Label-free characterization of Amyloid-β-plaques and associated lipids in brain tissues using stimulated Raman scattering microscopy
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Abstract
The brains of patients with neurodegenerative diseases such as Alzheimer’s Disease (AD) often exhibit pathological alterations that involve abnormal aggregations of proteins and lipids. Here, we demonstrate that high-resolution, label-free, chemically-specific imaging using Stimulated Raman Scattering Microscopy (SRS) provides novel insights into the biophysical properties and biochemical composition of such pathological structures. In brain slices of a mouse model of AD, SRS reveals large numbers of Amyloid-β plaques that commonly form a characteristic, three-dimensional core-shell structure, with a fibrillar proteinaceous core surrounded by a halo-like shell of lipid-rich deposits. SRS spectroscopic imaging allows for a clean, label-free visualization of the misfolded (β-sheet) Amyloid-β content in the plaque core. Surrounding lipid-rich deposits are found to contain comparatively high concentrations of membrane lipids (sphingomyelin, phosphatidylcholine), but lower levels of cholesterol than healthy white matter structures. Overall, the SRS spectra of plaque-associated lipids closely resemble those of nearby neurites, with the notable difference of a higher degree of lipid unsaturation compared to healthy brain structures. We hypothesize that plaque-associated lipid deposits may result from neuritic dystrophy associated with AD, and that the observed increased levels of unsaturation could help identify the kinds of pathological alterations taking place. Taken together, our results highlight the potential of Stimulated Raman Scattering microscopy to contribute to a deeper understanding of neurodegenerative diseases.
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There were no signs of sample damage.
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Optical configuration: 20x NA=0.75 W HC PL IRAPO objective, NA=1.4 oil condenser. Field of view: 450 x 450 micrometers, 2072x2072 pixels (216 nm pixel size). Acquisition time per pixel: 3.5 microseconds (achieved with 400 Hz unidirectional scanning, with 10x frame averaging). Power at the sample: High wavenumber region: ~20 milliwatts Pump, 40 milliwatts Stokes (Laser output: 150mW Pump, 300mW Stokes; half-wave plate transmission: 40%; estimated 33% system transmission to sample). Fingerprint region: ~50 milliwatts Pump, 100 milliwatts Stokes (Laser output: 150mW Pump, 300mW Stokes; half-wave plate transmission: 100%; estimated 33% system transmission to sample).
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SRS and CARS Microscopy
Was there any concern that the scanning would cause damage to the samples (e.g. through heating or photodamage), and how were these concerns mitigated?
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SRS images are acquired sequentially at a range of wavenumbers, as in the representative images shown in
What parameters (power, dwell time, scan duration) were used for this acquisition?
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