Fig 1.
Atherosclerotic models and plaque imaging by stimulated Raman loss.
Plaque samples were derived from three sources, (A) a study of male Macaques on a high fat diet for 24 weeks, inoculated with SIVmac239, or not, and kept on diet another 9 weeks before sacrifice and analysis, (B) HIV+ autopsy samples of the left anterior descending coronary artery, (C) a study of Ldlr-/- mice fed a high fat diet for 8 weeks. (D) Diagram of the mechanism of the stimulated Raman loss process, where ω1 and ω2 are the frequencies of the incoming photons, and Ω corresponds to the frequency of the molecular vibration. (E-F) Imaging modalities used and measures taken in the models.
Fig 2.
Control and SIV+ plaques display needle-like CCs as detected by SRS and SHG imaging.
Control plaque, top three panels, SIV+, bottom three, with SHG signal shown in (A & D, blue) SRS signal (B & E, orange) and composites (C & F). Arrow in F points to a macrophage attempting to phagocytose a CC. Scale bars are in micrometers (μm), and vessel lumen is denoted.
Fig 3.
Counts of CCs number and length by Otsu’s method.
(A) binary rendering of the SHG control plaque image shown in Fig 2A–2C, (B) after segmentation, (C) failed overlapping objects (D) passed independent objects. (E) Graph of CC number per unit area of control and SIV+ plaques, (F) Graph of CC length of control and SIV+ plaques. For E & F a total of 973 objects were counted from 5 animals (con-2, Siv+-3, 3–4 images/animal, p value <0.05 for E, and <0.0001 for F, by paired t- test). (G-I) A SIV+ plaque showing CCs in the luminal endothelial layer using SHG (G), SRS (H), and CARS (I).
Fig 4.
Hyperspectral scans of SIV+, and control, plaques detect a CE dominate signature.
(A & C) spectral traces for two needle objects (green and red ovals), a protein rich area of the shoulder region (blue) and an amorphous lipid rich shoulder region (yellow) are shown for the control plaque. (B & D) SRS spectral traces for a needle object (red), a protein region (blue), and an amorphous lipid droplet (green arrow) of a SIV+ plaque. The top two dashed black traces in C & D represent protein and cholesteryl-linoleate reference SRS spectra, respectively. Shaded spectral band indicates the spectral response of unsaturated acyl chains.
Fig 5.
A lipid rich HIV+ coronary artery plaque with aggregated cuboidal CCs.
SHG (A), SRS (B) and overlay (C) images of a lipid rich plaque. Panels (D), (E) and (F) show the same plaque at higher magnification, corresponding to the area enclosed by the green box in (A). SRS images were recorded at 2845 cm-1. Magenta indicates regions of high overlap in the overlay images. Vessel lumen of the left descending artery and magnification scales are indicated, and yellow arrow in panel D denotes cuboidal feature shown in higher detail in Fig 7D.
Fig 6.
Lipid rich plaque from HIV+ coronary artery showing needle CCs.
SHG (A), SRS (B) and overlay (C) images of a lipid rich plaque. Panels (D), (E) and (F) show the same plaque at higher magnification, corresponding to the area enclosed by the green box in (A). SRS images were recorded at 2845 cm-1. Magenta indicates regions of high overlap in the overlay images. Vessel lumen of the left descending artery and magnification scales are indicated, and yellow arrow in panel B indicates fat droplet in vessel lumen derived from extravascular adipose tissue.
Fig 7.
Hyperspectral scan of HIV+ plaque aggregated cuboidal CCs detect a CE dominate signature.
Left panels show the SHG (A, top) and SRS (B, bottom) with two regions of interest denoted (green and blue circles). Graphed right (C) are the SRS spectra of the individual objects (green and blue lines) and their average (red). The top black solid and dashed traces represent the SRS spectra of cholesteryl oleate and linoleate standards, respectively. Detail of green region 2 is shown in (D) for the SHG and SRS channels, and the cuboidal structure is indicated by the yellow arrow.
Fig 8.
Hyperspectral scan of HIV+ plaque needle CCs detect a CE dominate signature.
Left panels show the SHG (A, top) and SRS (B, bottom) with two object regions denoted (green and blue ovals with arrows indicating relative polarization direction). Graphed right (C) are the spectral traces of the individual objects (green and blue lines), their average (red), with the top black dashed trace showing the cholesteryl linoleate reference SRS spectrum.
Fig 9.
Hyperspectral scan of a Ldlr-/- plaque cuboidal CC detects a CE dominate signature.
Top panels (A-C) show SHG, SRS and composite image of a lipid rich aorta plaque with a cuboidal crystalline region denoted (C, blue oval). Graphed below (D) is the spectral traces of the CC object (blue line) as well as standard traces of cholesteryl oleate (green) and free cholesterol monohydrate (red). The finger print region of the traces in the graph is highlighted (blue rectangle).
Fig 10.
Ldlr-/- bone marrow derived macrophages from HFD fed Ldlr-/- mice display a lipid engorged phenotype.
Ldlr-/- mice were fed a chow or a HFD for 4 (panel A & C) or 8 weeks (B & D) and the bone marrow derived macrophages cultured for 5 days were imaged for auto-fluorescence (green) and counterstained for nuclei (blue) and lysosome (red). Circled area in C & D are shown in more detail in E and F, respectively.
Fig 11.
Cholesterol crystal uptake by J774A.1 macrophages induces auto-fluorescent puncta.
Cholesterol crystals (200 μg/ml) were added to cultured J774A.1 macrophage for 24 h and compared to vehicle treated cultures. A, Control treated cells, B, cholesterol crystal treated cell with left panels showing stained nuclei, and right panels showing auto-fluorescence and nuclei.
Fig 12.
AcLDL uptake by J774A.1 macrophages induces auto-fluorescent puncta distinct from the lipid droplet.
Vehicle (A, top panels), or AcLDL (B, bottom panels, 40 μg/ml), was added to cultures for 24 h and cells were imaged for auto-fluorescence (green) nuclei (blue) and lipid droplets (red) with right panels showing selected cells in more detail from the center panels.
Fig 13.
AcLDL induced auto-fluorescence puncta co-localize with auto-phagosome LC3 activity.
J774A.1 cells were transfected with LC3B-RFP, and treated with AcLDL (C, 40 μg/ml, 24 h), starved in PBS (B, positive control for autophagic induction, 2 h), or left in serum free media (A, negative control for autophagic induction), and imaged for auto-fluorescence shown in green, nuclei in blue, and LC3B-RFP distribution in red.