Article Figures & Data
Figures
- Fig. 1.
Preparation of skull for in vivoimaging. a, Gross appearance of skull through dissecting microscope before imaging. The pial vasculature is visible through the intact but thinned region of skull. Anterior midline sutures are also visible in the image. Scale marks are spaced 1 mm apart.b, Schematic diagram of the microscope objective during imaging. The thinned area of skull is bathed in a pool of ACSF (light gray) that is retained by a ring of bone wax (dark gray). A small break is made in the lateral wall of the thinned area to allow for thioflavine S entry. c,In vivo visualization of thioflavine S-positive amyloid in a 15-month-old Tg2576 mouse. A single optical section near the surface of the skull is shown. Thioflavine S-positive amyloid angiopathy is visible ringing the pial arteriole in this image. The fainter autofluorescence of the skull bone is visible in thebottomrightcorner; the fibrous autofluorescence of the dura is visible as aband at bottom right. d, Another optical section from the same z-series asc, but 50 μm deeper into the brain, showing a thioflavine S-positive amyloid deposit in layer 1 of the mouse cortex.e, Perpendicular volume rendering of the entire stack of images, with the skull visible at the top, the amyloid-encrusted pial vessel just beneath, and the thioflavine S-positive plaque deep in the living brain. The autofluorescent dura can also be seen as a faint layer between the vessel and the skull. The approximate levels of optical sections shown in c andd are represented by dotted lines. Scale bars, 25 μm.
- Fig. 2.
To confirm that the thioflavine S-positive structures were indeed senile plaques, thioflavine S and an anti-amyloid-β monoclonal antibody, cy3-labeled 10D5 (Elan Pharmaceuticals), were applied to the surface of a fixed but intact Tg2576 brain. a, Fluorescence emission in the range 380–480 nm shows thioflavine S staining the amyloid core of a plaque ∼40 μm deep into the brain. Scale bar, 10 μm.b, Emission in the 560–650 nm range shows the Cy3–10D5 staining of the same Aβ surrounding the thioflavine S-positive core.c, Glial fibrillary acidic protein immunoreactivity in a section through the area imaged by multiphoton microscopy 2 d previously. Sparse immunoreactive astrocytes, not substantially different from adjacent (nonimaged) cortex, suggest minimal tissue response to imaging. Scale bar, 100 μm.
- Fig. 3.
In vivo imaging of thioflavine S-positive amyloid deposition in a Tg2576 mouse. a, A 3 × 3 montage of 60× fields acquired on initial imaging day. Optical sections were obtained every 2 μm for a distance of 200 μm from the inner surface of the skull; images were aligned in thex, y, and z axes, then projected onto a single image revealing amyloid angiopathy and senile plaques. b, In vivo imaging of a thioflavine S-positive plaque ∼40 μm beneath the skull surface. This image is a single optical section through the body of the plaque. Scale bar, 10 μm. c, The same plaque as inb, re-imaged 2 d later under identical imaging conditions. d, Single optical section showing thioflavine S-positive amyloid angiopathy associated with a pial arteriole. e, The same arteriole as d, imaged after 2 d.
- Fig. 4.
Sequential imaging of a plaque over time. Images of a single, identified plaque were obtained at the initial imaging session and again 45 and 110 d later in a live mouse. Fine details of the plaque are clearly recognizable over these ranges of time. Scale bar, 20 μm.
- Fig. 5.
Analysis of variability of plaque measurements. Top, Percentage change (average ± SD) for all plaque measurements binned into 0.5 month groups shows no trend in either the average measure or the variability of measurement over the time interval examined. N values for each measurement are noted above the SD bars.Bottom, Linear regression plot of initial measurement and subsequent measurement for all time intervals, showing tight correlation for all plaque sizes. The slope of the line approaches unity (0.98) with a correlation coefficientR2 = 0.89.
- Fig. 6.
A subpopulation of plaques change size over time. The images are two-channel volume-rendered stacks of thioflavine S plaques (red) and fluorescein angiograms (green) taken from the same animal at the initial imaging session (left images) and 104 d later (right images). Four clearly imaged plaques can be seen in these volumes, labeled A–D. The autofluorescence of the dura appears at the upper edge of the volume stacks and appears slightly different in the images here and in Figure6 because the image stacks are not exactly coincident at their initial depth. The graph below represents the percentage change in diameter for each plaque. The plaques labeled A andB increase in size by ∼50%, plaque Cremains the same size, and plaque D decreases by 40%. Scale bar, 20 μm.
- Fig. 7.
Appearance of a novel plaque in the imaged region.a, Volume rendering of a set of three plaques during an initial imaging session. b, Volume rendering of the same region imaged 64 d later, showing the initial plaques joined by a novel thioflavine S-positive plaque. The fibrous autofluorescence atbottom left is dura mater. Scale bar, 50 μm.
