Jeffrey L. Suhalim

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

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.

Identification of cholesterol crystals in plaques of atherosclerotic mice using hyperspectral CARS imaging

The accumulation of lipids, including cholesterol, in the arterial wall plays a key role in the pathogenesis of atherosclerosis. Although several advances have been made in the detection and imaging of these lipid structures in plaque lesions, their morphology and composition have yet to be fully elucidated, particularly in different animal models of disease. To address this issue, we analyzed lipid morphology and composition in the atherosclerotic plaques of two animal models of disease, the low density lipoprotein receptor-deficient (LDLR−/−) mouse and the ApoE lipoprotein-deficient (ApoE−/−) mouse, utilizing hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy in combination with principal component analysis (PCA). Hyperspectral CARS imaging revealed lipid-rich macrophage cells and condensed needle-shaped and plate-shaped lipid crystal structures in both mice. Spectral analysis with PCA and comparison to spectra of pure cholesterol and cholesteryl ester derivatives further revealed these lipid structures to be pure cholesterol crystals, which were predominantly observed in the ApoE−/− mouse model. These results illustrate the ability of hyperspectral CARS imaging in combination with multivariate analysis to characterize atherosclerotic lipid morphology and composition with chemical specificity, and consequently, provide new insight into the formation of cholesterol crystal structures in atherosclerotic plaque lesions.

Picosecond spectral coherent anti-Stokes Raman scattering imaging with principal component analysis of meibomian glands

The lipid distribution in the mouse meibomian gland was examined with picosecond spectral anti-Stokes Raman scattering (CARS) imaging. Spectral CARS data sets were generated by imaging specific localized regions of the gland within tissue sections at consecutive Raman shifts in the CH(2) stretching vibrational range. Spectral differences between the location specific CARS spectra obtained in the lipid-rich regions of the acinus and the central duct were observed, which were confirmed with a Raman microspectroscopic analysis, and attributed to meibum lipid modifications within the gland. A principal component analysis of the spectral data set reveals changes in the CARS spectrum when transitioning from the acini to the central duct. These results demonstrate the utility of picosecond spectral CARS imaging combined with multivariate analysis for assessing differences in the distribution and composition of lipids in tissues.

Effect of Desiccating Stress on Mouse Meibomian Gland Function

PURPOSE Mice exposed to standardized desiccating environmental stress to induce dry eye-like symptoms have been used as a model to study the underlying mechanisms of evaporative dry eye. While studies have shown marked inflammatory and immune changes, the effect of such stress on meibomian gland function remains largely unknown. We sought to evaluate the effects of desiccating stress on meibocyte proliferation and meibum quality. METHODS Ten mice were treated with scopolamine and subjected to a drafty low humidity environment (30-35%). Five and ten days after treatment, eyelids were harvested and cryosections stained with Ki67 antibody to identify cycling cells. Sections were also imaged using stimulated Raman scattering (SRS) microscopy to characterize the gland compositional changes by detecting the vibrational signatures of methylene (lipid) and amide-I (protein). RESULTS Desiccating stress caused a 3-fold increase in basal acinar cell proliferation from 18.3 ± 11.1% in untreated mice to 64.4 ± 19.9% and 66.6 ± 13.4% after 5 and 10 days exposure, respectively (P < .001). In addition, SRS analysis showed a wider variation in the protein-to-lipid ratio throughout the gland, suggesting alterations in meibocyte differentiation and lipid synthesis. CONCLUSIONS These data are consistent with a model that a desiccating environment may have a direct effect on meibomian gland function, leading to a significant increase in basal acinar cell proliferation, abnormal meibocyte differentiation, and altered lipid production.

The need for speed

One of the key enabling features of coherent Raman scattering (CRS) techniques is the dramatically improved imaging speed over conventional vibrational imaging methods. It is this enhanced imaging acquisition rate that has guided the field of vibrational microscopy into the territory of real-time imaging of live tissues. In this feature article, we review several aspects of fast vibrational imaging and discuss new applications made possible by the improved CRS imaging capabilities. In addition, we reflect on the current limitations of CRS microscopy and look ahead at several new developments towards real-time, hyperspectral vibrational imaging of biological tissues. (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

Spaceflight Activates Lipotoxic Pathways in Mouse Liver

Spaceflight affects numerous organ systems in the body, leading to metabolic dysfunction that may have long-term consequences. Microgravity-induced alterations in liver metabolism, particularly with respect to lipids, remain largely unexplored. Here we utilize a novel systems biology approach, combining metabolomics and transcriptomics with advanced Raman microscopy, to investigate altered hepatic lipid metabolism in mice following short duration spaceflight. Mice flown aboard Space Transportation System -135, the last Shuttle mission, lose weight but redistribute lipids, particularly to the liver. Intriguingly, spaceflight mice lose retinol from lipid droplets. Both mRNA and metabolite changes suggest the retinol loss is linked to activation of PPARα-mediated pathways and potentially to hepatic stellate cell activation, both of which may be coincident with increased bile acids and early signs of liver injury. Although the 13-day flight duration is too short for frank fibrosis to develop, the retinol loss plus changes in markers of extracellular matrix remodeling raise the concern that longer duration exposure to the space environment may result in progressive liver damage, increasing the risk for nonalcoholic fatty liver disease.

Correction: Spaceflight Activates Lipotoxic Pathways in Mouse Liver

[This corrects the article DOI: 10.1371/journal.pone.0152877.].

Characterization of expressed human meibum using hyperspectral stimulated Raman scattering microscopy

PURPOSE This study examined whether hyperspectral stimulated Raman scattering (hsSRS) microscopy can detect differences in meibum lipid to protein composition of normal and evaporative dry eye subjects with meibomian gland dysfunction. METHODS Subjects were evaluated for tear breakup time (TBUT), staining, meibum expression and gland dropout. Expressed meibum was analyzed using SRS vibrational signatures in the CH stretching region (2800-3050 cm-1). Vertex component analysis and K-means clustering were used to group the spectral signatures into four fractions containing high lipid (G1) to high protein (G4). RESULTS Thirty-three subjects could be statistically analyzed using pooled meibum (13 with stable tear films (TBUTs > 10 s) and 20 with unstable tear films (TBUTs ≤ 10 s). Significant differences in meibum from subjects with unstable vs. stable TBUTs were found for the G1 fraction (medians 0.164 and 0.020, respectively; p = 0.012) and the G2 fraction (medians 0.244 and 0.272, respectively; p = 0.045). No differences were observed for the G3 and G4 fractions. Single orifice samples were not significantly different vs. pooled samples from the fellow eye, and eyelid sector samples (nasal, central and temporal) G2:G3 fractional components were not significantly different (p = 0.449). Spearman analysis suggested a significant inverse correlation between G1 fraction and TBUT (R = -0.351; p = 0.045). CONCLUSIONS hsSRS microscopy allows compositional analysis of expressed meibum from humans which correlated to changes in TBUT. These findings support the hypothesis that hsSRS may be useful in classifying meibum quality and evaluating the effects of therapy.

Picosecond CARS Spectral Imaging with Principal Component Analysis

We demonstrate the utility of picosecond spectral coherent anti-Stokes Raman scattering imaging with principal component analysis to rapidly map lipophilic components in cardiovascular tissues, facilitating the interrogation of atherosclerosis.