Intravital Microscopy in Atherosclerosis Research

Abstract

Atherosclerosis is a lipid-driven inflammatory disorder that narrows the arterial lumen and can induce life-threatening complications from coronary artery disease, cerebrovascular disease, and peripheral artery disease. On a mechanistic level, the development of novel cellular-resolution intravital microscopy imaging approaches has recently enabled in vivo studies of underlying biological processes governing disease onset and progress. In particular, multiphoton microscopy has emerged as a promising intravital imaging tool utilizing two-photon-excited fluorescence and second-harmonic generation that provides subcellular resolution and increased imaging depths beyond confocal and epifluorescence microscopy. In this chapter, we describe the state-of-the-art multiphoton microscopy applied to the study of murine atherosclerosis.

Keywords: Atherosclerosis; Gated microscopy; Inflammation; Intravital imaging; Intravital microscopy; Molecular imaging; Multiphoton microscopy.

Figure 1:. In vivo imaging of Cathepsin K (CatK) activity in carotid atherosclerotic plaques of ApoE^−/− mice.

Atheroma were surgically exposed and then underwent laser scanning intravital fluorescence microscopy 24 hours after injection of the CatK or control K imaging agent (5 nmol). Multiwavelength imaging allowed detection of the CatK signal and a spectrally resolved intravascular agent injected just before imaging. A cylindrical Cy5.5 dye-filled phantom (green) was placed under the carotid artery to facilitate localization. A) Fusion in vivo image of a carotid vessel (×5 magnification; 13×13-μm in-plane resolution; 10-μm slice thickness) demonstrating focal CatK signal (green) in an atherosclerotic lesion (arrowhead). The lesion was confirmed to be within the vascular space as defined by the intravascular agent (red). B) Fusion image of a carotid plaque (arrowhead) in the control group demonstrating minimal NIRF signal in the CatK channel. The plaque appears as a signal void or filling defect within the vascular space. C) and D) Projection images of the carotid plaques after injection of the CatK imaging agent (C) or control agent (D), demonstrating greater plaque TBRs (E) in the CatK vs control group (*P<0.05). Projection images processed and windowed identically. Scale bar, 250 μm. Reproduced from ref. [20] with permission from Wolters Kluwer Health, Inc.

Figure 2:. Intravital two-photon imaging and PET-based scanning reveal immune cell behavior in aortic arch grafts.

A) Representative picture showing monocytes/macrophages (green) and CD4+ T cells (red) in CBA aortic arch grafts 1 week after transplantation into B6 CX3CR1 GFP/+ mice (n=3) (GFP, green fluorescent protein). Male → male strain combination is depicted in this figure. Monocytes / macrophages (green) in male B6 ApoE^−/− aortic arch grafts (B) 1 month (n=3) and (C) 4 months (n=3) after transplantation into male B6 CX3CR1 GFP/+ mice. D) Reduction of density of CX3CR1 GFP+ cells within regressing plaques (212 ± 17 cells/area (220 x 240 μm2) at 1 month (blue) vs. 54 ± 16 cells/area at 4 months (red), n=3, **p < 0.01). E) Representative image of male B6 ApoE^−/− aortic arch grafts transplanted into male B6 LysM-GFP mice 1 day after engraftment (n=3). Collagen appears blue due to second harmonic generation. Blood vessels are labeled red after injection of quantum dots. Scale bars, 30 μm. F) Velocity of LysM-GFP cells in male B6 ApoE^−/− aortic arch grafts 24 hours after engraftment (mean velocity is 16.02 μm/min ± 1.08; analyzed cells pooled from 3 independent experiments. Reproduced from ref. [21] with permission from Wolters Kluwer Health, Inc.

Figure 3:. Proximal edge of am acute stasis deep vein thrombosis (DVT) formed in the murine saphenous vein following, fluorescin isothiocyanate (FITC) injection and FITC-based light illumination for 60 seconds.

A) FITC-dextran (green) image of vessel lumen and thrombus (white arrow) and leukocytes appearing as negative-contrast. B) Leukocytes and platelets visualized by Rhodamine 6G (red) injected after FITC-light thrombus induction. C) Merged FITC-dextran, Rhodamine 6G and SHG (blue) signals, with SHG visualizing primarily type I collagen in the vessel wall. Scale bar, 50 μm.