3D OPT imaging of DCX and nestin stained mouse brain.

<p>3D optical imaging with OPT was used to image a single hemisphere stained with DCX (red) and nestin (blue) simultaneously. Optical slices through the tissue are shown to represent the 3D nature of the sample (A). The slice orientation is shown on a cartoon. A 3D representation shows the distribution of DCX- and nestin-stained cells along the surface of the lateral ventricle (B).</p

3D OPT imaging of GFAP staining in the mouse brain.

<p>3D optical imaging with OPT was used to image a GFAP-stained brain hemisphere. Optical slices of the brain are shown and GFAP-positive cells are seen throughout the brain (A). A cartoon shows the location of the slices. A 3D representation shows the distribution of GFAP staining along the corpus callosum (B).</p

3D serial two-photon tomography imaging of GFAP staining in the mouse brain.

<p>Serial two-photon tomography was used to image a single hemisphere stained with GFAP. Optical slices through the brain show the staining pattern, which is widespread throughout the brain. The slice location is shown in the cartoon. The grayscale inset shows the typical astrocyte morphology, which is comparable to the 2D histology section (colour inset).</p

Comparison of serial two-photon tomography in GFAP-stained brains and traditional immunohistochemistry in sections.

<p>Slices from a 3D serial two-photon tomography data set (A, C) are compared with traditional sections (B, D). Expanded images are shown for comparable regions (E, G, I and F, H, J). Rostral sections (A, B and E, F) show comparable results. Staining in the hippocampus is lighter in the 3D stained section (G) than in the traditional section (H). At the periphery, the 3D stain produces more intense GFAP signal with a higher background (I) than does the traditional section (J). Examples of lightly-stained cells are highlighted with arrowheads in (G) and (J). The arrow in (I) highlights an example of the characteristic astrocyte morphology. Maps of cell density are shown in (K, M). Rostral cell densities (K, L) are similar between the two methods. In the caudal regions (M, N), the density in the dentate gyrus (white arrowheads) is reduced in the 3D stain while it is appears increased in some regions of the cortex (asterisks). Cell density maps (K–N) are shown with black and white corresponding to <30 cells/mm² and >1200 cells/mm² respectively.</p

3D serial two-photon tomography imaging of DCX staining in the mouse brain.

<p>Serial two-photon tomography was used to image a single hemisphere stained with DCX. Five individual slices are shown, with the slice location shown in the cartoon at lower right. For each slice, the grayscale image shows autofluorescence for anatomical reference and the color overlay shows the DCX staining. Neuronal processes can be distinguished (DCX in grayscale insets i, ii; arrowhead, inset i). The staining pattern is comparable to traditional 2D sections (insets with red color, iv and v), although with lower contrast to background signal.</p

3D serial two-photon tomography imaging of nestin staining in the mouse brain.

<p>Serial two-photon tomography was used to image a single brain hemisphere stained with nestin. The slice locations are shown in the cartoon. The staining pattern (grayscale inset) is comparable to the 2D histology section (color inset).</p

Validation of DCX staining.

<p>A traditional 2D stain of DCX (A) shows a reference distribution of migrating neuroblasts (red). The 3D staining method in a brain hemisphere (B) shows a comparable distribution (arrows, A and B), with the exception of some additional punctate fluorescence (arrowheads, B). Cryosections prepared from the 3D stained hemisphere were re-stained with another secondary antibody (green; C) to highlight primary antibody left unbound by secondary during the 3D stain; the primary antibody is bound by secondary in the 3D staining method (arrows). In (D), an additional cryosection from the same brain as (C) was re-stained with both primary and secondary antibody to highlight antigen binding sites not occupied by primary. Overlap of the 3D-stained and re-stained cells (arrowheads, D), indicate that no DCX cells have been missed in the 3D-stained images. LV = lateral ventricle, scalebars = 100 µm</p

3D OPT imaging of nestin staining in the mouse brain.

<p>3D optical imaging with OPT was used to image a nestin-stained brain hemisphere. Optical slices of the brain are shown and nestin-positive cells are visible surrounding the lateral ventricle. A cartoon shows the location of the slices.</p

3D OPT imaging of DCX staining in the mouse brain.

<p>3D OPT of a DCX-stained single hemisphere are shown as optical slices. DCX staining is visible surrounding the lateral ventricle (A; i–iii) and in the subgranular zone of the dentate gyrus (A; iv). A cartoon shows the location of the slices. The chains of DCX migrating neuroblasts along the lateral ventricle can be visualized on the ventricle surface in 3D (B). In (C), a maximum intensity projection of a full brain shows cells migrating along the RMS to the olfactory bulbs (shaded green).</p

Validation of 3D nestin staining.

<p>Cryosections cut from a stained brain hemisphere show nestin-positive cells (red; arrows, A). Some additional punctate fluorescence was present (arrowheads, A). The sections were re-probed with a secondary antibody (green; A) to highlight primary antibody left unbound by secondary antibody. No evidence of primary antibody left unbound by secondary antibody was found. Sections were also re-stained with both primary and secondary antibodies (B) to highlight primary-antibody antigen sites unoccupied. There is overlap of the original 3D nestin stain with the 2D re-stain (arrowheads). LV = Lateral ventricle, scalebars = 100 µm.</p