国立卫生研究院拨款R01 HL085369。

1。股票方案的高级准备<ol><li>准备提前在去离子水台氏液（16倍）的股票解决方案，它们存储在4 ° C时： <ol class="lalpha"><li>股票我（119.872克/ L的NaCl，3.056 g / L的氯化钙2（H 2 O），5.6 g / L的氯化钾，2.6274克/升的NaH 2 PO 4，3.408 g / L的氯化镁2（H 2 O），（费舍尔科学，公平的草坪，NJ））; </li><li>证券交易所II（26.88 g / L的碳酸氢钠，（费舍尔科学，公平的草坪，新泽西州））。 </li></ol></li><li>准备荧光染料的股票解决方案。为了避免反复冻融，我们店30μL分装于-20 ° C，这是一个实验是足够了两种染料： <ol class="lalpha"><li>电压敏感染料RH237（Invitrogen公司，卡尔斯巴德，加利福尼亚州）原液，1.25 mg / ml的二甲基亚砜溶液（DMSO，Sigma公司，圣路易斯，密苏里州）; </li><li>钙指标Rhod - 2AM（Invitrogen公司，卡尔斯巴德，加利福尼亚州）股票的解决方案，为1 mg / ml的溶液，在DMSO。 </li></ol></li><li>准备兴奋收缩解偶联剂blebbistatin原液（Tocris生物科学，圣路易斯，密苏里州，为2 mg / ml的溶液，在DMSO）提前溶解blebbistatin存储在4 ° C。 </li></ol> 2。准备灌注的解决方案和实验装置7 <ol start="4"><li>刚准备2L台氏液（128.2mM氯化钠，氯化钙为1.3mm 2（H 2 O），4.7毫米氯化钾，氯化镁1.05mM 2（H 2 O），1.19mM的NaH 2 PO 4，20MM 11.1mM碳酸氢钠3，D -葡萄糖在去离子水，pH值= 7.35 ± 0.05）。如果正在使用原液，使台氏液（足够一个实验）在我的股票，证券交易所二125毫升125毫升1750毫升的去离子水和混合，和4G的葡萄糖2L。 </li><li>打开两个泵灌注系统。设置蠕动泵（围之星，WPI的，萨拉索塔，美国）是逆行灌注40毫升/分钟。设置其他蠕动泵（科尔 - 帕默的Masterflex Peristalic泵升/秒，科尔 - 帕默仪器公司，伊利诺伊州弗农山，）使用灌流和返回灌流控股水库至80毫升/分钟。 </li><li> 2L去离子水30分钟，然后用70％乙醇的清洗灌注系统。 </li><li>一旦所有的去离子水是从室疏散，分发台氏液，并通过5微米过滤器（Millipore公司，比尔里卡，MA，USA）。灌流预热至37 °与水套循环（ThermoNESLAB EX7，现代城，美国）和冒泡到解决方案中的 O 2 / CO 2（95％/ 5％）气体氧气灌流。监测pH计（Oakton仪器，弗农山，IL）溶液的pH值和调整的O 2 / CO 2冒泡率保持在7.35 ± 0.05 pH值。继续监测实验过程中的pH值和温度。 </li><li>双光学测绘仪器由两个MiCAM终极- L的CMOS摄像头（SciMedia，科斯塔梅萨，CA），有较高的空间（100x100像素，每个像素的230 ± 20微米）和时间（1,000-3,000帧/秒）的分辨率。修正一个带通滤波器（590 ± 15纳米，Thorlabs，牛顿，新泽西州）在指定的钙成像相机面前;同时，长通滤波器（&gt; 700纳米，Thorlabs，牛顿，新泽西州）需要被定位在前面指定的电压成像相机。该相机是垂直排列的持有人，其中包含了分色镜（伯瑞特波罗（Brattleboro），欧米茄光，635 nm的截止，VT）。持有的双摄像头下方有一个镜头（尼康尼克尔55毫米1:1.4 235052），其重点是从心脏到分色镜发射光。工作距离大约是3厘米。 激发光所产生的卤素灯（新港奥丽尔仪器，斯特拉特福，CT; SciMedia，科斯塔梅萨，加利福尼亚），并通过热过滤器，快门，和带通滤波器（520 ± 45纳米）通过。一个灵活的导光引导到准备带通滤波的光，快门是用来确保该制剂是暴露在光线下图像采集期间，才避免了染料的漂白。 </li><li>准备提前起搏和传感2银/氯化银电极放在心脏前室安装。确保放大器和滤波器调整到适当水平。 </li></ol> 3。收获的小鼠心脏，cannulate，并成立了Langendorff灌流<ol start="10"><li>鼠标麻醉与氯胺酮/甲苯​​噻嗪（氯胺酮，80mg/kg体重;甲苯噻嗪，10毫克/公斤体重）和腹腔注射肝素（100单位）。保证适当的麻醉水平，缺乏痛觉反射。 </li><li>中旬胸骨正中切口后，迅速 ​​取出心脏和洗中含氧（95％O2，5％的CO 2），恒温（37 ± 1 ° C）台氏液。 </li><li>使用解剖显微镜迅速查明整个下面的右锁骨下动脉升主动脉主动脉和一个干净的切割。短款的主动脉，然后连接到一个自定义与燃烧尖21号套管。 4-0黑色编织丝线缝合（外科专业公司，读，PA）是用于固定到套管的心脏。后插管，心脏逆行灌注，并与台氏液灌流。逆行灌注率调整在2-5毫升/分钟，以保持动脉压60至80毫米汞柱（压力传感器，世界精密仪器公司（WPI），萨拉索塔，美国的范围;桥式放大器TBM4M，WPI，萨拉索塔美国）。 </li><li>是空心的心脏后，肺，胸腺和脂肪组织，然后解剖和删除。 </li><li>离体心脏灌注室底部的顶点（Sylgard涂层），以防止流诱导运动是固定的（精细科学的工具）。左，右心耳也拉伸和固定（精细的Foster City，CA公司，科学工具）腔的底部，它提供了心房的光学测量的最大表面面积。 很重要的！一个小的硅管插入左心室，通过肺静脉和丝线缝合固定在附近的结缔组织。这防止拥堵被困在左心室，这是抑制与兴奋收缩解偶联剂的室性早搏后显得尤为重要（见第4部分第19步）灌流和酸化的解决方案。 </li><li>自定义的电极放置在心脏表面进行起搏刺激，这是由主8“（AMP仪器有限公司，耶路撒冷，以色列）或PowerLab系统26T（公元仪器，悉尼，澳大利亚）。 </li><li>一个小玻璃盖是固定的解决方案，在振动的解决方案，以减少运动伪影的心脏表面上。 </li><li>重点对心脏的激发光。此外，调整双摄像头设备，使最高分辨率将获得心脏之间的距离。 </li><li>在房间里所有的灯关闭，并开始使用PowerLab系统26T的电气录音。 </li></ol> 4。负载电压和钙敏感染料和兴奋收缩解偶联剂<ol start="19"><li>热身0.6毫升blebbistatin。混合控股水库灌流blebbistatin 0.5毫升。台氏液，在1毫升稀释blebbistatin余下0.1毫升，慢慢注入通过套管附近的药物端口（超过20分钟的时间内）。观察作为blebbistatin逐渐减少了运动伪影。 </li><li>电压敏感染料RH237台氏液1毫升的原液稀释30微升，超过5-7分钟，慢慢注入相同的进样口作为blebbistatin。 </li><li> 30μL，钙指标Rhod凌晨2点是聚醚F127（Invitrogen公司，卡尔斯巴德，加利福尼亚，20％DMSO溶液）混合，然后在台氏液1毫升稀释，慢慢地通过相同的进样口的应用在5-10分钟1:1 。 </li><li>等待5-10分钟blebbistatin和染料达到细胞膜和细胞质。继续与运动时是完全压制的协议。 </li><li>连续监测心电图在整个过程的录音，以确保正常心脏的电气功能。 </li><li>开始使用SciMedia定制软件（SciMedia，科斯塔梅萨，CA）的荧光信号录音。 </li></ol> 5。代表性的成果： <img alt="图1" src="/files/ftp_upload/3275/3275fig1.jpg" /> 图1实验装置灌注，电气录音和光学测绘。 EM =排放; LP =长通<img alt="图2" src="/files/ftp_upload/3275/3275fig2.jpg" /> 图2。心室起搏期间录得的实验准备和信号的例子。左：心电图信号从Ag / AgCl电极圆盘电极（顶部）和S1S1刺激协议的一个例子是显示（底部）收集。中心的Langendorff小鼠心脏的准备。右：代表光的动作电位和钙瞬变信号，从心房（顶）和心室（下）所示。黄色箭头（上）点的荧光信号散射来自心室，这是在心房录音。 LV =左心室; RV =右心室; LA =左心房; RA =右心房<img alt="图3" src="/files/ftp_upload/3275/3275fig3.jpg" /> 图3。V M和猫从野生型小鼠心脏心室代表的光录音。 A.一个均匀分布的黑点标记，（c）中可以看出，其光学录音地点数组的实验准备。 B.范例跟踪从一个阵列上的中央位置（见方框 V M和CAT我N（C））。 C. V M（蓝色）和CAT（红色）从均匀分布的位置阵列。信号进行分级的3x3。 <img alt="图4" src="/files/ftp_upload/3275/3275fig4.jpg" /> 图4。激活地图和传导。答：例如，从横向（T）和纵向（L）的白色箭头指示的方向与野生型小鼠心脏激活的地图。 B. V M信号（上）和对应的dV / dt（下）看到这三个点（T1，T2，T3，L1，L2，L3）。 <img alt="图5" src="/files/ftp_upload/3275/3275fig5.jpg" /> 图5。动作电位和钙瞬变的持续时间分析。 A.行动在80％复极（APD80）和80％放宽（CaD80）的钙瞬变持续时间可能持续的时间地图显示从控制条件下的心脏（左）和30纳米异丙应用程序（右）后。在心室的黄/绿颜色（右）表示，异丙肾上腺素缩短APD80和CaD80。 B. APD80和CaD80例描从野生型小鼠心室（顶部）和心房（下）。

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没有利益冲突的声明。

在这个实验中，我们修改了加入一个小的硅管，这是特别重要的室性早搏抑制与兴奋收缩解偶联剂后Langendorff灌流方法。硅管是用来防止解充血，灌注液酸化，并在左心室缺血的发展。鼠标心脏是，以低温非常敏感，因此，整个心脏的温度变化会导致动作电位持续时间的人为差异。因此，灌注室加热系统实施，以保持一个温度恒定在37 ° C，在整个实验的 8 。的Langendorff模型由于不保留神经支配的心脏，需要考虑增加神经递质的灌流，以探讨生理和副交感神经的语气 9 。除了逆行灌注，灌流的心脏除了有助于保持适宜的环境参数，如pH值和温度。在此方法中的Langendorff灌流心脏水平放置。一个垂直Langendorff灌流设置，也可以用 10，但可能会导致在略有不同心脏力学11。除了​​CMOS摄像头，其他探测器也可，并可以应用到地图 V M和CAT 同时 12 。 高时空分辨率的CMOS摄像头的应用，保证了录音的准确性，但是，光的映射信号不是从一个单细胞。相反，每个荧光信号来自数百或数千个细胞，视光学倍率。心室荧光大得多，可以扭曲心房光散射信号;因此，细致的解释是需要的光学记录的信号。鼠标准备的另一个限制是由表面的曲率，由于体积小的心脏13引起的信号失真和噪声。不仅从鼠标心脏的曲率，但也可以从电极的极性和虚拟电极传导速度的测量，可以改变。为了实现传导速度，激活各向异性，和复极地图的准确性，正确的相机的聚焦在心脏表面是必不可少的。 在此方法中，实时心电图记录可以补充的心脏电生理光学调查。电压敏感染料（RH237）和钙（Rhod - 2AM）使用的协议，因为他们的反应速度快，类似的激励，和不同的发射光谱3,7。有替代染料，可用于测量V M和CAT比RH237和Rhod - 2AM 3组合。一种新型电压敏感染料，PGHI斯托克的一个大转变（&gt; 200nm的），被发现，以便更好的 V M和猫，因为更大的分离PGHI和Rhod上午02时14之间的发射波长信号。今后的改进重点放在探索新型的荧光探针，开发新的成像探测器，和改进的图像处理软件。较高的分辨率和光学成像方式，新型三维光映射也是重要的未来发展方向的光的映射5。

<table border="1"><tbody><tr><td> 化学 </td><td> 公司 </td><td> 产品编号 </td></tr><tr><td>氯化钠</td><td>费舍尔科学，公平的草坪，新泽西州</td><td> S271 - 1 </td></tr><tr><td>氯化钙2（H 2 O） </td><td>费舍尔科学，公平的草坪，新泽西州</td><td> C79 - 500 </td></tr><tr><td>氯化钾</td><td>费舍尔科学，公平的草坪，新泽西州</td><td> S217 - 500 </td></tr><tr><td>氯化镁2（H 2 O） </td><td>费舍尔科学，公平的草坪，新泽西州</td><td> M33 - 500 </td></tr><tr><td>的NaH 2 PO 4（H 2 O） </td><td>费舍尔科学，公平的草坪，新泽西州</td><td> S369 - 500 </td></tr><tr><td>碳酸氢钠3 </td><td>费舍尔科学，公平的草坪，新泽西州</td><td> S233 - 3 </td></tr><tr><td> D -葡萄糖</td><td>费舍尔科学，公平的草坪，新泽西州</td><td> D16 - 1 </td></tr><tr><td> Blebbistatin </td><td> Tocris生物科学，Ellisville，莫</td><td> 1760 </td></tr><tr><td> RH237 </td><td> Invitrogen公司，卡尔斯巴德，CA </td><td> S1109 </td></tr><tr><td> Rhod - 2AM </td><td> Invitrogen公司，卡尔斯巴德，CA </td><td> R1244 </td></tr><tr><td>聚醚F127 </td><td> Invitrogen公司，卡尔斯巴德，CA </td><td> P3000MP </td></tr><tr><td>二甲基亚砜（DMSO） </td><td> Sigma公司，圣路易斯，密苏里州</td><td> D2650 </td></tr></tbody></table>

optical mapping of action potentials and calcium transients in the mouse heart

鼠标心脏是一个流行的模式由于成本低，在这个物种的基因工程技术存在心血管研究。心血管鼠标心脏生理表型可以很容易地使用荧光成像技术，采用各种探头跨膜电位（V M），钙瞬变（CAT），以及其他参数。兴奋收缩偶联的特点是动作电位和细胞内钙离子的动态，因此，它是极其重要的映射 V M和CAT同时从同一位置上的心脏 1-4 。同时，从光映射的Langendorff灌注小鼠心脏有可能澄清心脏衰竭，心律失常，代谢性疾病，和其他心脏疾病的基本机制。可视化激活，传导速度减慢，动作电位时程，并在无数的网站的其他参数可以不实现从细胞水平调查， 但光学映射1,5,6解决。在本文中，我们目前的仪表安装和实验条件的同步光映射在小鼠心脏V M和CAT高时空决议，利用国家的最先进的CMOS成像技术。用这种方法得到一致的光录音说明的Langendorff灌注小鼠心脏，同时光的映射是可行和可靠的。

Watch this Scientific Journal Video about Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart at JoVE.com

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

本文详细解剖程序，工具设置，并在光学测绘跨膜电位（VM）和细胞内钙瞬变（CAT），在隔离的Langendorff完整的实验条件下灌注小鼠心脏。

在小鼠心脏的动作电位和钙瞬变的光学测绘

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

optical-mapping-action-potentials-calcium-transients-mouse

Research

JoVE Journal

Bioengineering

27.0K 次观看.  Washington University in St. Louis. 本文详细解剖程序，工具设置，并在光学测绘跨膜电位（VM）和细胞内钙瞬变（CAT），在隔离的Langendorff完整的实验条件下灌注小鼠心脏。

视频: Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

多参数的Langendorff灌注兔心脏的光学测绘

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

阿的Cre - LOX P为检测细胞融合重组方法在体内</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

在体外合成的原住民，纤维长间距“和”分段长间距胶原蛋白

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

光学检测<em&gt; E。大肠杆菌</em&gt;由细菌介孔硅生物传感器

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

在生物膜的力学性能原位制图粒子追踪Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

光片荧光显微术捕捉4维图像调制剪切应力对斑马鱼心脏发育的影响

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

光片荧光显微术在小鼠心脏研究中的应用

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

房室心脏瓣膜的双轴力学特性

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...