Characterization of Cholesterol Crystals in Atherosclerotic Plaques Using Stimulated Raman Scattering and Second-Harmonic Generation Microscopy

Abstract

Cholesterol crystals (ChCs) have been identified as a major factor of plaque vulnerability and as a potential biomarker for atherosclerosis. Yet, due to the technical challenge of selectively detecting cholesterol in its native tissue environment, the physiochemical role of ChCs in atherosclerotic progression remains largely unknown. In this work, we demonstrate the utility of hyperspectral stimulated Raman scattering (SRS) microscopy combined with second-harmonic generation (SHG) microscopy to selectively detect ChC. We show that despite the polarization sensitivity of the ChC Raman spectrum, cholesterol monohydrate crystals can be reliably discriminated from aliphatic lipids, from structural proteins of the tissue matrix and from other condensed structures, including cholesteryl esters. We also show that ChCs exhibit a nonvanishing SHG signal, corroborating the noncentrosymmetry of the crystal lattice composed of chiral cholesterol molecules. However, combined hyperspectral SRS and SHG imaging reveals that not all SHG-active structures with solidlike morphologies can be assigned to ChCs. This study exemplifies the merit of combining SRS and SHG microscopy for an enhanced label-free chemical analysis of crystallized structures in diseased tissue.

Study of the Development of the Mouse Thoracic Aorta Three-Dimensional Macromolecular Structure using Two-Photon Microscopy

“…There have been many different approaches used in preparing vessels for ex vivo microscopy reported in the literature. Some have included mounting variably sized vessel segments into a perfusion chamber (Megens et al 2007a;Megens et al 2007b), fresh and/or formalinfixed tissue preparations mounted between a glass slide and a coverslip (Kwon et al 2008;Lee et al 2009;Kim et al 2010, Lim et al 2010, Lim et al 2011Suhalim et al 2012), as well as agar gel-infused vascular casts that are segmented into thinly sliced molds and mounted on a glass slide (van Zandvoort et al 2004;Megens et al 2007a;Megens et al 2008;Le et al 2010). In vivo approaches have also been described but these are with the addition of mechanical techniques to help minimize motion artifact.…”

Accumulating advantages, reducing limitations: Multimodal nonlinear imaging in biomedical sciences – The synergy of multiple contrast mechanisms

“…Polarization sensitive SHG enables visualization of the fiber direction. Maximal SHG signal is emitted, when the polarization direction is orthogonal to the fiber axis, which allows for direction mapping of the fiber orientation in tendon as shown in Figure 2C [ 35], in the cornea [36], but also for determining the ordering of cholesterol crystals [37]. Similarly, also other techniques like SRS are sensitive to the input polarization, which provides additional information on the molecular ordering, e.g., of cholesterol crystals [37].…”

“…Hence, a variety of techniques for multispectral data analysis have been applied to spectral CRS and fluorescence data. Principal component analysis (PCA) known from linear Raman spectroscopy has been applied to spectral CARS data to visualize differences in the lipid distribution in meibomian glands [46], and to identify cholesterol crystals in both SRS and CARS spectral data [37,47]. Even more sophisticated methods are required to extract the Raman spectrum from multiplex CARS data, e.g., the maximum entropy method or the Kramers Kronig relations [48][49][50].…”

Multimodal Nonlinear Optical Microscopy for Simultaneous 3-D Label-Free and Immunofluorescence Imaging of Biological Samples

“…As a biological sample, we used the atherosclerotic aorta from an apolipoprotein E-deficient (ApoE -/-) mouse with the goal of obtaining simultaneous label-free and targeted molecular images. Recently, atherosclerosis has been studied as one of the medical applications of multimodal NLO microscopy [18][19][20][21][22]. Atherosclerosis is a chronic, progressive arterial disease associated with lipid deposition and chronic inflammation [23,24].…”

“…Atherosclerotic lesions have biomolecules associated with endothelial cells, extracellular lipid droplets, lipid-rich cells, low-density lipoprotein aggregates, collagen, and elastin. TPEF, SHG, and CARS have been demonstrated for imaging extracellular elastin, type-I collagen fibrils, and lipid-rich molecules, respectively, for non-stained atherosclerotic plaque [18][19][20][21][22]. However, analysis of label-free visualizations of elastin, collagen, and lipids are limited by complicated morphological or chemical changes in atherosclerotic progress.…”