Name: light-sheet fluorescence microscopy for the study of the murine heart
Uploaded: 2018-09-15T14:00:00
Description: Watch this Scientific Journal Video about Light-sheet Fluorescence Microscopy for the Study of the Murine Heart at JoVE.com

作者希望向加州大学洛杉矶分校的邵阮和淳浩表示感谢, 他们为小鼠提供了图像样本。这项研究得到了 NIH HL118650 (粽子 k Hsiai) 的资助, HL083015 (粽子 k Hsiai), HD069305 (到北卡罗来纳州和粽子 k Hsiai), HL111437 (粽子 k. Hsiai 和北卡罗来纳州), HL129727 (粽子 k Hsiai), 德克萨斯大学系统明星资助 (Juhyun李)。

所有涉及使用动物的程序都已被加州大学洛杉矶分校的机构审查委员会 (IACUC) 批准。
1. 成像系统设置
注: 见图 1和图 2。
<ol><li>检索3波长的连续波 (CW) 激光器: 405 nm、473 nm 和 532 nm。将2镜 (M1 和 M2) 分开150毫米, 并将其与在45°上的镜面平面对准光束。 注意: 执行此步骤可将激光从双面照明设置中重定向。产生...

<ol>
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这里描述的 LSFM 系统和技术利用双侧照明光束和光清除相结合, 在产后和成人阶段对鼠标心脏进行图像处理。传统的单一光照光束受光子散射和吸收通过更厚和更大的组织样本1,3。双面光束为样品提供了更均匀的光照, 从而最大限度地减少了条带和其他工件在单面光照产生的图像中经常看到的效果。该技术还可以通过调整双高斯光束8的...

光片荧光显微镜是1903年首次开发的一种技术, 目前作为一种研究基因表达的方法, 也用于生产3维或4维组织样本1,2,3的模型。这种成像方法使用一层薄薄的光来照亮样本的单个平面, 以便只有该平面被探测器捕获。然后, 可以在轴向方向移动样本以捕获每一层, 一次一节, 并在获得的图像4后处理后呈现一个3维模型。然而, 由于光子的吸收和散射, LSFM 仅限于几微米厚或光学上透明1的样品。
LSFM 的局限性导致了对生物体的广泛研究, 这些有机体具有光学上透明的组织, 如斑马鱼。有关心脏发育和分化的研究往往是对斑马鱼, 因为有保守的基因之间的人和斑马鱼5,6。尽管这些研究已经导致了与心肌6,7相关的心脏研究的进展, 但仍有必要对哺乳动物等高级生物进行类似的研究。
哺乳动物的心脏组织提出了一个挑战, 由于组织的厚度和不透明, 红细胞中血红蛋白的吸收, 以及在传统的 LSFM 方法下, 由于样品单面光照产生的条带.1,8. 为了弥补这些限制, 我们建议使用双侧照明和简化版的清晰度技术9结合折射率匹配解决方案 (轮辋)。因此, 该系统允许对大于 10 x 10 x 10 毫米3的样本进行成像, 同时在轴向和侧面平面上保持良好的质量分辨率8。
该系统是第一次校准使用荧光珠安排在不同的配置在玻璃管。然后, 该系统用于图像产后和成年鼠心脏。首先, 产后鼠心脏被成像7天 (P7), 以揭示心室腔, 心室壁的厚度, 瓣膜结构, 和 trabeculation 的存在。其次, 研究了在1天 (P1) 用培养的心肌细胞和黄色荧光蛋白 (YFP), 通过使用产后小鼠心脏, 来识别将会分化为细胞的干细胞。最后, 7.5 月的成年小鼠在基因治疗后观察到肾外髓质钾 (ROMK) 通道的存在,8。

<table border="0" cellpadding="0" cellspacing="0" style="width:967px;" width="967">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">CW Laser</td>
			<td style="width:136px;">Laserglow Technologies</td>
			<td style="width:393px;">LMM-GBV1-PF3-00300-05</td>
			<td style="width:191px;">Excitation of fluorophores</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Neutral density filter</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">NDC-50C-4M</td>
			<td style="width:191px;">Controls amount of light entering system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Achromatic beam expander</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">GBE05-A</td>
			<td style="width:191px;">Expands the beam of light</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Mechanical slit</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">VA100C</td>
			<td style="width:191px;">Controls width of beam</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Beam splitter</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">BS013</td>
			<td style="width:191px;">Forms dual-illumination beam</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Stereo microscope with 1X objective lense</td>
			<td style="width:136px;">Olympus</td>
			<td style="width:393px;">MVX10</td>
			<td style="width:191px;">Used for observation of sample</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">ORCA-Flash4.0 LT sCMOS camera</td>
			<td style="width:136px;">Hamamatsu Photonics</td>
			<td style="width:393px;">C11440-42U</td>
			<td style="width:191px;">Used to capture Images</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Acrylonitrile butadiene styrene (ABS)</td>
			<td style="width:136px;">Stratasys</td>
			<td style="width:393px;">uPrint</td>
			<td style="width:191px;">Material used to 3-D print a sample holder</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Fluorescent polystyrene beads</td>
			<td style="width:136px;">Spherotech Inc</td>
			<td style="width:393px;">PP-05-10</td>
			<td style="width:191px;">Used for imaging system calibration</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Borosilicate glass tubing</td>
			<td style="width:136px;">Corning</td>
			<td style="width:393px;">Pyrex 7740</td>
			<td style="width:191px;">Tubing for sample embedding</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Glycerol</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:393px;">BP229-4</td>
			<td style="width:191px;">Fill for sample chamber</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Phosphate-buffered saline</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:393px;">BP39920</td>
			<td style="width:191px;">Rinse solution for mouse hearts</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Paraformaldehyde</td>
			<td style="width:136px;">Electron microscopy sciences</td>
			<td style="width:393px;">RT-15700</td>
			<td style="width:191px;">First incubation solution</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:247px;">Acrylamide</td>
			<td style="width:136px;">Wako Chemicals</td>
			<td style="width:393px;">AAL-107</td>
			<td style="width:191px;">Mixed with 2,2&#39;-Azobis dihydrochloride for second incubation solution for mouse hearts</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">2,2&#39;-Azobis dihydrochloride</td>
			<td style="width:136px;">Wako Chemicals</td>
			<td style="width:393px;">VA-044</td>
			<td style="width:191px;">Mixed with Acrylamide for second incubation solution for mouse hearts</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Sodium dodecyl sulfate&#160;</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:393px;">71725</td>
			<td style="width:191px;">Mixed with Boric acid for third incubtion solution for mouse hearts</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Boric acid</td>
			<td style="width:136px;">Fischer Scientific</td>
			<td style="width:393px;">A74-1</td>
			<td style="width:191px;">Mixed with Sodium dodecyl sulfate for third incubtion solution for mouse hearts</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:247px;">Sigma D2158</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:393px;">D2158</td>
			<td style="width:191px;">Mixed with PB, Tween-20, and Sodium azide as a refractive index matching solution</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:247px;">Tween-20</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td>11332465001</td>
			<td style="width:191px;">Mixed with Sigma D2158, PB, and Sodium azide as a refractive index matching solution</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Sodium azide</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td>S2002</td>
			<td style="width:191px;">Mixed with Sigma D2158, PB, and Tween-20 as a refractive index matching solution</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Adeno-associated virus vector 9 with a cardiac-specific Troponin T promoter tagged with GFP</td>
			<td style="width:136px;">Vector Biolabs</td>
			<td style="width:393px;">VB2045</td>
			<td style="width:191px;">Expresses GFP when bound to ROMK</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">DC Servo Motor Actuator</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">Z825B</td>
			<td style="width:191px;">Used for movement of sample in axial direction within light sheet</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">K-Cube Brushed DC Servo Motor Controller</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">KDC101</td>
			<td style="width:191px;">Connects to motor actuator and controls movement of the actuator</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Amira</td>
			<td style="width:136px;">FEI Software</td>
			<td style="width:393px;">N/A</td>
			<td style="width:191px;">Visualization software for producing 2-D and 3-D images</td>
		</tr>
	</tbody>
</table>

light-sheet fluorescence microscopy for the study of the murine heart

光片荧光显微技术已广泛应用于从微米到毫米尺度的高轴向分辨率的快速图像采集。传统的光片技术涉及在样品组织中使用单一光照束定向正交。使用单一光照光束制作的大型样品的图像含有条纹或工件, 由于组织的散射和吸收, 导致分辨率降低。本研究采用双侧光照光束和简化的小鼠心脏清晰度光清除技术。这些技术可以通过去除心脏的脂质和产生一个大的成像领域, 大于 10 x 10 x 10 毫米3, 更深层次的成像。因此, 这一策略使我们能够量化的心室尺寸, 跟踪心脏谱系, 并本地化的心脏特异性蛋白和离子通道的空间分布, 从产后到成年小鼠心脏有充分的对比度和分辨率。

这里描述的技术使用了双侧照明光束结合光清除鼠标心脏组织样本, 以达到更深的成像深度和更大的成像量, 有足够的成像分辨率 (图 1)。为了校准系统, 荧光珠被放置在玻璃管和成像系统内。然后将珠子在 x-y 平面、y z 平面和 x z 平面上进行成像和可视化, 以获得系统8的点扩展函数 (PSF)。系统标定产生了在 d侧向≈2.8 µm 的横...

Watch this Scientific Journal Video about Light-sheet Fluorescence Microscopy for the Study of the Murine Heart at JoVE.com

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

本研究采用双侧光照光片荧光显微镜 (LSFM) 技术, 结合光清除法对小鼠心脏进行研究。

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. ...

这项技术的主要优点是，它允许对鼠标心脏进行快速成像。它可以用来观察基因治疗后空气通道的存在。虽然此方法可以提供对心脏病学发展有深入的见解，但它也可以应用于其他系统，如神经学和肺病学。一般来说，个人将很难与这种技术，因为安装成人小鼠心脏成像可能很困难，它不同于其他传统技术，如共和和倒置显微镜。放置三个波长为 405 纳米、473 纳米和 532 纳米的连续波激光器。然后，粘贴一面镜子，并在 45 度时将其与镜面对齐。这将将激光引导到光束分路器，从而形成双面照明设置。接下来，通过一个直径为50毫米的中性密度滤波器、一个光束膨胀器和一个直径为25毫米的针孔，所有位置彼此150毫米。通过 50-50 光束分路器，从针孔放置 150 毫米。接下来，将两个 150 毫米的镜面从光束分离器放置，在 90 度处放置到正向光束，并对齐它，使其镜面与光束保持 45 度角。然后，将镜子从镜子二处放置三个 100 毫米，并对齐镜子，使其镜面与从镜像二反射的光束保持 45 度角。使用此反射光束形成双照明灯片的一侧。在梁分离器的远侧，放置镜面 5，以便其镜面在 45 度处，以向前进方向发射的光束。使用镜子五发出的光束形成双照明灯片的第二面。其次，以系统的方式建立双面照明系统。将圆柱透镜两个 150 毫米远离镜子三，另一个相同的圆柱透镜 150 毫米远离镜子五，在另一侧的双照明设置。接下来，放置两面镜子，每面与圆柱镜一样，距离为 50 毫米，以 90 度反射光束。在光片的两侧，从一对透镜中形成一个无色双片，第一个镜头从前一面镜子放置100毫米，直径1英寸，焦距为100毫米。第二个镜头应放置在镜头一的160毫米，直径为一英寸，焦距为60毫米。然后，将照明目标从以前的无色双精度线放置 1 和 2 个 150 毫米，以便它们与光束一致。从目标发出的光束形成用于成像样品的光片。首先，在预热折射率匹配溶液中稀释0.53微米珠溶液（1至15万）来制备荧光珠样品，用1%的低熔体指向阿加糖。接下来，使用一块内径为 12 毫米、外径为 18 毫米、长度为 30 毫米的硅酸盐玻璃管。在硅酸盐管中移液珠子，使红糖在室温下凝固。现在，用 99.5% 的陀螺仪溶液填充 3D 打印 ABS 腔室。将含有珠子的硅酸盐玻璃管放在室内。将三维电动平移级连接到硅酸盐玻璃管，以控制 ABS 腔室中样品的移动和方向。然后，使用自定义设计的软件以每秒 30 帧的速度使用 sCMOS 摄像机获取图像。使用电机控制器，将样品在横向方向移动一毫米，并在每一毫米增量中获取图像，直到对整个样品进行成像。使用可视化软件堆叠采集的图像，并使用这些珠子图像测量系统的点传播功能。使用 pH 为 7.5 的折射率匹配溶液，将 1% 的 agarose 溶解到匹配溶液中。将清除的成年小鼠心脏样本放入折射率匹配溶液中，溶解百分之一的阿加罗斯。然后，将样品插入硅酸盐玻璃管中，使糖在室温下凝固。将三维电动平移级连接到硅酸盐玻璃管，以控制三维打印 ABS 腔室中样品的移动和方向。放置样品，使样品位于由双照明系统创建的高斯光束的中心。然后，将 sCMOS 摄像机的采集速率设置为每秒 30 帧。现在，使用电机控制器，将样品在轴向方向上移动一毫米，并在每一毫米增量下获取图像。继续，直到整个样本被成像。使用可视化软件堆叠获取的图像。使用这些图像堆栈开发三有图像。为此，可以解构以前确定的点传播函数，并用于获取的图像堆栈。最后，设置像素阈值强度值以观察心脏的轮廓，并基于此灰度强度向图像添加伪色。在产后的第一天，人们可以可视化的阀门，中庭，心室，腹膜肌肉，和撕裂，以及其他功能。在产后第七天，特征更加明确。心房、心室、心室腔尺寸和心室壁厚都很明显。为了研究心脏内的心肌细胞分化，异质细胞敲击小鼠被贴上了微环标签，以显示心肌细胞。标记的单元格显示在这里，在每个平面方向。三个平面被合并，以创建心脏的三D渲染。内嵌内的红色圆圈区域显示两个半透明的小鼠心后，通过进行清晰度技术，并插入到硅酸盐玻璃管。除了研究产后小鼠，成像系统还可用于研究成年小鼠心脏。在这里，GFP 标记的 ROMK 频道显示在 7.5 个月。这些主要发现在心室壁。一旦掌握了，这项技术可以在一到两分钟内完成。尝试此过程时，必须清除管中样品周围的所有气泡。其他方法（如图像堆栈的自动分段）可以执行，以回答与心血管损伤和再生相关的其他问题。研究者在发育后，利用该技术研究小鼠产后和成人发育阶段的心脏结构。

light-sheet-fluorescence-microscopy-for-the-study-of-the-murine-heart

Research

JoVE Journal

Bioengineering

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

科研

生物工程

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3d轨道跟踪的改进双光子显微镜之应用：细胞内的囊泡的跟踪

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

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 ...

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 ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

血流成像与超快多普勒

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

体内 使用双光子荧光和受激拉曼散射显微镜对生物组织进行成像

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

虚拟现实交互分析 Zebrafish 心脏收缩

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for gaining insights into structural and functional changes in response to injuries. However, obtaining high-resolution and high-speed 4-dimensional (4D, 3D spatial + 1D temporal) images of the zebrafish heart to assess cardiac architecture and contractility remains challenging. In this context, an in-house light-sheet microscope (LSM) and customized computational analysis are&#160;used to overcome these technical limitations. This strategy, involving LSM system construction, retrospective synchronization, single cell tracking, and user-directed analysis, enables one to investigate the micro-structure and contractile function across the entire heart at the single-cell resolution in the transgenic Tg(myl7:nucGFP) zebrafish larvae. Additionally, we are able to further incorporate microinjection of small molecule compounds to induce cardiac injury in a precise and controlled manner. Overall, this framework allows one to track physiological and pathophysiological changes, as well as the regional mechanics at the single-cell level during cardiac morphogenesis and regeneration.

作者聚焦：斑马鱼中的高分辨率 4D 光片成像和虚拟现实用于心脏功能的单细胞分析

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

斑马鱼心脏收缩的 4D 光片成像

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for ...