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Showing content from https://pubmed.ncbi.nlm.nih.gov/33859370/ below:

Multimodal, label-free fluorescence and Raman imaging of amyloid deposits in snap-frozen Alzheimer's disease human brain tissue

doi: 10.1038/s42003-021-01981-x. Multimodal, label-free fluorescence and Raman imaging of amyloid deposits in snap-frozen Alzheimer's disease human brain tissue

Affiliations

¹ Department of Physics and Astronomy, LaserLaB Amsterdam, VU Amsterdam, Amsterdam, The Netherlands. ben.lochocki@vu.nl.
² Department of Pathology, Amsterdam Neuroscience, Amsterdam UMC-location VUmc, Amsterdam, The Netherlands.
³ Department of Physics and Astronomy, LaserLaB Amsterdam, VU Amsterdam, Amsterdam, The Netherlands.

PMID: 33859370
PMCID: PMC8050064
DOI: 10.1038/s42003-021-01981-x

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Multimodal, label-free fluorescence and Raman imaging of amyloid deposits in snap-frozen Alzheimer's disease human brain tissue

Benjamin Lochocki et al. Commun Biol. 2021.

doi: 10.1038/s42003-021-01981-x. Affiliations

¹ Department of Physics and Astronomy, LaserLaB Amsterdam, VU Amsterdam, Amsterdam, The Netherlands. ben.lochocki@vu.nl.
² Department of Pathology, Amsterdam Neuroscience, Amsterdam UMC-location VUmc, Amsterdam, The Netherlands.
³ Department of Physics and Astronomy, LaserLaB Amsterdam, VU Amsterdam, Amsterdam, The Netherlands.

PMID: 33859370
PMCID: PMC8050064
DOI: 10.1038/s42003-021-01981-x

Item in Clipboard

Abstract

Alzheimer's disease (AD) neuropathology is characterized by hyperphosphorylated tau containing neurofibrillary tangles and amyloid-beta (Aβ) plaques. Normally these hallmarks are studied by (immuno-) histological techniques requiring chemical pretreatment and indirect labelling. Label-free imaging enables one to visualize normal tissue and pathology in its native form. Therefore, these techniques could contribute to a better understanding of the disease. Here, we present a comprehensive study of high-resolution fluorescence imaging (before and after staining) and spectroscopic modalities (Raman mapping under pre-resonance conditions and stimulated Raman scattering (SRS)) of amyloid deposits in snap-frozen AD human brain tissue. We performed fluorescence and spectroscopic imaging and subsequent thioflavin-S staining of the same tissue slices to provide direct confirmation of plaque location and correlation of spectroscopic biomarkers with plaque morphology; differences were observed between cored and fibrillar plaques. The SRS results showed a protein peak shift towards the β-sheet structure in cored amyloid deposits. In the Raman maps recorded with 532 nm excitation we identified the presence of carotenoids as a unique marker to differentiate between a cored amyloid plaque area versus a non-plaque area without prior knowledge of their location. The observed presence of carotenoids suggests a distinct neuroinflammatory response to misfolded protein accumulations.

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Conflict of interest statement

The authors declare the following competing interests. Heidelberg Engineering was part of the consortium funding the research (I-READ).

Figures
Fig. 1. Experimental work flow.

Brain autopsy…

Fig. 1. Experimental work flow.

Brain autopsy is performed within 9 h post-mortem and the…

Fig. 1. Experimental work flow.

Brain autopsy is performed within 9 h post-mortem and the tissue is immediately snap-frozen to −80 °C. Next, the tissue is cut at 20 µm and mounted on a CaF₂ microscope slides. Afterwards, the section is imaged under the fluorescence microscope. Subsequently, Raman maps are recorded. Hereafter, the tissue is measured across the amide-I protein peak using the SRS microscope. Following SRS, the tissue section is stained with thioflavin-S and imaged under the fluorescence microscope for plaque confirmation. Finally, the data are processed and analyzed.

Fig. 2. Fluorescence images of AD cases…

Fig. 2. Fluorescence images of AD cases 1 and 2, before and after staining, at…
Fig. 2. Fluorescence images of AD cases 1 and 2, before and after staining, at ×20 and ×40 magnification.

a Auto-fluorescence images of freshly cut tissue. Greenish dense cores (#1a and #1b) and greenish patches (#2) are clearly visible. The uniformly distributed yellow spots (blue arrows, just one is identified in each image) are lipofuscin deposits. b Fluorescence images of the same tissue sections and areas stained with thioflavin-S. Here, the yellow bright spot areas are the positively stained amyloid accumulations. All images were taken with the full-field fluorescence microscope and an illumination source of 470 nm. Yellow and red dashed boxes mark the measured plaque areas. # refers to AD donor number and plaque in Table 1. Scale bars: 100 µm. The ×40 image pairs of the red dashed boxes are shown in the Supplementary Information, Fig. S2. Enlarged images of each plaques are shown in Fig. 3. The image pairs of cases 3–5 are shown in Fig. S3.

Fig. 3. Side-by-side comparison of plaque locations…

Fig. 3. Side-by-side comparison of plaque locations (as indicated by the yellow dashed boxes in…
Fig. 3. Side-by-side comparison of plaque locations (as indicated by the yellow dashed boxes in Fig. 2 for cases #1a, #1b, and 2) of auto-fluorescence images and the same location after staining with thioflavin-S.

The blue arrows point to lipofuscin deposits, which are not visible in the thioflavin-S stained images since the fluorescence of thioflavin-S is stronger. Scale bars: 40 µm.

Fig. 4. Fluorescence emission spectra of plaque…

Fig. 4. Fluorescence emission spectra of plaque areas excited with a 488 nm source.

Top…

Fig. 4. Fluorescence emission spectra of plaque areas excited with a 488 nm source.

Top row: cored amyloid deposit. Bottom: fibrillary amyloid deposit. Symbols connected with a dashed line are the measured data. The solid lines are the corresponding third-order polynomial fits; peak position and R² values are indicated. The right half shows the corresponding z-projection of the emission images and the locations where the data points for plaque (P), lipofuscin (L), and background were taken, next to it the corresponding full-field auto-fluorescence images. Corresponding thioflavin-S images are not shown. Scale bar: 40 µm.

Fig. 5. Overview of fluorescence and Raman…

Fig. 5. Overview of fluorescence and Raman images.

Each row represents one tissue section. First…

Fig. 5. Overview of fluorescence and Raman images.

Each row represents one tissue section. First column: Auto-fluorescence images of the presumed plaque (green) areas. Second column: The total intensity image of the spectral Raman data after data pre-processing. Third and fourth column: Raman peak intensity images of the protein (1666 cm⁻¹) and lipid (1445 cm⁻¹) bands. Fifth and sixth column: Raman peak intensity images at two prominent carotenoid wavenumbers (1518 and 1154 cm⁻¹). Seventh column: four-cluster images of the Raman data after MCR-ALS computing, highlighting the plaque locations in orange (#1) and red (#2 and #4c). Last column (eighth): Fluorescence image of the thioflavin-S stained tissue, confirming anticipated plaque locations by yellow fluorescence. Note for cored plaques #1a, #1b, #3a, #3c, and #5 how well the carotenoid peak images and the cluster analysis match the stained plaque depositions. Scale bars: 40 µm; color coding for columns 2 to 6: blue (low) to yellow (high).

Fig. 6. Four-cluster images of all plaques…

Fig. 6. Four-cluster images of all plaques and their corresponding spectra after complete data processing.
Fig. 6. Four-cluster images of all plaques and their corresponding spectra after complete data processing.

The orange solid lines, obtained from the cored plaques and from fibrillar plaque #3b, exhibit a distinguishable spectrum with clear carotenoid-associated peaks. However, in plaques #2 and #4 (a–c) no carotenoid peaks were detectable. The red area, which largely overlaps with the later confirmed plaque location, corresponds to the spectrum (red line) on the right which is not associated with carotenoids. (The whole recorded spectra of #1a including the CH-stretch range can be found in the Supplementary Information, Fig. S11.) Scale bars: 40 µm.

Fig. 7. SRS images obtained while scanning…

Fig. 7. SRS images obtained while scanning across the protein peak for each plaque.

Auto-fluorescence…

Fig. 7. SRS images obtained while scanning across the protein peak for each plaque.

Auto-fluorescence images overlaid on SRS max. intensity projection images (@ 1666 cm⁻¹) with the plaque locations shown as green spots. Next to the images are the SRS response graphs of averaged sweeps across the protein peak per plaque. A clear shift from the “normal” 1659 cm⁻¹ protein peak towards the beta-sheet peak at 1666 cm⁻¹ is only observed in plaques featuring a dense cored plaque. This shift was not observed in the plaque #2 and less visible in #1a (which was measured with bigger step sizes). The yellow circles indicate areas where data were taken for the background and the red circles indicate the areas for plaques. Scale bar: 50 µm.