Atherosclerosis

2019

DOI: 10.1016/j.atherosclerosis.2019.04.223

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

Jose J. Rico-Jimenez

¹

,

Michael J. Serafino

²

,

Sebina Shrestha

³

et al.

Abstract: 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 fluo… Show more

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Analysis Of the Spatial Distributions Of Fluorescence Lifetimes1

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Cited by 12 publications

(13 citation statements)

References 36 publications

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“…The histological analysis of coronary specimens has indicated the presence of collagen, proteoglycans as well as extracellular lipids, thus the direct identification of the molecular specie responsible for the observed LT1 increase was not possible. The FLIm results for the third emission channel (green spectral band) were found consistent with previous reports that linked lifetime increase in this band with mFCs accumulation ( 6 , 32 ). The results for the second emission channel (blue spectral band) show that for these samples, the computed lifetime is to a large extent a linear combination of the violet and green bands, leading to its exclusion from the analysis.…”

Section: Discussionsupporting

confidence: 91%

Investigating Origins of FLIm Contrast in Atherosclerotic Lesions Using Combined FLIm-Raman Spectroscopy

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³

et al. 2020

Front. Cardiovasc. Med.

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Background: Fluorescence lifetime imaging (FLIm) is a spectroscopic imaging technique able to characterize the composition of luminal surface of arterial vessels. Studies of human coronary samples demonstrated that distinct atherosclerotic lesion types are characterized by FLIm features associate with distinct tissue molecular makeup. While conventional histology has provided indications about potential sources of molecular contrast, specific information about the origin of FLIm signals is lacking. Here we investigate whether Raman spectroscopy, a technique able to evaluate chemical content of biological samples, can provide additional insight into the origin of FLIm contrast. Methods: Six human coronary artery samples were imaged using FLIm (355 nm excitation)-Raman spectroscopy (785 nm excitation) via a multimodal fiber optic probe. The spatial distribution of molecular contrast in FLIm images was analyzed in relationship with histological findings. Raman data was investigated using an endmember technique and compared with histological findings. A descriptive modeling approach based on multivariate regression was used to identify Raman bands related with changes in lifetime in four spectral channels (violet: 387/35 nm, blue: 443/29 nm, green: 546/38 nm, and red: 628/53 nm). Results: Fluorescence lifetime variations in the violet, blue and green spectral bands were observed for distinct areas of each tissue sample associated with distinct pathologies. Analysis of Raman signals from areas associated with normal, pathological intimal thickening, and fibrocalcific regions demonstrated the presence of hydroxyapatite, collagenous proteins, carotene, cholesterol, and triglycerides. The FLIm and Raman descriptive modeling analysis indicated that lifetime increase in the violet spectral band was associated with increased presence of cholesterol and carotenes, a new finding consistent with LDL accumulation in atherosclerotic lesions, and not with collagen proteins, as expected from earlier studies. Conclusions: The systematic, quantitative analysis of the multimodal FLIm-Raman dataset using a descriptive modeling approach led to the identification of LDL accumulation as the primary source of lifetime contrast in atherosclerotic lesions in the violet spectral range. Earlier FLIm validation studies relying on histopathological findings had associated this contrast to increased collagen content, also present in advanced lesions, thus demonstrating the benefits of alternative validation methods.

“…The histological analysis of coronary specimens has indicated the presence of collagen, proteoglycans as well as extracellular lipids, thus the direct identification of the molecular specie responsible for the observed LT1 increase was not possible. The FLIm results for the third emission channel (green spectral band) were found consistent with previous reports that linked lifetime increase in this band with mFCs accumulation ( 6 , 32 ). The results for the second emission channel (blue spectral band) show that for these samples, the computed lifetime is to a large extent a linear combination of the violet and green bands, leading to its exclusion from the analysis.…”

Section: Discussionsupporting

confidence: 91%

Investigating Origins of FLIm Contrast in Atherosclerotic Lesions Using Combined FLIm-Raman Spectroscopy

¹

,

²

,

³

et al. 2020

Front. Cardiovasc. Med.

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Background: Fluorescence lifetime imaging (FLIm) is a spectroscopic imaging technique able to characterize the composition of luminal surface of arterial vessels. Studies of human coronary samples demonstrated that distinct atherosclerotic lesion types are characterized by FLIm features associate with distinct tissue molecular makeup. While conventional histology has provided indications about potential sources of molecular contrast, specific information about the origin of FLIm signals is lacking. Here we investigate whether Raman spectroscopy, a technique able to evaluate chemical content of biological samples, can provide additional insight into the origin of FLIm contrast. Methods: Six human coronary artery samples were imaged using FLIm (355 nm excitation)-Raman spectroscopy (785 nm excitation) via a multimodal fiber optic probe. The spatial distribution of molecular contrast in FLIm images was analyzed in relationship with histological findings. Raman data was investigated using an endmember technique and compared with histological findings. A descriptive modeling approach based on multivariate regression was used to identify Raman bands related with changes in lifetime in four spectral channels (violet: 387/35 nm, blue: 443/29 nm, green: 546/38 nm, and red: 628/53 nm). Results: Fluorescence lifetime variations in the violet, blue and green spectral bands were observed for distinct areas of each tissue sample associated with distinct pathologies. Analysis of Raman signals from areas associated with normal, pathological intimal thickening, and fibrocalcific regions demonstrated the presence of hydroxyapatite, collagenous proteins, carotene, cholesterol, and triglycerides. The FLIm and Raman descriptive modeling analysis indicated that lifetime increase in the violet spectral band was associated with increased presence of cholesterol and carotenes, a new finding consistent with LDL accumulation in atherosclerotic lesions, and not with collagen proteins, as expected from earlier studies. Conclusions: The systematic, quantitative analysis of the multimodal FLIm-Raman dataset using a descriptive modeling approach led to the identification of LDL accumulation as the primary source of lifetime contrast in atherosclerotic lesions in the violet spectral range. Earlier FLIm validation studies relying on histopathological findings had associated this contrast to increased collagen content, also present in advanced lesions, thus demonstrating the benefits of alternative validation methods.

“…175,205 Pixel-level FLIM analysis has been previously used to quantify lipid membrane integrity and heterogeneity, immune cell heterogeneity, cell development, protein conformation and organization, and other phenomena. [206][207][208][209][210][211]…”

Section: Pixel-level Analysismentioning

confidence: 99%

“…51,80,205,209,243,[249][250][251][252] Furthermore, tissue-level lifetime images can distinguish cellular compartments across diverse tissue types (e.g., stroma, endothelium, epithelium, and cancerous tissue). 11,208,241,253,254 Quantitative metrics of spatial heterogeneity have also been developed for FLIM. Spatial statistical analyses have quantified cell-level spatial heterogeneity in autofluorescence lifetimes across in vitro and in vivo tumor models.…”

Section: Analysis Of the Spatial Distributions Of Fluorescence Lifetimesmentioning

confidence: 99%

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

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et al. 2020

J. Biomed. Opt.

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Significance: Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to distinguish the unique molecular environment of fluorophores. FLIM measures the time a fluorophore remains in an excited state before emitting a photon, and detects molecular variations of fluorophores that are not apparent with spectral techniques alone. FLIM is sensitive to multiple biomedical processes including disease progression and drug efficacy. Aim: We provide an overview of FLIM principles, instrumentation, and analysis while highlighting the latest developments and biological applications. Approach: This review covers FLIM principles and theory, including advantages over intensitybased fluorescence measurements. Fundamentals of FLIM instrumentation in time-and frequencydomains are summarized, along with recent developments. Image segmentation and analysis strategies that quantify spatial and molecular features of cellular heterogeneity are reviewed. Finally, representative applications are provided including high-resolution FLIM of cell-and organelle-level molecular changes, use of exogenous and endogenous fluorophores, and imaging protein-protein interactions with Förster resonance energy transfer (FRET). Advantages and limitations of FLIM are also discussed. Conclusions: FLIM is advantageous for probing molecular environments of fluorophores to inform on fluorophore behavior that cannot be elucidated with intensity measurements alone. Development of FLIM technologies, analysis, and applications will further advance biological research and clinical assessments.

“… 11 In particular, fluorescence lifetime imaging microscopy (FLIM) is robust to fluorescence intensity fluctuations and able to measure the relative concentration of lipids and collagen. 12 – 14 The measure and identification of lipoproteins is important for finding so-called “vulnerable” plaques that are prone to rupture causing a sudden coronary event.…”

Section: Introductionmentioning

confidence: 99%

Dual-modality optical coherence tomography and frequency-domain fluorescence lifetime imaging microscope system for intravascular imaging

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²

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³

et al. 2020

J. Biomed. Opt.

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Significance: Detailed biochemical and morphological imaging of the plaque burdened coronary arteries holds the promise of improved understanding of atherosclerosis plaque development, ultimately leading to better diagnostics and therapies. Aim: Development of a dual-modality intravascular catheter supporting swept-source optical coherence tomography (OCT) and frequency-domain fluorescence lifetime imaging (FD-FLIM) of endogenous fluorophores with UV excitation. Approach: We instituted a refined approach to endoscope development that combines simulation in a commercial ray tracing program, fabrication, and a measurement method for optimizing ball-lens performance. With this approach, we designed and developed a dual-modality catheter endoscope based on a double-clad fiber supporting OCT through the core and fluorescence collection through the first cladding. We varied the relative percent of UV excitation launched into the core and first cladding to explore the potential resolution improvement for FD-FLIM. The developed catheter endoscope was optically characterized, including measurement of spatial resolution and fluorescent lifetimes of standard fluorophores. Finally, the system was demonstrated on fresh ex vivo human coronary arteries. Results: The developed endoscope was shown to have optical performance similar to predictions derived from the simulation approach. The FLIM resolution can be improved by over a factor of 4 by primarily illuminating through the core rather than the first cladding. However, time-dependent solarization losses need to be considered when choosing the relative percentage. We ultimately chose to illuminate with 7% of the power transmitting through the core. The resulting catheter endoscope had 40-μm lateral resolution for OCT and <100 μm lateral resolution for FD-FLIM. Images of ex vivo coronary arteries are consistent with expectations based on histopathology. Conclusions: The results demonstrate that our approach for endoscope simulation produces reliable predictions of endoscope performance. Simulation results guided our development of a multimodal OCT/FD-FLIM catheter imaging system for investigating atherosclerosis in coronary arteries.

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