Neal Shepherd

Doppler velocity detection limitations in spectrometer-based versus swept-source optical coherence tomography

Recent advances in Doppler techniques have enabled high sensitivity imaging of biological flow to measure blood velocities and vascular perfusion. Here we compare spectrometer-based and wavelength-swept Doppler OCT implementations theoretically and experimentally, characterizing the lower and upper observable velocity limits in each configuration. We specifically characterize the washout limit for Doppler OCT, the velocity at which signal degradation results in loss of flow information, which is valid for both quantitative and qualitative flow imaging techniques. We also clearly differentiate the washout effect from the separate phenomenon of phase wrapping. We demonstrate that the maximum detectable Doppler velocity is determined by the fringe washout limit and not phase wrapping. Both theory and experimental results from phantom flow data and retinal blood flow data demonstrate the superiority of the swept-source technique for imaging vessels with high flow rates.

In vivo spectral domain optical coherence tomography volumetric imaging and spectral Doppler velocimetry of early stage embryonic chicken heart development

Progress toward understanding embryonic heart development has been hampered by the inability to image embryonic heart structure and simultaneously measure blood flow dynamics in vivo. We have developed a spectral domain optical coherence tomography system for in vivo volumetric imaging of the chicken embryo heart. We have also developed a technique called spectral Doppler velocimetry (SDV) for quantitative measurement of blood flow dynamics. We present in vivo volume images of the embryonic heart from initial tube formation to development of endocardial cushions of the same embryo over several stages of development. SDV measurements reveal the influence of heart tube structure on blood flow dynamics.

Synthetic wavelength based phase unwrapping in spectral domain optical coherence tomography

Phase sensing implementations of spectral domain optical coherence tomography (SDOCT) have demonstrated the ability to measure nanometer-scale temporal and spatial profiles of samples. However, the phase information suffers from a 2pi ambiguity that limits observations of larger sample displacements to lengths less than half the source center wavelength. We introduce a synthetic wavelength phase unwrapping technique in SDOCT that uses spectral windowing and corrects the 2pi ambiguity, providing accurate measurements of sample motion with information gained from standard SDOCT processing. We demonstrate this technique by using a common path implementation of SDOCT and correctly measure phase profiles from a phantom phase object and human epithelial cheek cells which produce multiple wrapping artifacts. Using a synthetic wavelength for phase unwrapping could prove useful in Doppler or other phase based implementations of OCT.

Temperature‐sensitive TREK currents contribute to setting the resting membrane potential in embryonic atrial myocytes

TREK channels belong to the superfamily of two‐pore‐domain K+ channels and are activated by membrane stretch, arachidonic acid, volatile anaesthetics and heat. TREK‐1 is highly expressed in the atrium of the adult heart. In this study, we investigated the role of TREK‐1 and TREK‐2 channels in regulating the resting membrane potential (RMP) of isolated chicken embryonic cardiac myocytes. At room temperature, the average RMP of embryonic day (ED) 11 atrial myocytes was −22 ± 2 mV. Raising the temperature to 35°C hyperpolarized the membrane to −69 ± 2 mV and activated a large outwardly rectifying K+ current that was relatively insensitive to conventional K+ channel inhibitors (TEA, 4‐AP and Ba2+) but completely inhibited by tetracaine (200 μm), an inhibitor of TREK channels. The heat‐induced hyperpolarization was mimicked by 10 μm arachidonic acid, an agonist of TREK channels. There was little or no inwardly rectifying K+ current (IK1) in the ED11 atrial cells. In marked contrast, ED11 ventricular myocytes exhibited a normal RMP (−86.1 ± 3.4 mV) and substantial IK1, but no temperature‐ or tetracaine‐sensitive K+ currents. Both RT‐PCR and real‐time PCR further demonstrated that TREK‐1 and TREK‐2 are highly and almost equally expressed in ED11 atrium but much less expressed in ED11 ventricle. In addition, immunofluorescence demonstrated TREK‐1 protein in the membrane of atrial myocytes. These data indicate the presence and function of TREK‐1 and TREK‐2 in the embryonic atrium. Moreover, we demonstrate that TREK‐like currents have an essential role in determining membrane potential in embryonic atrial myocytes, where IK1 is absent.

Synthetic wavelength-based phase unwrapping in Fourier domain optical coherence tomography

Phase-sensitive adjuncts to optical coherence tomography (OCT) including Doppler and polarization-sensitive implementations allow for quantitative depth-resolved measurements of sample structure and dynamics including fluid flows and orientation of birefringent structures. The development of Fourier-domain OCT (FDOCT), particularly spectrometer-based spectral-domain systems with no moving parts (spectral-domain OCT or SDOCT), have greatly enhanced the phase stability of OCT systems particularly when implemented in a common-path geometry. The latter combination has given rise to a new class of nm-scale sensitive quantitative phase microscopies we have termed spectral domain phase microscopy. However, the phase information in all of these techniques suffers from a 2π ambiguity that limits resolvable pathlength differences to less than half the source center wavelength. This is problematic for situations such as cellular imaging, Doppler velocimetry, or polarization sensitive applications where it may be necessary to monitor sample profiles, displacements, phase differences, or refractive index variations which vary rapidly in space or time. A technique previously introduced in phase shifting interferometry uses phase information from multiple wavelengths to overcome this limitation. We show that by appropriate spectral windowing of the broadband light source already used in OCT, particularly by reshaping the source spectrum about two different center wavelengths, the resulting phase variation may be cast in terms of a much longer synthetic wavelength chosen to span the phase variation of interest. We show theoretically that the optimal choice of synthetic wavelength depends upon a tradeoff between the minimum resolvable phase and the length of unambiguous phase measurement. We demonstrate this technique using a broadband source centered at 790 nm by correctly reconstructing the phase profile from a phantom sample containing multiple 2π wrapping artifacts at the center wavelength and compare our result with atomic force microscopy.

Rapid fusion between mesenchymal stem cells and cardiomyocytes yields electrically active, non-contractile hybrid cells

Cardiac cell therapies involving bone marrow-derived human mesenchymal stem cells (hMSCs) have shown promising results, although their mechanisms of action are still poorly understood. Here, we investigated direct interactions between hMSCs and cardiomyocytes in vitro. Using a genetic Ca2+ indicator gCaMP3 to efficiently label hMSCs in co-cultures with neonatal rat ventricular myocytes (NRVMs), we determined that 25–40% of hMSCs (from 4 independent donors) acquired periodic Ca2+ transients and cardiac markers through spontaneous fusion with NRVMs. Sharp electrode and voltage-clamp recordings in fused cells showed action potential properties and Ca2+ current amplitudes in between those of non-fused hMSCs and NRVMs. Time-lapse video-microscopy revealed the first direct evidence of active fusion between hMSCs and NRVMs within several hours of co-culture. Application of blebbistatin, nifedipine or verapamil caused complete and reversible inhibition of fusion, suggesting potential roles for actomyosin bridging and Ca2+ channels in the fusion process. Immunostaining for Cx43, Ki67, and sarcomeric α-actinin showed that fused cells remain strongly coupled to surrounding NRVMs, but downregulate sarcomeric structures over time, acquiring a non-proliferative and non-contractile phenotype. Overall, these results describe the phenotype and mechanisms of hybrid cell formation via fusion of hMSCs and cardiomyocytes with potential implications for cardiac cell therapy.

Calcium Dependent CAMTA1 in Adult Stem Cell Commitment to a Myocardial Lineage

The phenotype of somatic cells has recently been found to be reversible. Direct reprogramming of one cell type into another has been achieved with transduction and over expression of exogenous defined transcription factors emphasizing their role in specifying cell fate. To discover early and novel endogenous transcription factors that may have a role in adult-derived stem cell acquisition of a cardiomyocyte phenotype, mesenchymal stem cells from human and mouse bone marrow and rat liver were co-cultured with neonatal cardiomyocytes as an in vitro cardiogenic microenvironment. Cell-cell communications develop between the two cell types as early as 24 hrs in co-culture and are required for elaboration of a myocardial phenotype in the stem cells 8–16 days later. These intercellular communications are associated with novel Ca2+ oscillations in the stem cells that are synchronous with the Ca2+ transients in adjacent cardiomyocytes and are detected in the stem cells as early as 24–48 hrs in co-culture. Early and significant up-regulation of Ca2+-dependent effectors, CAMTA1 and RCAN1 ensues before a myocardial program is activated. CAMTA1 loss-of-function minimizes the activation of the cardiac gene program in the stem cells. While the expression of RCAN1 suggests involvement of the well-characterized calcineurin-NFAT pathway as a response to a Ca2+ signal, the CAMTA1 up-regulated expression as a response to such a signal in the stem cells was unknown. Cell-cell communications between the stem cells and adjacent cardiomyocytes induce Ca2+ signals that activate a myocardial gene program in the stem cells via a novel and early Ca2+-dependent intermediate, up-regulation of CAMTA1.

Developmental expression of a functional TASK-1 2P domain K+ channel in embryonic chick heart

BackgroundBackground K+ channels are the principal determinants of the resting membrane potential (RMP) in cardiac myocytes and thus, influence the magnitude and time course of the action potential (AP).MethodsRT-PCR and in situ hybridization are used to study the distribution of TASK-1 and whole-cell patch clamp technique is employed to determine the functional expression of TASK-1 in embryonic chick heart.ResultsChicken TASK-1 was expressed in the early tubular heart, then substantially decreased in the ventricles by embryonic day 5 (ED5), but remained relatively high in ED5 and ED11 atria. Unlike TASK-1, TASK-3 was uniformly expressed in heart at all developmental stages. In situ hybridization studies further revealed that TASK-1 was expressed throughout myocardium at Hamilton-Hamburger stages 11 and 18 (S11 & S18) heart. In ED11 heart, TASK-1 expression was more restricted to atria. Consistent with TASK-1 expression data, patch clamp studies indicated that there was little TASK-1 current, as measured by the difference currents between pH 8.4 and pH 7.4, in ED5 and ED11 ventricular myocytes. However, TASK-1 current was present in the early embryonic heart and ED11 atrial myocytes. TASK-1 currents were also identified as 3 μM anandamide-sensitive currents. 3 μM anandamide reduced TASK-1 currents by about 58% in ED11 atrial myocytes. Zn2+ (100 μM) which selectively inhibits TASK-3 channel at this concentration had no effect on TASK currents. In ED11 ventricle where TASK-1 expression was down-regulated, IK1 was about 5 times greater than in ED11 atrial myocytes.ConclusionFunctional TASK-1 channels are differentially expressed in the developing chick heart and TASK-1 channels contribute to background K+ conductance in the early tubular embryonic heart and in atria. TASK-1 channels act as a contributor to background K+ current to modulate the cardiac excitability in the embryonic heart that expresses little IK1.

Doppler velocity detection limitations in spectrometer and swept-source Fourier-domain optical coherence tomography

Recent advances in Doppler and variance techniques have enabled high sensitivity imaging in regions of biological flow to measure blood velocities and vascular perfusion. In recent years, the sensitivity and imaging speed benefits of Fourier domain OCT have become apparent. Spectrometer-based and wavelength-swept implementations have both undergone rapid development. Comparative analysis of the potential benefits and limitations for the various configurations would be useful for matching technology capabilities to specific clinical problems. Here we take a first step in such a comparative analysis by presenting theoretical predictions and experimental results characterizing the lower and upper observable velocity limits in spectrometer-based versus swept-source Doppler OCT. Furthermore, we characterize the washout limit, the velocity at which signal degradation results in loss of flow information. We present comparative results from phantom flow data as well as retinal data obtained with a commercial spectrometer OCT system and a custom high-speed swept-source retinal OCT system.

Quantitative Measurement of Hemodynamics during Early Cardiovascular Development Using Spectral Doppler Velocimetry

The study of hemodynamic effects on embryonic cardiovascular development has been limited by technology. Here we present an extension to Doppler OCT which may provide insight in the relationship between blood flow and heart development.