Whole-cell-analysis of live cardiomyocytes using wide-field interferometric phase microscopy.

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2010-08-23

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Abstract

We apply wide-field interferometric microscopy techniques to acquire quantitative phase profiles of ventricular cardiomyocytes in vitro during their rapid contraction with high temporal and spatial resolution. The whole-cell phase profiles are analyzed to yield valuable quantitative parameters characterizing the cell dynamics, without the need to decouple thickness from refractive index differences. Our experimental results verify that these new parameters can be used with wide field interferometric microscopy to discriminate the modulation of cardiomyocyte contraction dynamics due to temperature variation. To demonstrate the necessity of the proposed numerical analysis for cardiomyocytes, we present confocal dual-fluorescence-channel microscopy results which show that the rapid motion of the cell organelles during contraction preclude assuming a homogenous refractive index over the entire cell contents, or using multiple-exposure or scanning microscopy.

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10.1364/BOE.1.000706

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Shaked, Natan T, Lisa L Satterwhite, Nenad Bursac and Adam Wax (2010). Whole-cell-analysis of live cardiomyocytes using wide-field interferometric phase microscopy. Biomed Opt Express, 1(2). pp. 706–719. 10.1364/BOE.1.000706 Retrieved from https://hdl.handle.net/10161/8427.

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Scholars@Duke

Satterwhite

Lisa L Satterwhite

Assistant Research Professor in the Department of Civil and Environmental Engineering

Molecular epidemiology of environmental exposures, pesticide exposure and Parkinson’s disease, Cyanobacterial harmful algal blooms and ALS, and creating climate change resilient rural communities.

Bursac

Nenad Bursac

Professor of Biomedical Engineering

Bursac's research interests include: Stem cell, tissue engineering, and gene based therapies for heart and muscle regeneration; Cardiac electrophysiology and arrhythmias; Organ-on-chip and tissue engineering technologies for disease modeling and therapeutic screening; Small and large animal models of heart and muscle injury, disease, and regeneration.

The focus of my research is on application of pluripotent stem cells, tissue engineering, and gene therapy technologies for: 1) basic studies of striated muscle biology and disease in vitro and 2) regenerative therapies in small and large animal models in vivo. For in vitro studies, micropatterning of extracellular matrix proteins or protein hydrogels and 3D cell culture are used to engineer rodent and human striated muscle tissues that replicate the structure-function relationships present in healthy and diseased muscles. We use these models to separate and systematically study the roles of structural and genetic factors that contribute cardiac and skeletal muscle function and disease at multiple organizational levels, from single cells to tissues. Combining cardiac and skeletal muscle cells with primary or iPSC-derived non-muscle cells (endothelial cells, smooth muscle cells, immune system cells, neurons) allows us to generate more realistic models of healthy and diseased human tissues and utilize them to mechanistically study molecular and cellular processes of tissue injury, vascularization, innervation, electromechanical integration, fibrosis, and functional repair. Currently, in vitro models of Duchenne Muscular Dystrophy, Pompe disease, dyspherlinopathies, and various cardiomyopathies are studied in the lab. For in vivo studies, we employ rodent models of volumetric skeletal muscle loss, cardiotoxin and BaCl2 injury as well as myocardial infarction and transverse aortic constriction to study how cell, tissue engineering, and gene (viral) therapies can lead to safe and efficient tissue repair and regeneration. In large animal (porcine) models of myocardial injury and arrhythmias, we are exploring how human iPSC derived heart tissue patches and application of engineered ion channels can improve cardiac function and prevent heart failure or sudden cardiac death.

 

Wax

Adam P. Wax

Professor of Biomedical Engineering

Dr. Wax's research interests include optical spectroscopy for early cancer detection, novel microscopy and interferometry techniques.

The study of intact, living cells with optical spectroscopy offers the opportunity to observe cellular structure, organization and dynamics in a way that is not possible with traditional methods. We have developed a set of novel spectroscopic techniques for measuring spatial, temporal and refractive structure on sub-hertz and sub-wavelength scales based on using low-coherence interferometry (LCI) to detect scattered light. We have applied these techniques in different types of cell biology experiments. In one experiment, LCI measurements of the angular pattern of backscattered light are used to determine non-invasively the structure of sub-cellular organelles in cell monolayers, and the components of epithelial tissue from freshly excised rat esophagus. This work has potential as a diagnostic method for early cancer detection. In another experiment, LCI phase measurements are used to examine volume changes of epithelial cells in a monolayer in response to environmental osmolarity changes. Although cell volume changes have been measured previously, this work demonstrates for the first time the volume of just a few cells (2 or 3) tracked continuously and in situ.


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