Automated detection of superficial macrophages in atherosclerotic plaques using autofluorescence lifetime imaging.

BACKGROUND AND AIMS: Macrophages play an important role in the development and destabilization of advanced atherosclerotic plaques. Hence, the clinical imaging of macrophage content in advanced plaques could potentially aid in identifying patients most at risk of future clinical events. The lifetime of the autofluorescence emission from atherosclerotic plaques has been correlated with lipids and macrophage accumulation in ex vivo human coronary arteries, suggesting the potential of intravascular endogenous fluorescence or autofluorescence lifetime imaging (FLIM) for macrophage imaging. The aim of this study was to quantify the accuracy of the coronary intima autofluorescence lifetime to detect superficial macrophage accumulation in atherosclerotic plaques. METHODS: Endogenous FLIM imaging was performed on 80 fresh postmortem coronary segments from 23 subjects. The plaque autofluorescence lifetime at an emission spectral band of 494 ±â€¯20.5 nm was used as a discriminatory feature to detect superficial macrophage accumulation in atherosclerotic plaques. Detection of superficial macrophage accumulation in the imaged coronary segments based on immunohistochemistry (CD68 staining) evaluation was taken as the gold standard. Receiver Operating Characteristic (ROC) curve analysis was applied to select an autofluorescence lifetime threshold value to detect superficial macrophages accumulation. RESULTS: A threshold of 6 ns in the plaque autofluorescence lifetime at the emission spectral band of 494 ±â€¯20.5 nm was applied to detect plaque superficial macrophages accumulation, resulting in ∼91.5% accuracy. CONCLUSIONS: This study demonstrates the capability of endogenous FLIM imaging to accurately identify superficial macrophages accumulation in human atherosclerotic plaques, a key biomarker of atherosclerotic plaque vulnerability.

Simultaneous morphological and biochemical endogenous optical imaging of atherosclerosis.

AIMS: The aim of this study was to validate novel imaging technology for simultaneous morphological and biochemical endogenous optical imaging of coronary atherosclerotic plaque. METHODS AND RESULTS: Optical coherence tomography (OCT) generates high-resolution 3D images of plaque morphology and endogenous fluorescence lifetime imaging microscopy (FLIM) characterizes biochemical composition. Both imaging modalities rely on plaque's intrinsic optical characteristics, making contrast agents unnecessary. A multimodal OCT/FLIM system was utilized to generate luminal biochemical maps superimposed on high-resolution (7 µm axial and 13 µm lateral) structural volumetric images. Forty-seven fresh postmortem human coronary segments were imaged: pathological intimal thickening (PIT, n = 26), fibroatheroma (FA, n = 12), thin-cap FA (TCFA, n = 2), and fibrocalcific plaque (CA, n = 7), determined by histopathology. Multimodal images were evaluated, and each plaque identified as PIT, FA, TCFA, or CA based on expert OCT readers, and as having high-lipid (HL), high-collagen (HC), or low-collagen/low-lipid (LCL) luminal composition based on linear discriminant analysis of FLIM. Of 47 plaques, 89.4% (42/47) of the plaques were correctly identified based on OCT/FLIM evaluation using tissue histopathology and immunohistochemistry as the gold standard. Four of the misclassifications corresponded to confusing PIT with HL luminal composition for FA with HL cap. The other corresponded to confusing FA with a HC cap for FA with an LCL cap. CONCLUSION: We have demonstrated the feasibility of accurate simultaneous OCT/FLIM morphological and biochemical characterization of coronary plaques at spatial resolutions and acquisition speeds compatible with catheter-based intravascular imaging. The success of this pilot study sets up future development of a multimodal intravascular imaging system that will enable studies that could help improve our understanding of plaque pathogenesis.

Biochemical imaging of human atherosclerotic plaques with fluorescence lifetime angioscopy.

A prototype angioscopy system with fluorescence lifetime imaging microscopy (FLIM) capabilities was built and applied for biochemical imaging of human coronary atherosclerotic plaques. The FLIM angioscopy prototype consisted of a thin flexible angioscope suitable for UV-excited autofluorescence imaging, and a FLIM detection system based on a pulse sampling approach. The angioscope was composed of an imaging bundle attached to a gradient index objective lens and surrounded by a ring of illumination fibers (2 mm outer diameter, 50 microm spatial resolution). For FLIM detection based on the pulse sampling approach, a gated-intensified charge-couple device camera (200 ps temporal resolution) was used. Autofluorescence was excited with a pulsed UV laser (337 nm) and FLIM images were acquired at three emission bands (390/40 nm, 450/40 nm, 550/88 nm). The system was characterized on standard fluorophores and then used to image postmortem human coronary arteries. The FLIM angioscope allowed us to distinguish elastin-dominant plaques (peak emission at 450 nm, approximately 1.5 ns lifetimes) from collagen-dominant plaques (peak emission at 390 n, approximately 2-3 ns lifetimes) based on their intrinsic fluorescence spectral and lifetime differences. This study demonstrates the potential of FLIM angioscopy for biochemical imaging of human coronary atherosclerotic plaques.