Fig 1.
Principle of standard UM consisting of a slit aperture and a single cylindrical lens on each illumination side. The transparent brain is illuminated perpendicular to the observation pathway by a laser forming a thin sheet of light. The emitted fluorescence light is projected to a camera target by an objective, while a matched optical filter blocks the excitation light. By moving the specimen through the light sheet a stack of images is recorded. From these data a 3D-reconstruction is calculated by software. 1) Optical system (OS), which generates a magnified parallel beam. This beam is divided into two equal parts using a beam splitter. 2) Rectangular-slit aperture, 3) cylindrical lens, 4) clamp, 5) brain, 6) Sy: computer-controlled elevation (y-direction), 7) Sz: computer-controlled linear stage (z-direction), 8) immersion cap, 9) objective, 10) tube lens(es), 11) camera target, 12) computer with imaging software, 13) computer with 3D reconstruction software.
Fig 2.
UM´s example images of APPPS1.
Cross sections in the orthogonal directions are used to analyze the β-amyloid plaque distribution in the entire mouse brain of APPPS1. The β-amyloid plaques appear as bright dots in the neocortex (example of an animal of the young group is shown). A) Transversal plane (x,y); B) transversal plane (x,y); C) Computed sagittal plane (yz); D) computed coronal plane (xz). ctx: cortex; cb: cerebellum; hc: hippocampus, bs: brainstem; ob: olfactory bulb.
Fig 3.
Comparison between a young (2.3-month-old) and adult (7.5-month-old) APPPS1 tg brain.
3D reconstructions demonstrate an overall age-related increase in β-amyloid plaques. The maximum intensity projections of reconstructed images show a higher plaque density with increasing age. Brain from a mouse at 2.3 months of age viewed from the top (A), from the right side (B) and left side of (C). D) Brain from a mouse at 7.5 months of age viewed from the top (D), from the right side (E) of and left side (F). Scale bar: 1mm.
Fig 4.
Location of measured test-cubes.
β-Amyloid plaques (yellow dots) in the right hemisphere of the frontal cortex in the APPPS1 mouse model, side view: example from the young (2.7 month-old) group (A) and the adult (7.8 month-old) group (B). Positioning of the six cubed-shaped areas (purple color) in the frontal cortex for measuring the β-amyloid plaque volumes by applying a threshold segmentation technique. C-D) Top view of the frontal cortex: example from the young group (C) and old group (D). After segmentation amyloid plaque volumes of the six cubed shaped areas are represented in various colors. Scale bar 500 μm.
Fig 5.
Quantification of the number of amyloid plaques per mm3.
The total β-amyloid plaque number per mm3 was obtained from six sample cubes, acquired within the frontal cortex of young (2.5 months) and adult (7–8.5 months) APPPS1 tg mice. The total number of plaque per mm3 increases with age.
Fig 6.
Quantification of the 3D β-amyloid plaque load.
The β-amyloid plaque load (volume %) was obtained from six sample cubes, acquired within the frontal cortex of young (2.5 months) and adult (7–8.5 months) APPPS1 tg mice. The β-amyloid plaque load in the adult group is significantly higher compared to the young group (p<0.001, t-test).
Fig 7.
Histograms of plaque diameters.
The plaque sizes were binned into size groups of 10 μm for the young and the adult mice. In total, the number of β-amyloid plaques in all size classes was increased in the adult group. All plaques within the young group and the adult group, respectively were counted for histogram generation.
Fig 8.
Variations in the fraction of plaques for different diameters.
In the β-amyloid plaque diameter categories 40–50μm plaques were relatively more frequent in the adult animals. However, in the diameter group of 20–30 μm plaques were relatively more frequent in the young animals. For the β-amyloid plaque diameter categories 0–20 μm and 30–40 μm no significant differences could be shown. It cannot by excluded that plaques > 50 μm may be due to agglomeration of smaller plaques or artefacts.
Table 1.
Determined Plaque diameters compared to a previous study.