[Paper Review] Multimodal microscopy for characterization of amyloid-${\unicode[Times]{x3B2}}$ plaques biomarkers in animal model of Alzheimer's disease

Renan Cunha, Lucas Lafetá+6 more|arXiv (Cornell University)|Feb 7, 2021

Spectroscopy Techniques in Biomedical and Chemical Research56 references21 citations

TL;DR

This study introduces a label-free multimodal microscopy approach combining two-photon excitation autofluorescence (TPEA), second harmonic generation (SHG), spontaneous Raman scattering (SpRS), coherent anti-Stokes Raman scattering (CARS), and stimulated Raman scattering (SRS) to characterize amyloid-β (Aβ) plaques in an Alzheimer’s disease mouse model. The key contribution is the identification of phenylalanine and amide B as novel, high-resolution SRS biomarkers for the Aβ plaque core, with amide B enabling rapid imaging and strong potential for diagnostic applications due to its distinct spatial correlation with the plaque core.

ABSTRACT

Given the long subclinical stage of Alzheimer's disease (AD), the study of biomarkers is relevant both for early diagnosis and the fundamental understanding of the pathophysiology of AD. Biomarkers provided by amyloid-${\unicode[Times]{x3B2}}$ (A${\unicode[Times]{x3B2}}$) plaques have led to an increasing interest in characterizing this hallmark of AD due to its promising potential. In this work, we characterize A${\unicode[Times]{x3B2}}$ plaques by label-free multimodal imaging: we combine two-photon excitation autofluorescence (TPEA), second harmonic generation (SHG), spontaneous Raman scattering (SpRS), coherent anti-Stokes Raman scattering (CARS), and stimulated Raman scattering (SRS) to describe and compare high-resolution images of A${\unicode[Times]{x3B2}}$ plaques in brain tissues of an AD mouse model. Comparing single-laser techniques images, we discuss the origin of the SHG, which can be used to locate the plaque core reliably. We study both the core and the halo with vibrational microscopy and compare SpRS and SRS microscopies for different frequencies. We also combine SpRS spectroscopy with SRS microscopy and present two core biomarkers unexplored with SRS microscopy: phenylalanine and amide B. We provide high-resolution SRS images with the spatial distribution of these biomarkers in the plaque and compared them with images of the amide I distribution. The obtained spatial correlation corroborates the feasibility of these biomarkers in the study of A${\unicode[Times]{x3B2}}$ plaques. Furthermore, since amide B enables rapid imaging, we discuss its potential as a novel fingerprint for diagnostic applications.

Motivation & Objective

• To overcome limitations of label-based imaging by developing a label-free multimodal approach for high-resolution characterization of Aβ plaques in Alzheimer’s disease.
• To identify and validate novel endogenous vibrational biomarkers in the core and halo regions of Aβ plaques using multimodal optical techniques.
• To compare the spatial distribution of established (amide I) and newly identified (phenylalanine, amide B) biomarkers for improved plaque delineation.
• To evaluate the feasibility of amide B as a rapid imaging fingerprint for potential diagnostic applications.
• To validate label-free techniques against the gold standard TPEF-ThioS staining for plaque identification.

Proposed method

• Combined TPEA, SHG, SpRS, CARS, and SRS microscopy on brain tissue sections from an Alzheimer’s disease mouse model.
• Used 810 nm laser for TPEA and SHG; 532 nm CW laser for SpRS; and tunable OPO-based pump and Stokes beams for SRS and CARS.
• Performed spectral analysis and image correlation between SpRS and SRS at various vibrational frequencies to identify biomarkers.
• Subtracted SHG contribution from TPEA to isolate autofluorescence signals.
• Validated all label-free images against TPEF-ThioS staining as the gold standard for plaque localization.
• Employed lock-in amplification and polarization modulation to enhance SRS signal-to-noise ratio.

Experimental results

Research questions

• RQ1 What is the origin of the SHG signal in Aβ plaques, and can it reliably localize the plaque core?
• RQ2 How do the spatial distributions of phenylalanine and amide B compare with amide I in the Aβ plaque core?
• RQ3 Can SpRS and SRS yield comparable vibrational images of Aβ plaques across different scattering geometries?
• RQ4 Do unsaturated lipid vibrations in the halo region correlate with other vibrational modes in the plaque?
• RQ5 Can amide B serve as a rapid, high-contrast biomarker for Aβ plaque imaging in diagnostic applications?

Key findings

• SHG signal originates from the β-sheet-rich core of Aβ plaques and provides reliable core localization, validated against TPEF-ThioS staining.
• Phenylalanine, a previously unexplored biomarker in SRS microscopy, shows strong spatial specificity to the Aβ plaque core, with high correlation to amide I.
• Amide B vibrational mode exhibits a distinct spatial distribution in the plaque core and enables rapid imaging due to its high Raman cross-section.
• SRS and SpRS images show nearly identical correspondence across different vibrational frequencies and scattering geometries, confirming consistency of the technique.
• Unsaturated lipid vibrations in the halo region show strong spatial correlation with other lipid modes, supporting the multimodal characterization of plaque microenvironment.
• The spatial correlation between amide B and the plaque core, combined with its rapid imaging capability, establishes amide B as a promising novel fingerprint for diagnostic applications.