Name: coordinate mapping of hyolaryngeal mechanics in swallowing
Uploaded: 2014-05-06T15:30:00
Description: Watch this Scientific Journal Video about Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing at JoVE.com

作者承认Kendrea福赫特，CSCD，CCC-SLP和伊夫林特拉梅尔研究院语音和吞咽在南卡罗来纳医科大学分享MBS用来证明这种方法的成像文件。从国家中心的推进转化科学和授权号1K24DC12801（PI：马丁 - 哈里斯）：这些MBS数据是通过授权号TL1TR000061（福赫特PI）的资助外支持从收集国立耳聋与其他交流障碍，并肌壁间支持马克和伊夫林特拉梅尔信任。这些方法最初是由主要研究者开发的，而通过授权号F31DC011705从国立耳聋与其他交流障碍的支持。内容完全是作者的责任，并不一定代表国立耳聋与其他交流障碍或国家机构的官方意见健康的。

1，配置一台电脑<ol><li>对于Macintosh，下载下面的开源或免费软件：ImageJ的，MacX视频转换器免费版（苹果机）和QuickTime（见表格材料/设备）。 </li><li>对于一台PC，下载下面的开源或免费软件：ImageJ的，MPEG Streamclip（PC）和QuickTime（见表格材料/设备）。 </li></ol> 2，准备视频剪辑<ol><li>文件转换。将原始的视频文件转换为。MOV在ImageJ的数据收集。注：可用的视频应该包括下颌骨，...

<ol>
	<li>Leonard, R. J., Kendall, K. A., McKenzie, S., Gon&#231;alves, M. I., Walker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Displacements+in+Normal+Swallowing:+A+Videofluoroscopic+Study.">Structural Displacements in Normal Swallowing: A Videofluoroscopic Study.</a> Dysphagia. 15, 146-152 (2000).</li><li>Molfenter, S. M., Steele, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+Variability+in+the+Deglutition+Literature:+Hyoid+and+Laryngeal+Kinematics.">Physiological Variability in the Deglutition Literature: Hyoid and Laryngeal Kinematics.</a> Dysphagia. 26, 67-74 (2010).</li><li>Pearson, W. G., Hindson, D. F., Langmore, S. E., Zumwalt, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+Swallowing+Muscles+Essential+for+Hyolaryngeal+Elevation+by+Using+Muscle+Functional+Magnetic+Resonance+Imaging.">Evaluating Swallowing Muscles Essential for Hyolaryngeal Elevation by Using Muscle Functional Magnetic Resonance Imaging.</a> International Journal of Radiation Oncology* Biology* Physics. 85, 735-740 (2013).</li><li>Pearson, W. G., Langmore, S. E., Yu, L. B., Zumwalt, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Analysis+of+Muscles+Elevating+the+Hyolaryngeal+Complex.">Structural Analysis of Muscles Elevating the Hyolaryngeal Complex.</a> Dysphagia. 27, 445-451 (2012).</li><li>Kim, Y., McCullough, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximum+hyoid+displacement+in+normal+swallowing.">Maximum hyoid displacement in normal swallowing.</a> Dysphagia. 23, 274-279 (2008).</li><li>Steele, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+hyoid+and+laryngeal+displacement+and+swallowing+impairment.">The relationship between hyoid and laryngeal displacement and swallowing impairment.</a> Clin. Otolaryngol. 36, 30-36 (2011).</li><li>Logemann, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+Biomechanical+Characteristics+of+Oropharyngeal+Swallow+in+Younger+and+Older+Men.">Temporal and Biomechanical Characteristics of Oropharyngeal Swallow in Younger and Older Men.</a> Journal of Speech, Language and Hearing Research. 43, 1264-1274 (2000).</li><li>Pearson, W., Langmore, S., Zumwalt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+Structural+Properties+of+Suprahyoid+Muscles+and+their+Potential+for+Moving+the+Hyoid.">Evaluating the Structural Properties of Suprahyoid Muscles and their Potential for Moving the Hyoid.</a> Dysphagia. 26, 345-351 (2011).</li><li>Hopkins, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measures+of+reliability+in+sports+medicine+and+science.">Measures of reliability in sports medicine and science.</a> Sports Med. 30, 1-15 (2000).</li><li>Bingjie, L., Zhang, T., Sun, X., Xu, J., Jiang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+videofluoroscopic+analysis+of+penetration-aspiration+in+post-stroke+patients.">Quantitative videofluoroscopic analysis of penetration-aspiration in post-stroke patients.</a> Neurol. India. 58, 42-47 (2010).</li><li>Webster, M., Sheets, H. D., Alroy, J., Hunt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+introduction+to+landmark-based+geometric+morphometrics.">A practical introduction to landmark-based geometric morphometrics.</a> Quantitative Methods in Paleobiology. Paleontological Society Papers. 16, 163-188 (2010).</li><li>Inamoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+swallowing+using+320-detector-row+multislice+CT.+Part+II:+Kinematic+analysis+of+laryngeal+closure+during+normal+swallowing.">Evaluation of swallowing using 320-detector-row multislice CT. Part II: Kinematic analysis of laryngeal closure during normal swallowing.</a> Dysphagia. 26, 209-217 (2011).</li></ol>

这项研究表明，利用坐标解剖标志数据来计算吞咽hyolaryngeal运动的多个运动测量的方法的有效性。六评价者，其中包括两名新手评价者，对于坐标和​​计算变量间信度为强（ICC&gt; 0.90）。来自健康非吞咽困难的成年人的随机样本的代表性结果表明响应于2丸剂类型的几个运动学参数的差异。我们还发现，使用的参考不同的轴，用于计算舌骨的偏移产生的未很强的相关性的结果。 <p class="jove_cont...

吞咽的咽部阶段是一个涉及超过20的肌肉和骨骼的多个元件，以从口腔传送推注到食道，同时保护气道的复杂过程。前咽收缩时，hyolaryngeal复杂的元素（舌骨，喉，及相关结构，包括食管上括约肌）被移动到一个呼吸管转换成消化道。喉部被重新位于前方远离迎面而来的大丸剂的轨迹，和上部食道括约肌被拉伸开。从视频透视吞咽研究（也称为MBS或修改吞钡研究）采取动态测量的主要研究方法为定量hyolaryngeal配合物1的多个动作。 而定量的视频透视测量是测量吞咽功能，参考不同轴有用的测定结果的标量中的发现是其中运动测量2的各种方法不兼容。根据临床医生手动控制的患者和透视的运动也颠倒测量这种复杂的生理过程的准确性。更重要的是，运动测量不一定反映结构到功能关系的评价无序吞咽重要。特别舌骨的运动学已设计为在参考与椎骨对准的解剖平面的前部或向上的方向跟踪的移动。然而，这种配置并不代表肌肉暂停舌骨的作用线。 hyolaryngeal仰角在吞咽的咽部阶段的双吊索机制已被确定（ 图1）3,4。舌骨上肌群包括前肌吊索，以及长咽肌包括在posterior肌肉吊索。这些肌肉提升hyolaryngeal复杂的各种元素，包括舌骨，喉，并且形成上部食道括约肌结构。 hyolaryngeal力学的坐标映射利用9容易辨认解剖标志映射的前部和后部肌肉吊索（ 图2）的hyolaryngeal复杂相当于附着点三个骨骼杠杆和功能。在吞咽时，hyolaryngeal复杂的每个骨骼杆，特点是在运动中。通过收集坐标，该系统可以被捕获在任何时间帧。坐标的三角转换可用于吞咽时产生hyolaryngeal运动的多个运动测量。变量可以计算出与文献中报道的结果作比较，或用于生成表示感兴趣结构到功能关系的新的测量。 本文的主要目的是展示从生成一组改自吞钡（MBS）的​​研究中收集的解剖标志坐标计算多个运动测量的方法。我们通过使用组内相关系数来确定的6种不同的调查，包括一位专家，三评价者与经验，以及两个新手的间信度记录此方法的可靠性。从运动成绩，通过团一致性吞咽机制的差异进行评估。最后，关于在测量舌骨运动中使用的参考轴的重要性提出Molefenter和Steele的问题得到解决。处理这个问题，我们比较在参照椎骨和在参考了下颌骨，由两个大丸剂类型相同的一组坐标的计算舌骨漂移的测量结果。如果测量舌骨移动的这两种方法表示相同的结构的功能关系髋关节，那么结果应该密切相关。 在这项研究中，40侧面观的MBS研究随机抽取的批准下，由摄政佐治亚大学机构审查委员会，并与伊夫林特拉梅尔研究所语音研究合作和吞咽时医科大学研究方案139正常学习的集合中选择南卡罗来纳州。为了证明这种方法的效用，十变量表征hyolaryngeal运动学计算从同一组的坐标数据（ 表1）。这些测量计算的七个先前已在文献中，包括使用：前部和参照椎骨5优越的舌骨距离测量;前壁和优越舌骨位移为C2-4长度的比，也参照椎骨6;在参考了椎骨7喉部优越的运动; hyolaryngeal approximat离子1;并在参考椎骨1最大舌骨游览。此外，分别计算三种新型测量：咽缩短逼近palatopharyngeus肌肉的附 ​​件，喉海拔下面有一行的行动代表茎突和舌骨漂移近似舌骨肌4,8的附件。 专家头部和颈部解剖（WP），3名调查员的有限经验，采取测量（CJ，SR，TT），和两个新手调查（RS，JT）获得使用下面描述的协议坐标映射数据。专家（WP）训练三个评价者与经验，而这些反过来又训练有素的两个新手评价者。按主题由坐标计算的坐标数据和结果间信度是由组内相关系数9确定。双尾t检验对各变量进行判断statisticall在弹丸类型Ÿ显著差异。 Pearson相关系数和决定系数被用来评估与椎骨作为参考与舌骨游览与下颌骨作为对5毫升薄燕液和5ml的参考轴的轴计算舌骨漂移的结果之间的协议布丁燕子。

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coordinate mapping of hyolaryngeal mechanics in swallowing

表征hyolaryngeal运动是很重要的吞咽困难的研究。在此之前方法需要多次测量，以获得一个动态的测量，而使用的坐标改良吞钡（MBS），使用一组坐标来计算利息多个变量的hyolaryngeal力学的映射。出于演示的目的，是从一组坐标生成10运动测量，以确定在吞咽两种不同类型的丸差异。对椎骨和下颌骨舌骨偏移的计算是相关的确定的基准轴的重要性。 为了证明坐标映射方法，40 MBS研究随机抽取健康正常人的数据集选择的，没有已知的吞咽障碍。将5ml薄的液体推注和5毫升的布丁燕子从每个对象进行测量。九坐标，绘制颅底，下颌骨，椎骨和孝的元素喉癌复杂的，被记录在最小和最大hyolaryngeal偏移的帧。坐标进行数学转换成hyolaryngeal力学十个变量。 间信度是由组内相关系数（ICC）的评估。双尾t-检验来评估通过推粘度运动学的差异。针对参考不同轴舌骨游览测量相关。在六个评估者为18坐标间信度介于ICC = 0.90 - 0.97。十大运动测量值的石板是由六个评价者之间的被摄体相比。一个异常被拒绝，其余的可靠性分数的平均值为ICC = 0.91，0.84 - 0.96，95％CI。双尾t-检验与比较10运动学变量（5毫升薄液与5毫升布丁燕子）邦费罗尼修正显示，舌骨游览，喉上运动，咽shortenin统计学显著差异G（P &lt;0.005）。舌骨偏移测量从参考两个不同的轴皮尔逊相关系数分别为：R = 0.62，R 2 = 0.38，（薄液）; R = 0.52，R 2 = 0.27，（布丁）。 取得具有里程碑意义的坐标是一种可靠的方法来生成视频透视图像的吞咽困难研究有用的多运动学变量。

组内相关系数由六个调查谁独立分析80视频透视文件（从40名受试者2丸试行）采集坐标（ICC）介于ICC = 0.90 - 0.97。国际刑事法院的坐标由A组分类如下：坐标＃1 - 5（至少hyolaryngeal游览骨骼元素）平均= 0.93，0.91 - 0.95，95％CI;坐标＃6 - 9（hyolaryngeal复杂最小hyolaryngeal偏移）平均= 0.94，092 - 0.96，95％CI;坐标＃10 - 14（最大hyolaryngeal偏移骨骼元素）平均= 0.93，0.91 - 0.95，95％CI;并协调＃15 - 18（hyola...

Watch this Scientific Journal Video about Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing at JoVE.com

Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing

坐标映射记录是在吞咽的咽部阶段hyolaryngeal生物力学的显着特征的方法。此方法使用图像分析软件来记录的解剖标志坐标。这些坐标被导入到一​​个Excel宏并翻译成兴趣在吞咽障碍的研究有用的运动学变量。

协调Hyolaryngeal力学映射在吞咽

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement ...

coordinate-mapping-of-hyolaryngeal-mechanics-in-swallowing

Research

JoVE Journal

Medicine

Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. 
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins&#x2019; circulations. 
Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

导丝辅助导管和彩色染料注射的双人单绒毛膜胎盘血管映射

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC ...

科研

医学

High-Resolution Endocardial and Epicardial Optical Mapping in a Sheep Model of Stretch-Induced Atrial Fibrillation

Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance seen in clinical practice and its prevalence is expected to increase in the coming years.3 Increased intra-atrial pressure and dilatation have been long recognized to lead to AF,1,4 which highlights the relevance of using animal models and stretch to study AF dynamics. Understanding the mechanisms underlying AF requires visualization of the cardiac electrical waves with high spatial and temporal resolution. While high-temporal resolution can be achieved by conventional electrical mapping traditionally used in human electrophysiological studies, the small number of intra-atrial electrodes that can be used simultaneously limits the spatial resolution and precludes any detailed tracking of the electrical waves during the arrhythmia. The introduction of optical mapping in the early 90's enabled wide-field characterization of fibrillatory activity together with sub-millimeter spatial resolution in animal models5,6 and led to the identification of rapidly spinning electrical wave patterns (rotors) as the sources of the fibrillatory activity that may occur in the ventricles or the atria.7-9 Using combined time- and frequency-domain analyses of optical mapping it is possible to demonstrate discrete sites of high frequency periodic activity during AF, along with frequency gradients between left and right atrium. The region with fastest rotors activates at the highest frequency and drives the overall arrhythmia.10,11 The waves emanating from such rotor interact with either functional or anatomic obstacles in their path, resulting in the phenomenon of fibrillatory conduction.12 Mapping the endocardial surface of the posterior left atrium (PLA) allows the tracking of AF wave dynamics in the region with the highest rotor frequency. Importantly, the PLA is the region where intracavitary catheter-based ablative procedures are most successful terminating AF in patients,13 which underscores the relevance of studying AF dynamics from the interior of the left atrium. Here we describe a sheep model of acute stretch-induced AF, which resembles some of the characteristics of human paroxysmal AF. Epicardial mapping on the left atrium is complemented with endocardial mapping of the PLA using a dual-channel rigid borescope c-mounted to a CCD camera, which represents the most direct approach to visualize the patterns of activation in the most relevant region for AF maintenance.

This report provides a detailed description of the methodology and results of simultaneous endocardial and epicardial optical mapping of electrical excitation in the intact left atrium of a Langendorff-perfused sheep heart during stretch-induced atrial fibrillation.

高分辨率的心内膜和心外膜光学在拉伸诱发心房颤动的羊模型的映射

 Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance ...

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

在使用定量免疫肿瘤的异质性蛋白表达的映射

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.
cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.
Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).

Contactless dielectrophoresis (cDEP) achieves sorting and enrichment of particles via their intrinsic dielectric properties. Fluidic electrode channels replace metallic electrodes traditional to DEP, suiting cDEP to non-damaging sterile characterization and sorting of biological particles. We demonstrate how to prepare a cDEP microdevice and conduct cell characterization and sorting experiments.

无标记的分离和细胞的富集通过非接触式介电泳

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to ...

MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. Therefore, the ability of the blood vessels to dilate or constrict, known as Cerebral-Vascular-Reactivity (CVR), represents an important domain of vascular function. An imaging marker representing this dynamic property will provide new information of cerebral vessels under normal and diseased conditions such as stroke, dementia, atherosclerosis, small vessel diseases, brain tumor, traumatic brain injury, and multiple sclerosis. In order to perform this type of measurement in humans, it is necessary to deliver a vasoactive stimulus such as CO2 and/or O2 gas mixture while quantitative brain magnetic resonance images (MRI) are being collected. In this work, we presented a MR compatible gas-delivery system and the associated protocol that allow the delivery of special gas mixtures (e.g., O2, CO2, N2, and their combinations) while the subject is lying inside the MRI scanner. This system is relatively simple, economical, and easy to use, and the experimental protocol allows accurate mapping of CVR in both healthy volunteers and patients with neurological disorders. This approach has the potential to be used in broad clinical applications and in better understanding of brain vascular pathophysiology. In the video, we demonstrate how to set up the system inside an MRI suite and how to perform a complete experiment on a human participant.

Non-invasive imaging of the brain vasculature&#8217;s ability to dilate or constrict may allow a better understanding of cerebrovascular pathophysiology in various neurological diseases. The present report describes a reproducible and patient-comfortable protocol to perform vascular reactivity imaging in humans using magnetic resonance imaging (MRI).

脑血管反应性通过气体吸入挑战MRI映射

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

胃食管反流与小鼠手术模型

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

高分辨率光学制图对小鼠心房电生理的评价

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

以质子磁共振成像和氧为造影剂对人肺特异性通气进行定量定位

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

使用电子地图的光映射数据的高通量分析

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins &#8212;&#160;&#945;-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) &#8212; and one M-line protein &#8212; Myomesin-2 (Myom2) &#8212;&#160;were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.

This method can be used to examine sarcomere shortening using pluripotent stem cell-derived cardiomyocytes with fluorescent-tagged sarcomere proteins.

使用荧光标记肉瘤蛋白缩短多能干细胞衍生心肌细胞的肉瘤。

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells ...