3D-SIM Imaging of Brain Sections

SIM analysis of brain sections was performed on a Nikon SIM system. Tissues were imaged at laser excitation of 488 (for A11), 561 (for CD68) nm with a 3D-SIM acquisition protocol. Sixteen-bit images sized 1024×1024 pixels with a single pixel of 0.030 µm were acquired in a gray-level range of 0–16000 to exploit the linear range of the camera (iXon ultra DU-897U, Andor) and to avoid saturation. Three-dimensional Z-stacks were scanned with a 0.125 µm step size over 2–3 µm. Raw and reconstructed images were validated with the SIM-check plugin of ImageJ [59 (link)] and only those providing satisfactory image diagnosis were included in the study. Images were finally elaborated with GIMP (Gnu Image Manipulation Program).