Lipid signature of advanced human carotid atherosclerosis assessed by mass spectrometry imaging.

Carotid atherosclerosis is a risk factor for ischemic stroke, one of the main causes of mortality and disability worldwide. The disease is characterized by plaques, heterogeneous deposits of lipids, and necrotic debris in the vascular wall, which grow gradually and may remain asymptomatic for decades. However, at some point a plaque can evolve to a high-risk plaque phenotype, which may trigger a cerebrovascular event. Lipids play a key role in the development and progression of atherosclerosis, but the nature of their involvement is not fully understood. Using matrix-assisted laser desorption/ionization mass spectrometry imaging, we visualized the distribution of approximately 200 different lipid signals, originating of >90 uniquely assigned species, in 106 tissue sections of 12 human carotid atherosclerotic plaques. We performed unsupervised classification of the mass spectrometry dataset, as well as a histology-directed multivariate analysis. These data allowed us to extract the spatial lipid patterns associated with morphological plaque features in advanced plaques from a symptomatic population, revealing spatial lipid patterns in atherosclerosis and their relation to histological tissue type. The abundances of sphingomyelin and oxidized cholesteryl ester species were elevated specifically in necrotic intima areas, whereas diacylglycerols and triacylglycerols were spatially correlated to areas containing the coagulation protein fibrin. These results demonstrate a clear colocalization between plaque features and specific lipid classes, as well as individual lipid species in high-risk atherosclerotic plaques.

Label-free analytic histology of carotid atherosclerosis by mid-infrared optoacoustic microscopy.

Background and aims: Analysis of atherosclerotic plaque composition is a vital tool for unraveling the pathological metabolic processes that contribute to plaque growth. Methods: We visualize the constitution of human carotid plaques by mid-infrared optoacoustic microscopy (MiROM), a method for label-free analytic histology that requires minimal tissue preparation, rapidly yielding large field-of-view en-face images with a resolution of a few micrometers. We imaged endarterectomy specimens (n = 3, 12 sections total) at specific vibrational modes, targeting carbohydrates, lipids and proteins. Additionally, we recorded spectra at selected tissue locations. We identified correlations in the variability in this high-dimensional data set using non-negative matrix factorization (NMF). Results: We visualized high-risk plaque features with molecular assignment. Consistent NMF components relate to different dominant tissue constituents, dominated by lipids, proteins, and cholesterol and carbohydrates respectively. Conclusions: These results introduce MiROM as an innovative, stain-free, analytic histology technology for the biochemical characterization of complex human vascular pathology.

Micro Spectroscopic Photoacoustic (µsPA) imaging of advanced carotid atherosclerosis.

Atherosclerosis is a lipid-driven and an inflammatory disease of the artery walls. The composition of atherosclerotic plaque stratifies the risk of a specific plaque to cause a cardiovascular event. In an optical resolution photoacoustic microscopy setup, of 45 µm resolution, we extracted plaque lipid photoacoustic (PA) spectral signatures of human endarterectomy samples in the range of 1150-1240 nm, using matrix assisted laser desorption ionization mass spectrometry imaging as a reference. We found plaque PA signals to correlate best with sphingomyelins and cholesteryl esters. PA signal spectral variations within the plaque area were compared to reference molecular patterns and absorption spectra of lipid laboratory standards. Variability in the lipid spectroscopic features extracted by principal component analysis of all samples revealed three distinct components with peaks at: 1164, 1188, 1196 and 1210 nm. This result will guide the development of PA-based atherosclerosis disease staging capitalizing on lipidomics of atherosclerotic tissue.

Three-phase vaporization theory for laser-activated microcapsules.

Precision control of vaporization, both in space and time, is critical for numerous applications, including medical imaging and therapy, catalysis and energy conversion, and it can be greatly improved through the use of micro- or nano-sized light absorbers. Ultimately, optimization of these applications also requires a fundamental understanding of the vaporization process. Upon laser irradiation, polymeric microcapsules containing a dye can vaporize, leading to the growth of a vapor bubble that emits a strong acoustic signature. Here, we compare laser-activated capsules containing either a volatile or a non-volatile oil core. We theoretically explore the vaporization of the capsules based on a three-phase thermodynamics model, that accounts for the partial vaporization of both the surrounding fluid and the oil core as well as for the interaction between heat transfer and microbubble growth. The model is compared to ultra-high-speed imaging experiments, where we record the cavitation events. Theory and experiments are in convincing agreement.

Laser-activated microparticles for multimodal imaging: ultrasound and photoacoustics.

The increasing personalization of medical treatment demands refined imaging and increased monitoring capabilities, as well as an improved efficacy through targeted drug delivery. Such a transition in health care can be facilitated by the use of multimodal contrast agents. In this paper, we present a novel type of multimodal contrast agents, that enhances contrast both in ultrasound and in photoacoustic imaging, while at the same time being capable of triggered drug delivery. Upon pulsed laser irradiation, polymeric microparticles-containing a dye and an oil core-can create a cavitation bubble that subsequently emits a strong acoustic wave. We investigated different formulations of these particles, by changing the oil content, dye concentration and probing conditions using a combination of pulsed laser excitation and an ultrasound chirp. We demonstrated that capsules with a core containing a low boiling point oil give the highest photoacoustic and acoustic response. The laser activation threshold for this system is high in the visible range, but within the near infrared medical limits. The same system also produces a stable bubble. US scattering by these stable bubbles results in medically relevant frequencies, making the particles of interest for biomedical and pre-clinical imaging. Finally, the system has potential to carry a functional drug-load, and a route to these applications is discussed.

Data Processing Pipeline for Lipid Profiling of Carotid Atherosclerotic Plaque with Mass Spectrometry Imaging.

Atherosclerosis is a lipid and inflammation-driven disease of the arteries that is characterized by gradual buildup of plaques in the vascular wall. A so-called vulnerable plaque, consisting of a lipid-rich necrotic core contained by a thin fibrous cap, may rupture and trigger thrombus formation, which can lead to ischemia in the heart (heart attack) or in the brain (stroke). In this study, we present a protocol to investigate the lipid composition of advanced human carotid plaques using matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI), providing a framework that should enable the discrimination of vulnerable from stable plaques based on lipid composition. We optimized the tissue preparation and imaging methods by systematically analyzing data from three specimens: two human carotid endarterectomy samples (advanced plaque) and one autopsy sample (early stage plaque). We show a robust data reduction method and evaluate the variability of the endarterectomy samples. We found diacylglycerols to be more abundant in a thrombotic area compared to other plaque areas and could distinguish advanced plaque from early stage plaque based on cholesteryl ester composition. We plan to use this systematic approach to analyze a larger dataset of carotid atherosclerotic plaques.