Selected aspects of thinned and open skull preparations. (A) Example image showing a maximum-intensity side projection of eGFP-positive cells in the neocortex of a heterozygous Cx3cr1-eGFP mouse. Images were acquired through the thinned, autofluorescent skull (visible on top). Note that microglia somata (closed arrowheads) and their fine processes can be resolved clearly in layer 1 (L1) and parts of layer 2/3 (L2/3). Image quality degrades rapidly with depth. (B) (Left) Fluorescence images showing a transcranially imaged microglial cell in L1. (Right) The same microglial cell after skull removal. Fine processes can be resolved in both cases. (C) Example image showing a maximum-intensity side projection of enhanced yellow fluorescent protein (eYFP)-expressing neurons (red, open arrowheads) and eGFP-positive microglia (green, closed arrowheads) in the neocortex of a Thy1-eYFP × Cx3cr1-eGFP mouse implanted with an open skull window. Note increased depth penetration and spatial resolution compared to (A). (D) (Left) Side projection showing neuronal dendrites (red) and microglial cells (green) in the neocortex of a Thy1-eYFP × Cx3cr1-eGFP mouse immediately after thinned skull preparation. (Right) The same tissue volume imaged 1 d later without skull rethinning. Improper skull thinning can lead to inflammation in regions near the skull surface (yellow dashed lines). (A,B, Modified from supplementary online material from Nimmerjahn et al. 2005.)