Plaque-associated lipids in Alzheimer's diseased brain tissue visualized by nonlinear microscopy

Abstract

By simultaneous coherent anti-Stokes Raman scattering (CARS) and 2-photon fluorescence microscopy of Thioflavin-S stained Alzheimer´s diseased human brain tissues, we show evidence of lipid deposits co-localizing with fibrillar β-amyloid (Aβ) plaques. Two lipid morphologies can be observed; lamellar structures and coalescing macro-aggregates of sub-micron sizes to ~25 μm. No significant lipid deposits were observed in non-fibrillar, diffuse plaques identified by Aβ immuno-staining. CARS microscopy of unlabeled samples confirms the lamellar and macro-aggregate lipid morphologies. The composition of the plaques was analyzed by CARS microspectroscopy and Raman microscopy; vibrational signatures of lipids with long acyl chains co-localize with the β-sheet vibrations. The lipid fluidity was evaluated from the CARS spectra, illustrating that the lipid composition/organization varies throughout the plaques. Altogether this indicates close amyloid-lipid interplay in fibrillar Aβ plaques, rendering them more dynamic compositions than previously believed and, hence, potential sources of toxic oligomers.

Figure 1. Visualization of Aβ and lipid distributions in core plaques by fluorescence/CARS microscopy.

(a–c) Thioflavin-S fluorescence; (d–f) CARS images at 2840 cm⁻¹; (g–i) profile plots along the lines indicated in (a,d), (b,e) and (c,f) respectively; (j) a 3D rendering of a CARS microscopy image (yellow) of lipids distributed in the AD plaque region, visualizing the different morphologies: crystalline needle-like structures (left) and more diffusely distributed (right); (k) z-stack representation of the CARS signal from the plaque in (d). Scale bars, 25 μm.

Figure 2. Visualization of the heterogeneity in lipid aggregates composition in AD brain tissue.

(a) CARS (2840 cm⁻¹) image of lipid aggregate; (b) CARS spectra collected at positions marked by symbols in c, curves are displayed with an offset for clarity; (c) CARS ratio image, intensities at 2840 cm⁻¹ vs. 2870 cm⁻¹, representing variations in lipid fluidity; (d) profile plots along the lines indicated in (a,c). Scale bars, 25 μm.

Figure 3. Raman spectra from lipid aggregates in human AD brain tissue.

(a) Raman spectra in the finger print region highlighting the Amide I band; (b) Zoom-in on the Amide I band, from (a). Spectra were collected in the center and on the border of lipid aggregate, as well as from the tissue outside the lipid aggregate.

Figure 4. Differences in lipid aggregate morphology found in human AD brain tissues investigated by CARS microscopy (2840 cm⁻¹) including two main categories; lamellar structures (e.g. in a, e and g) and coalescent structures of different sizes, possibly originating from lipid micelles initially decorating the Aβ fibrils or ApoE particles, alternatively from lipid microvesicles shedded by microglia (see discussion).

(a–d) Patient №1; (e–h) patient №2. Scale bars, 25 μm. Note the different scales in the images.