Figure 3
STED microscopy in different cortical layers using Lifeact and Actin-Chromobody to highlight filamentous actin. (a) Lifeact-mNeptune2 expressed in a superficial neuron in layer 1 (L1) shows a bright actin label while the surrounding spines are rather dark (# indicates a cell body). Apical dendrite of layer 5 which passes layer 4 (L4) is rich in actin bundles in the dendritic shaft and spines. Layer 5 (L5) exhibits dendrites with densely packed dendritic spines. All images were recorded with the same settings and scaled to the same maximum and minimum photon counts for comparison of the fluorescence expression levels. White boxes show a widefield overview image with the area of measurement indicated by a star. (b) Actin-Chromobody fused to mNeptune2 is highly expressed in rare neurons of L1 and dark in surrounding spines (left). STED microscopy reveals bright apical dendrites in L4 (middle) and densely packed dendritic spines in L5 (right). (c,d) Magnified view of the yellow boxed area (a,b respectively), showing the superresolved spine morphology in greater detail. (e) Magnification of the overview images in (a), showing an overlap of the bright-field (grey) and fluorescence wide-field (red) microscopy image.
