y.t.y. 愿意承认科技部在赠款数量最多 105-2221-007-MY3 和国立清华大学的资助支持下, 105N518CE1 和106N518CE1。

1. 光学设置 注意: 光学设置的原理见图 1 。
<ol> <li> 按照它们的手册设置光学镊子套件 (参见 材料表 ) 和荧光模块 (参见 材料表 )。将 470 nm 蓝光发光二极管 (LED) 光源连接至荧光模组. </li>
	<li> 更换高数值孔径 (na) (na = 1.25, 放大倍数 100x) 油浸目标由一个长的工作距离 (wd) 显微镜目标 (焦距3.6 毫米, wd = 10.6 毫米, NA = 0.5). </li>
	<li> 卸下组装套件的光束扩展段中的透镜, 以实现激光束的松散聚焦. </li>
	<li> 启用波长为 980 nm 的激光二极管的电源和电流, 并使用带电耦合器件 (CCD) 摄像机来确保激光束正确对准. 
	注: 如果激光束是定向的, CCD 摄像机将会读取一个高斯点. </li>
</ol> 2。纳米 <ol> <li> 标记制作. 
	注意: 标记将有助于在制造过程和随后的陷印实验中定位 nanoplasmonic 阵列。详细的过程在 补充图 1 中说明。
	<ol> <li> 在厚度为 0.17 mm 的片上沉积 40 nm 氧化铟锡 (ITO) 薄膜. 
		注: ITO 薄膜将有助于在随后的电子束光刻过程中释放电子. </li>
		<li> 自旋涂层8和 #181; m 层的正光刻胶与自旋速度 4000 rpm 和时间三十年代与旋转涂布机. </li>
		<li> 软烘烤样品在90和 #176; C 为 5 min, 并将样品与掩对齐, 并将样品暴露于八十年代的 UV 光器. </li>
		<li> 将样品浸泡在光刻胶显影液中 130s. </li>
		<li> 使用热蒸发将2纳米铬层和一层40纳米的金沉积在样品上。 18 </li> <li> 将样品浸泡在丙酮中, 并将其放置在43赫和 150 W 的超声波清洗器中, 以进行5分钟的升降。
	</ol> </li> <li> 制作 Nanoplasmonic 阵列 <ol> <li> 自旋涂层一层电子束抗 PMMA 120K, 自旋速度 5000 rpm 三十年代在旋转涂布机。烘烤样品在160和 #176; C 为3分钟在一个热的盘子. </li>
		<li> 自旋涂层的另一层电子束抵御 PMMA 960K 与自旋速度 5000 rpm 三十年代在一个旋转涂布机。烘烤样品在160和 #176; C 为3分钟在一个热的盘子. </li>
		<li> 使用电子束写入器以加速电压30伏和剂量 400 C/cm 2 来公开电子束抵抗. </li>
		<li> 在热蒸发器中沉积40纳米金层. </li>
		<li> 将样品浸泡在丙酮中, 并将其放在超声波清洗机中5分钟以进行升降. </li>
	</ol> </li> </ol> 3。样品冷却系统及其温度标定 注意: 在 补充图 2 中显示了示例冷却阶段的设计. 
<ol> <li> 为采样冷却的驱动程序电路 <ol> <li> 将电阻、双极结点晶体管和电源金属氧化物场效应晶体管放在自定义电路板上, 按照 补充图3中的电路图进行操作.用烙铁焊接所有这些部件. </li>
		<li> 连接电路板控制端口和电子控制板之间的导线。连接电路板的输出端口和热电冷却 (TEC) 元件之间的导线。将 TEC 元素放在样品台上, 并热沉. 
		#8203; 注意: TEC 元素在中心有一个孔, 允许激光束通过. </li>
		<li> 将电路板上的导线连接到 5 V 电源。使用 forward-looking 红外摄像机监视温度以检查热电冷却是否正确冷却. </li>
	</ol> </li> <li> forward-looking 红外摄像机和电阻温度检测器 (RTD) 温度计中测量温度的校准。
	<ol> <li> 将 rtd 温度计放在空白片上, 并在其上应用少量的热贴, 以确保 rtd 温度计和片之间有适当的热接触. </li>
		<li> 通过改变脉冲宽度调制设置的占空比, 将电子控制电路的输出功率设置更改为 TEC 元件, 并等待3分钟, 以确保达到稳定状态温度。使用 RTD 温度计读取温度. </li>
		<li> 打开前瞻式红外摄像机并监视温度。重复这个在不同的输出功率设置, 以获得温度校准曲线。有代表性的温度校准曲线显示在 补充图 4 中. 
		注意: 在 RTD 温度计和前向红外摄像机之间进行校准是至关重要的, 因为 forward-looking 红外摄像机的温度读数必须准确, 以确保达到正确的温度. </li>
	</ol> </li> </ol> 4。微粒捕集 <ol> <li> 将直径2和 #181 的微聚苯乙烯微粒稀释在去离子水中, microcentrifugetube 具有适当容积率. 
	注: 微颗粒浓度可根据实验目的进行调整。较低浓度允许单粒子俘获事件的采样时间间隔, 较高浓度将缩短多粒子捕获的时间。对于单粒子俘获, 典型浓度为 ~ 0.05% (w/v). </li>
	<li> 将样品与 nanoplasmonic 阵列放在舞台上, 并打开 470 nm LED 作为荧光光源, 并手动将功率设置为5兆瓦的明亮场成像. </li>
	<li> 使用标记来定位 nanoplasmonic 阵列, 对齐样本, 并使用 CCD 摄像机确保阵列位于计算机屏幕上的感兴趣区域的中心. </li>
	<li> 免除10和 #181; L 对直径2和 #181 的稀释微粒子进行取样, 并在样品上使用微吸管. </li>
	<li> 打开当前电源到波长 980 nm 的激光二极管以激发阵列的浆共振, 其功率范围为1兆瓦至10兆瓦. </li>
	<li> 手动打开电子控制板上的电源以使样品冷却至稳定状态温度〜4和 #176; C. </li>
	<li> 在查看器软件中, 单击 #34; 录制视频和 #34; 序列以打开录制对话框。单击 "#34"; 记录和 #34; 按钮启动视频录制1.5 的微粒子的运动的帧速率为10帧/秒的影响下, 激光光束使用 CCD 相机。单击 #34; 停止和 #34; 按钮以停止录制。请参见 视频 1 . </li>
</ol>

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作者没有什么可透露的。

此处描述的过程使读者能够可靠地每天复制陷印。一个通用的经验指南, 设计一个可用的晶格是使用一个可比大小的浆阵, interdisc 距离, 和被困粒子的大小。与单一的, 孤立的浆纳米结构相结合, 光学晶格设计与高光功率所提供的冷却样品到〜4° c 使用, 大大提高了捕获概率。如果分离性好, 浆纳米结构被用作诱捕点, 你需要等待很长时间才能将微粒迁移到浆纳米结构附近的有效俘获体积中。同时, 增...

微尺度微粒的光学捕获最初是由亚瑟在二十世纪七十年代代初提出的。自其发明以来, 该技术已被开发为微型和纳米1,2的多功能工具。传统的基于远场聚焦原理的光阱, 其空间约束中的衍射固有地受到限制, 其中俘获力显著减小 (在一个半径为粒子的a3定律之后)a)3. 为了克服这种衍射极限, 研究人员利用浆金属纳米结构, 开发了基于倏逝光场的近场光学俘获技术, 此外, 将纳米级物体捕获到单蛋白分子已被证明4,5,6,7,8,9,10,11。此外, 浆光学晶格是由周期性浆纳米结构的阵列创建的, 以赋予微纳米粒子和多粒子堆叠的长距离传输11,12。在光学晶格中干扰俘获的一个主要障碍是光热对流, 并已努力通过几个组14、15、16、17来阐明其效果。使用格林函数, Baffou et al.通过将每个浆纳米结构建模为点加热器来计算温度剖面, 然后通过实验验证了它们的模型14。汤森的小组也测量了等离子体诱导对流的粒子测速仪15。作者的小组也描绘了近场和对流运输并且显示了一项工程战略抑制光热对流16,17。
在这里, 我们提出了一个光学装置的设计和详细的程序, 专门用于诱捕实验浆光学晶格。光的潜力是通过照亮一组金 nanodiscs 与一个松散的聚焦高斯光束。这里还描述了一种通过将样品冷却到低温 (~ 4 ° c) 来抑制光热对流以获得最佳捕获的方案, 这里也介绍了17。在方程近似下, 由 u ~l2 gβΔT / v给出了自然对流速度u的数量级估计, 其中L是热源的长度刻度, δT是由于加热而引起的相对于参照的温度升高。 g和β分别是重力加速度和热膨胀系数。在接近4° c 的温度下, 水介质的密度表现出反常的温度依赖性, 这就转化为近零的热膨胀系数, 因此, 微乎其微小的光热效应对流。

<table style="border-collapse:collapse;width:629pt" width="839">
	<colgroup>
		<col style="width:207pt" width="276" />
		<col style="width:131pt" width="174" />
		<col style="width:122pt" width="162" />
		<col style="width:170pt" width="227" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Thermoelectric cooling element</td>
			<td class="xl69" style="width:131pt" width="174">Thorlabs</td>
			<td class="xl69" style="width:122pt" width="162">TEC 1.4-6</td>
			<td class="xl70" style="width:170pt" width="227">TEC element for sample cooling</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl69" height="42" style="width:207pt;height:31.5pt" width="276">RTD thermometer</td>
			<td class="xl69" style="width:131pt" width="174">Omega Engineering</td>
			<td class="xl69" style="width:122pt" width="162">RTD Thermometer 969C</td>
			<td class="xl70"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Forward looking infrared camera</td>
			<td class="xl69" style="width:131pt" width="174">FLIR&#160;</td>
			<td class="xl69" style="width:122pt" width="162">FLIR One</td>
			<td class="xl70">IR camera for temperature monitoring</td>
		</tr>
		<tr height="46" style="height:34.5pt">
			<td class="xl69" height="46" style="width:207pt;height:34.5pt" width="276">light emitting diode light source</td>
			<td class="xl69" style="width:131pt" width="174">Touchbright</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl69" style="width:170pt" width="227">Light source for illumination for fluorescent imaging</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Long working distance objective</td>
			<td class="xl69" style="width:131pt" width="174">Olympus</td>
			<td class="xl69" style="width:122pt" width="162">LMPLFLN</td>
			<td class="xl70">For illuminating the sample and imaging</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Optical trap kit</td>
			<td class="xl69" style="width:131pt" width="174">Thorlabs</td>
			<td class="xl69" style="width:122pt" width="162">OTKB/M</td>
			<td class="xl70"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl71" height="21" style="height:15.75pt">Cover slip</td>
			<td class="xl69" style="width:131pt" width="174"></td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl69" style="width:170pt" width="227">thickness 0.17 mm</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl71" height="21" style="height:15.75pt">Scanning electron microscope</td>
			<td class="xl69" style="width:131pt" width="174">Hitachi</td>
			<td class="xl69" style="width:122pt" width="162">SEM-Hitachi S3400N</td>
			<td class="xl69" style="width:170pt" width="227"></td>
		</tr>
		<tr height="44" style="height:33.0pt">
			<td class="xl71" height="44" style="height:33.0pt">Electron beam blanker</td>
			<td class="xl69" style="width:131pt" width="174">DEBEN</td>
			<td class="xl69" style="width:122pt" width="162">PCD beam blanker</td>
			<td class="xl69" style="width:170pt" width="227">the blanker is added to the scanning electron microscope&#160;</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl71" height="21" style="height:15.75pt">Thermal evaporator</td>
			<td class="xl69" style="width:131pt" width="174">SYSKEY Technology</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl69" style="width:170pt" width="227"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl71" height="21" style="height:15.75pt">Mask aligner</td>
			<td class="xl69" style="width:131pt" width="174">Karl Suss</td>
			<td class="xl69" style="width:122pt" width="162">MJB 3</td>
			<td class="xl69" style="width:170pt" width="227">For marker fabrication</td>
		</tr>
		<tr height="24" style="height:18.0pt">
			<td class="xl69" height="24" style="width:207pt;height:18.0pt" width="276">Electron beam resist</td>
			<td class="xl69" style="width:131pt" width="174">Sigma Alrich</td>
			<td class="xl69" style="width:122pt" width="162">PMMA 120K</td>
			<td class="xl69" style="width:170pt" width="227">For e-beam lithography</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Electron beam resist</td>
			<td class="xl69" style="width:131pt" width="174">Sigma Alrich</td>
			<td class="xl69" style="width:122pt" width="162">PMMA 960K</td>
			<td class="xl70">For e-beam lithography</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl69" height="42" style="width:207pt;height:31.5pt" width="276">Fluoresent labeled polystyrene microspheres</td>
			<td class="xl69" style="width:131pt" width="174">Polyscience</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl70">2 um diameter</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Bipolar transistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162">2N3904</td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl69" height="28" style="width:207pt;height:21.0pt" width="276">Bipolar transistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162">2N3906</td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">MOSFET power transistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162">IRF5305</td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">MOSFET power transistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162">IRF131ON</td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">10 kOhm resistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl70">quantity 6 for TEC driver circuit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">910 Ohm resistor</td>
			<td class="xl69" style="width:131pt" width="174">Mouser</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl70">quantity 2 for TEC driver circuit</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Photoresist</td>
			<td class="xl69" style="width:131pt" width="174">Microchemicals</td>
			<td class="xl69" style="width:122pt" width="162">AZ4620</td>
			<td class="xl70">For marker fabrication</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl69" height="21" style="width:207pt;height:15.75pt" width="276">Acetone</td>
			<td class="xl69" style="width:131pt" width="174">Sigma Alrich</td>
			<td class="xl69" style="width:122pt" width="162"></td>
			<td class="xl70">For marker fabrication</td>
		</tr>
		<tr height="41" style="height:30.75pt">
			<td class="xl69" height="41" style="width:207pt;height:30.75pt" width="276">Fluorescence Module for the OTKB/M, Metric Threads</td>
			<td class="xl69" style="width:131pt" width="174">Thorlabs</td>
			<td class="xl69" style="width:122pt" width="162">OTKB-FL/M</td>
			<td class="xl69" style="width:170pt" width="227"></td>
		</tr>
		<tr height="49" style="height:36.75pt">
			<td class="xl69" height="49" style="width:207pt;height:36.75pt" width="276">Fluorescent filter set</td>
			<td class="xl69" style="width:131pt" width="174">Thorlabs</td>
			<td class="xl69" style="width:122pt" width="162">MDF-FITC</td>
			<td class="xl70">For Fluorescein Isothiocyanate (FITC)</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl70" height="21" style="height:15.75pt">Ultrasonic cleaner</td>
			<td class="xl69" style="width:131pt" width="174">Delta</td>
			<td class="xl70">DC150H</td>
			<td class="xl70">For the lift off step</td>
		</tr>
	</tbody>
</table>

trapping of micro particles in nanoplasmonic optical lattice

为了克服传统远场光学镊子的衍射极限, 研制了浆光学镊子。浆光学晶格由一组纳米结构组成, 它表现出各种俘获和传输行为。我们报告的实验程序, 捕获微粒子在一个简单的正方形 nanoplasmonic 光学晶格。我们还描述了 nanoplasmonic 阵列的光学设置和纳米。光学电位是通过照亮一组 980 nm 波长的高斯光束和令人兴奋的等离子体共振的金 nanodiscs 来产生的。通过荧光成像对颗粒的运动进行监测。此外, 还描述了一种抑制光热对流的方案, 以增加可用的光学功率, 从而获得最佳捕获。通过将试样冷却到低温, 并利用水介质的近零热膨胀系数, 实现了对流的抑制。这里报告了单粒子传输和多粒子捕获。

在我们的实验中记录了一个 CCD 摄像机的单粒子轨迹, 然后用一个自定义程序来处理图像, 以提取每个粒子的轨迹16。有代表性的结果显示在图 3 和视频 1中, 直径为2µm 的微球体. 观察了光学晶格内的多个粒子的修饰。从粒子的代表性运动视频中提取的连续图像显示在图 4中。对于直径2µm 的微粒, 可?...

Watch this Scientific Journal Video about Trapping of Micro Particles in Nanoplasmonic Optical Lattice at JoVE.com

Trapping of Micro Particles in Nanoplasmonic Optical Lattice

我们描述了在 nanoplasmonic 光学晶格中光阱微粒子的过程。

Nanoplasmonic 光学晶格中微粒子的俘获

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical ...

trapping-of-micro-particles-in-nanoplasmonic-optical-lattice

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

6.5K 次观看.  National Tsing Hua University. 我们描述了在 nanoplasmonic 光学晶格中光阱微粒子的过程。

视频: Trapping of Micro Particles in Nanoplasmonic Optical Lattice

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 ...

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

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

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. ...

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 ...

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

微观粒子图像测速技术，微血液流动的速度剖面测量

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

Intra-lymph Node Injection of Biodegradable Polymer Particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g.&#160;size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.

Lymph nodes are the immunological tissues that orchestrate immune response and are a critical target for vaccines. Biomaterials have been employed to better target lymph nodes and to control delivery of antigens or adjuvants. This paper describes a technique combining these ideas to inject biocompatible polymer particles into lymph nodes.

可生物降解聚合物颗粒的淋巴内结注射

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

疏水货物封装有针对性的质膜交付在液晶纳米颗粒载体

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

Nanoplasmonic 光学晶格中微粒子的俘获

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