45 µm around the lesion site over time (Rx(t)). Distal fluorescence of the first time point was determined within a diameter of 45 µm to 90 µm around the lesion site for normalization (Ry(0)). Microglial responses were represented as R(t) = (Rx(t)-Rx(0))/Ry(0). For the quantification of baseline surveillance, cells of interest were individually selected by manually drawing a region of interest (ROI) around an area including all their process extensions throughout the 20 min movie and erasing data around that ROI. Starting with the second frame, we subtracted from each binarized frame the preceding frame and counted the number of pixels < 0 (retracting = PR) and > 0 (extending = PE). The surveillance index for each frame is then given by the sum of PR ad PE. The surveillance index of a given cell was then calculated by averaging the indices of the first 20 images in the movie. For ramification index (RI), we used the equation RI = (peri/area)/(2*sqrt(pi/area)), where peri and area are respectively the perimeter and area of a given cell in pixels. For the quantification of these two parameters, the ImageAnalyzeParticles operation in IGOR Pro 6.37 was applied on binarized images in which all analyzed microglia were manually examined and, if necessary, somata and processes connected.