Non-destructive two-photon excited fluorescence imaging identifies early nodules in calcific aortic-valve disease

Baugh, Lauren; Liu, Zhiyi; Quinn, Kyle P.; Osseiran, Sam; Evans, Conor L.; Huggins, Gordon S.; Hinds, Philip W.; Black, Lauren D.; Georgakoudi, Irene

doi:10.1038/s41551-017-0152-3

Cited by 33 publications

(38 citation statements)

References 78 publications

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“…Previously, TPEF was used to monitor the progression of osteogenic differentiation in human mesenchymal stem cells (MSCs) 18,21,22 and ORR and mitochondrial FD was shown to correlate with the osteogenic differentiation of human MSCs 18 . Recently, TPEF imaging was utilized in vitro and on ex vivo tissue explants to detect mineralization, a key hallmark of CAVD 23 . In the context of VICs, we have previously shown that ORR correlated with cellular proliferative potential when VICs were cultured under different media conditions 24 .…”

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confidence: 99%

Label-free metabolic biomarkers for assessing valve interstitial cell calcific progression

Tandon

Kolenc

Cross

et al. 2020

Sci Rep

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Calcific aortic valve disease (CAVD) is the most common form of valve disease where the only available treatment strategy is surgical valve replacement. Technologies for the early detection of CAVD would benefit the development of prevention, mitigation and alternate therapeutic strategies. Two-photon excited fluorescence (TPEF) microscopy is a label-free, non-destructive imaging technique that has been shown to correlate with multiple markers for cellular differentiation and phenotypic changes in cancer and wound healing. Here we show how specific TPEF markers, namely, the optical redox ratio and mitochondrial fractal dimension, correlate with structural, functional and phenotypic changes occurring in the aortic valve interstitial cells (VICs) during osteogenic differentiation. The optical redox ratio, and fractal dimension of mitochondria were assessed and correlated with gene expression and nuclear morphology of VICs. The optical redox ratio decreased for VICs during early osteogenic differentiation and correlated with biological markers for CAVD progression. Fractal dimension correlated with structural and osteogenic markers as well as measures of nuclear morphology. Our study suggests that TPEF imaging markers, specifically the optical redox ratio and mitochondrial fractal dimension, can be potentially used as a tool for assessing early CAVD progression in vitro. Calcific aortic stenosis or calcific aortic valve disease (CAVD) is a progressive disease involving multiple signaling pathways, endothelial dysfunction, cytokine infiltration, collagen remodeling, as well as lipid and calcium deposition 1-4. The symptoms and markers for CAVD manifest via both degenerative (apoptotic) and active (osteogenic) mechanisms 5,6. Aortic valve endothelial and interstitial cells differentiate into an osteoblast-like phenotype, the extracellular matrix becomes thicker and stiffer and calcium mineralization occurs throughout the tissue 7,8. Aortic stenosis and sclerosis have an increased prevalence in the elderly and contribute to a 50% elevated risk of infarction and other potentially fatal cardiovascular pathologies 2,9,10. Currently, valve replacement is the preferred treatment method, as other strategies, such as the retardation of calcific progression, prevention and early diagnosis, are non-existent 11. Diagnostic techniques like echocardiography, cardiac MRI and cardiac CT are widely used, but are only sensitive during later stages of the disease once there is tissue mineralization, and hemodynamic and geometric impairment 12,13. Aortic valve interstitial cells (VICs) are the primary cells in the heart valves and are involved in tissue maintenance, repair and remodeling 14. VICs exist in a quiescent state under healthy conditions and are activated due to injury or disease 15 , potentially differentiating into an osteogenic-like phenotype to potentiate calcification 2. Current in vitro biochemical techniques to assess CAVD are typically destructive as they involve cell lysis or fixation and do not facilitate the longitu...

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confidence: 99%

Label-free metabolic biomarkers for assessing valve interstitial cell calcific progression

Tandon

Kolenc

Cross

et al. 2020

Sci Rep

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“…In this case, 2 alternative sets of filters are employed: 1 consisting of a filter FF01‐470/28 (Semrock Inc) and the other 1 of 2 filters—FF500‐Di01 and FF01‐510/84 (Semrock Inc)—placed in cascade. In fact, fluorescence signal was disaggregated in 2 spectral windows—one centered near to the emission maxima of collagen and elastin , the other for detecting fluorescence emission from smooth muscle cells and calcified tissues —in order to test (1) which band has the best discrimination capability and (2) if they provide complementary information to each other. Thus, Table summarizes the spectral ranges observed for each imaging modality.…”

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confidence: 99%

Improved label‐free diagnostics and pathological assessment of atherosclerotic plaques through nonlinear microscopy

Baria

Nesi

Santi

et al. 2018

Journal of Biophotonics

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Coronary heart disease is the most common type of heart disease caused by atherosclerosis. In fact, an arterial wall lesion centered on the accumulation of cholesterol-rich lipids and the accompanying inflammatory response generates a plaque, whose rupture may result in a thrombus with fatal consequences. Plaque characterization for assessing the severity of atherosclerosis is generally performed through standard histopathological examination based on hematoxylin/eosin staining, which is operator-dependent and requires relatively long procedures. In this framework, nonlinear optical microscopy is a valid, label-free alternative to standard diagnostic methods. We combined second-harmonic generation (SHG), two-photon excited fluorescence (TPEF) and fluorescence lifetime imaging microscopy in a multimodal scheme for obtaining morphological and molecular information on human carotid ex vivo specimens affected by atherosclerosis. In this study, discrimination between different tissues within the atherosclerotic plaque was achieved based on both lifetime, TPEF-to-SHG ratio, and image pattern analysis. The presented methodology aims to be a starting point for future fully automated and fast characterization of atherosclerotic biopsies; moreover, it could be extended to the study of other tissues and pathologies. Combined TPEF/SHG mapping of a carotid specimen affected by atherosclerosis.

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“…Collagen emits a strong second-harmonic generation (SHG) signal due to its non-centrosymmetric structure with high second-order nonlinear susceptibility, while elastin, among different endogenous chromophores, such as keratin and melanin, can be visualized by another NLO technique, two-photon excitation fluorescence (TPF) [ 24 ]. In addition, TPF was found to be suitable for the detection of calcium depositions in artery walls and to track calcified nodule growth in aortic valves ex vivo [ 25 , 26 ]. In our present study, we visualized calcification and demonstrated the fragmentation of elastic fibers in the skin of PXE patients utilizing NLO microscopy imaging that allows the histologic diagnosis of the disease.…”

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confidence: 99%

Nonlinear optical microscopy is a novel tool for the analysis of cutaneous alterations in pseudoxanthoma elasticum

et al. 2020

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Pseudoxanthoma elasticum (PXE, OMIM 264800) is a rare autosomal recessive disorder with ectopic mineralization and fragmentation of elastin fibers. It is caused by mutations of the ABCC6 gene that leads to decreased serum levels of inorganic pyrophosphate (PPi) anti-mineralization factor. The occurrence of severe complications among PXE patients highlights the importance of early diagnosis so that prompt multidisciplinary care can be provided to patients. We aimed to examine dermal connective tissue with nonlinear optical (NLO) techniques, as collagen emits second-harmonic generation (SHG) signal, while elastin can be excited by two-photon excitation fluorescence (TPF). We performed molecular genetic analysis, ophthalmological and cardiovascular assessment, plasma PPi measurement, conventional histopathological examination, and ex vivo SHG and TPF imaging in five patients with PXE and five age- and gender-matched healthy controls. Pathological mutations including one new variant were found in the ABCC6 gene in all PXE patients and their plasma PPi level was significantly lower compared with controls. Degradation and mineralization of elastin fibers and extensive calcium deposition in the mid-dermis was visualized and quantified together with the alterations of the collagen structure in PXE. Our data suggests that NLO provides high-resolution imaging of the specific histopathological features of PXE-affected skin. In vivo NLO may be a promising tool in the assessment of PXE, promoting early diagnosis and follow-up.

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Non-destructive two-photon excited fluorescence imaging identifies early nodules in calcific aortic-valve disease

Cited by 33 publications

References 78 publications

Label-free metabolic biomarkers for assessing valve interstitial cell calcific progression

Label-free metabolic biomarkers for assessing valve interstitial cell calcific progression

Improved label‐free diagnostics and pathological assessment of atherosclerotic plaques through nonlinear microscopy

Nonlinear optical microscopy is a novel tool for the analysis of cutaneous alterations in pseudoxanthoma elasticum

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