Multimodal Imaging of Amyloid Plaques: Fusion of the Single-Probe Mass Spectrometry Image and Fluorescence Microscopy Image

Abstract

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. The formation of amyloid plaques by aggregated amyloid beta (Aβ) peptides is a primary event in AD pathology. Understanding the metabolomic features and related pathways is critical for studying plaque-related pathological events (e.g., cell death and neuron dysfunction). Mass spectrometry imaging (MSI), due to its high sensitivity and ability to obtain the spatial distribution of metabolites, has been applied to AD studies. However, limited studies of metabolites in amyloid plaques have been performed due to the drawbacks of the commonly used techniques such as matrix-assisted laser desorption/ionization MSI. In the current study, we obtained high spatial resolution (∼17 μm) MS images of the AD mouse brain using the Single-probe, a microscale sampling and ionization device, coupled to a mass spectrometer under ambient conditions. The adjacent slices were used to obtain fluorescence microscopy images to locate amyloid plaques. The MS image and the fluorescence microscopy image were fused to spatially correlate histological protein hallmarks with metabolomic features. The fused images produced significantly improved spatial resolution (∼5 μm), allowing for the determination of fine structures in MS images and metabolomic biomarkers representing amyloid plaques.

Figure 1.

Fluorescence microscopy images of brain slices of (A) 5xFAD and (B) control mice.

Figure 2.

Optical and MS images of FAD mouse brain. Optical image of (A) a coronal section of mouse brain and (B) the zoomed-in region containing the area for MSI measurement (enclosed in the red rectangle). MS images of (C) [PC(36:1) + H]⁺ and (D) [PC(38:1) + H]⁺) representing metabolites primarily distributed in the white matter. MS images of (E) [PC(38:4) + K]⁺ and (F) [PC(38:6) + K]⁺) representing metabolites primarily distributed in the gray matter. All metabolites were identified using MS² from the tissue slice, and the results were compared with METLIN (Table S4).

Figure 3.

Fusion of the fluorescence microscopy image and the MS image. (A) Fluorescence microscopy image of a 5XFAD mouse brain slice stained using Thioflavin S. (B) Original MS images of metabolites ([PC(34:1) + H]⁺ (m/z 760.5851) (top), [PC(38:6) + H]⁺ (m/z 844.5218) (middle), and [LPC(18:0) + H]⁺ (m/z 524.3693) (bottom)) and (C) their fused images. All metabolites were identified using MS² from the tissue slice, and the results were compared with METLIN (Figure S3).

Figure 4.

Pixel selection and average spectra (MS positive ion mode). (A) Fused image and the pixels representing Aβ plaques and their surrounding region. (B) Averaged mass spectra of an Aβ plaque and its surrounding region.

Figure 5.

Representative metabolites (MS positive ion mode) with significantly different abundances between Aβ plaques and their surrounding regions. Results were obtained from three Aβ plaques. The error bar indicates the standard deviation of the relative intensities obtained from the selected pixels. All metabolites were identified using MS² from the tissue slice, and the results were compared with METLIN (Figure S3) (from t test: ***, <0.001).

Figure 6.

Image fusion of fluorescence microscopy and MS images (MS negative ion mode). (A) Fluorescence microscopy image of 5xFAD mouse brain. (B) MS image of [PA(O-32:0)-2H]²⁻ and (C) its fused image. (D) MS image of dodecenoic acid [M+K-2H]⁻ and (E) its fused image.

Figure 7.

Pixel selection and ion abundance comparison (MS negative ion mode). (A) Pixels representing Aβ plaques and their surrounding areas in the fused images. (B) Representative metabolites possessing significantly different abundances between Aβ plaques and their surrounding regions (from t test: ***, < 0.001). All metabolites were identified using MS² from the tissue slice, and the results were compared with METLIN (Figure S4).