作者感谢弗吉尼亚理工大学卡里里昂研究所所长迈克尔·弗里德兰德博士鼓励我们的研究努力。该项目得到了S.M.M和D.F.K.的发展基金的支持，部分得到了弗吉尼亚理工大学关键技术和应用科学研究所的纳米生物倡议的支持。

1. 准备亲和力捕获设备16
<ol><li>清洁氮化硅E芯片，在15毫升乙酮中孵育2分钟，然后用15毫升甲醇2分钟（图1A）。允许芯片在层压气流下干燥。</li>
	<li>在加热搅拌板上（不搅拌）上孵化干屑，在 150 °C 下 1.5 小时，然后让它们在使用前冷却到室温。</li>
	<li>使用汉密尔顿注射器在小玻璃管中合成脂质混合物，以包含 25% 的氯仿， 55% DLPC （1，2-迪劳罗伊尔-磷胆碱） 在氯仿 （1 毫克/毫升） 和 20% 尼-NTA 脂质 （1，2 - 二恶英 - 二恶英 - 二恶英酸-苏奇尼尔-镍盐） 在氯仿 （1 毫克/毫升） 总容量为 40 μl.</li>
	<li>在潮湿的培养皿（图1B）中的一片Parafilm上，在每15微克的米利-Q水上涂抹1μl的混合物。在冰上孵化样品至少60分钟。</li>
</ol>2. 捕获巨分子17
<ol><li>将带有 150 nm 集成垫片的 E 芯片放在单层样品顶部，孵育 1 分钟（图 1C）。</li>
	<li>轻轻将芯片从样品中取出，并在室温下加入3μl的蛋白质A.孵化剂，在室温下孵化1分钟（图1D）。</li>
	<li>使用 Whatman #1滤纸擦去多余的滴液，并立即添加 3μl 抗体溶液。在室温下孵化1分钟。</li>
	<li>使用汉密尔顿注射器去除多余的溶液，并立即在缓冲溶液中加入 1μl 的轮状病毒 DLP （0.1 毫克/毫升），其中包含 50 mM HEPES （pH 7.5）、150 mM NaCl、10 毫克 MgCl2和 10 mM CaCl2。在室温下孵化至少2分钟。DLP的编制工作已于18日进行。
</li>
</ol>3. 组装微流体室并加载 原位 标本支架
<ol><li>将含有病毒样本的湿 E 芯片加载到微流体标本支架的尖端。发光放电第二个扁平电子芯片1分钟，然后加载在垫片顶部（图2， 面板1-5）。</li>
	<li>或者，为了增加生物大分子的对比度，重金属污渍（如0.2%的尿素前体）可以在将第二个电子芯片放在湿标本上之前添加到湿标本中。但是，包含标本的电子芯片在添加对比试剂之前需要用 Milli-Q 水清洗。</li>
	<li>将整个装配夹在一起，形成密封的外壳，由 3 个黄铜螺丝（图2，面板 6-8）机械地放在支架内。</li>
	<li>组装后，支架的尖端使用涡轮泵干泵站泵至 10-6 托尔，然后将支架放入 TEM 内。</li>
</ol>4. 使用传输电子显微镜进行液体成像
<ol><li>将原位 标本支架装载到配备拉布6 灯丝的传输电子显微镜 （FEI 公司）中，并在 120 kV 下运行。</li>
	<li>打开 TEM 灯丝，利用摆动器功能将样品从 -15° 来回倾斜至柱子中的 +15° ，从而调整显微镜阶段与标本的中心高度。此过程调整 z 方向的阶段，以帮助考虑液室的适当厚度。此步骤还可确保在录制图像时使用准确的放大倍数。</li>
	<li>首先记录沿边缘和微流体室的角落区域的图像。这些区域通常包含最薄的解决方案。使用CCD相机记录低剂量条件下标本的图像（1-3电子/+2）。使用 6，000X - 30，000X 的名义放大倍数。</li>
	<li>通过聚焦在流体室的边缘来确定适当的去菌值。使用 -1.5μm 值以 30，000 倍的放大倍数记录图像。如果遇到厚溶液，或者样品制备中未使用对比剂，请使用 -2 至 -4 μm 范围内的较高除毒素值。</li>
	<li>为了确保在整个实验过程中，微流体中都包含溶液，将电子束对焦，直到在设备内的液体中形成气泡。</li>
</ol>

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作者马德琳·杜克斯是原芯片公司的员工。

在我们的介绍工作中，我们采用亲和力捕获方法将轮状病毒 DLP 系在微流体平台上。这允许在液体微环境下对大分子复合物进行 原位 成像。捕获 方法对于其他微流体成像技术具有重要意义 ，因为它将生物标本定位到成像窗口，以否定在以液体中记录图像时出现的大型扩散问题。然而， 我们协议中最关键的步骤 之一是将脂质生物膜转移到微芯片表面。如果脂质单层...

<a href="https://app.jove.com/t/6605">This corrects</a> the article <a href="https://dx.doi.org/10.3791/50936">10.3791/50936</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/50936">In situ TEM of Biological Assemblies in Liquid</a>. The Representative Results and References sections were&#160;updated.

Erratum: In situ TEM of Biological Assemblies in Liquid

生物学家和工程师的共同目标是了解分子机器的内部工作。传输电子显微镜 （TEM） 是以近原子分辨率1-2可视化这些复杂细节的理想仪器。为了维持TEM的高真空系统，生物样本通常嵌入在薄膜的微薄冰3，糖4，重金属盐5，或其中的一些组合6。因此，嵌入式标本的图像可能只显示动态过程的有限快照。
帕森斯及其同事利用差分泵送阶段，对环境液体室中水合的生物标本进行了早期维护尝试。未染色催化晶体的电子衍射模式在水合状态7-8中被成功记录到3的分辨率。此外，相分离的脂质域可以在人类红细胞9-10的水合膜中检查。然而，由于扩散液体及其对电子束的干扰引起的运动导致严重分辨率损失，直到最近才尝试使用生物标本进行进一步实验。
新开发的微流体标本持有者已引进，利用半导体微芯片形成微尺度环境室。这些设备可以在将样品放置在 TEM 列11-12中时，将样品保持在液体中。TEM成像技术上的这一突破使研究人员首次能够在分子水平13上看到渐进事件。我们称这种新模式为"原位 分子显微镜"，因为现在可以在EM列14-15的"内部"进行实验。该方法的总体目标是在液体中对生物组件进行成像，以便以纳米分辨率观察其动态行为。开发技术背后的理由是记录实时观测，并检查生物机械在溶液中的新特性。这种方法扩展了TEM在细胞和分子生物学12-16中用于更广泛的目的。
在当前的视频文章中，我们提出了一个全面的协议，以组装和使用市售的微流体标本支架。这些专业支架利用用集成垫片生产的氮化硅微芯片形成一个液体腔室，将微量溶液包围起来。薄而透明的窗户被蚀刻在微芯片中，用于成像目的12。我们演示了使用微流体支架使用 TEM 检查液体中的模拟轮状病毒双层颗粒 （DLP） 的正确用途。为了确保生物组件（如DLP）在成像时不会在很远的距离上快速扩散，我们采用亲和力捕获方法将它们系在微流体16的表面。与液体中生物标本成像 的替代技术而言 ，这种分子捕获步骤具有重大优势，因为它允许获取用于下游处理程序的图像。这个捕获步骤与微流体成像相结合，是我们程序17所独有的。使用 TEM 或微流体成像室使用结构生物学应用程序的读者可能会考虑在分子水平上的动态观测是最终目标时使用亲和力捕获技术。

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	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:237px;">E-chips, spacer chip</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;">EPB-52TBD</td>
			<td style="width:167px;">400 &#956;m x 50 &#956;m window</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:237px;">E-chip, top chip</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;">EPT-45W</td>
			<td style="width:167px;">400 &#956;m x 50 &#956;m window</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Ni-NTA lipid</td>
			<td style="width:183px;">Avanti Polar Lipids</td>
			<td style="width:245px;">790404P</td>
			<td style="width:167px;">Powder form</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">DLPC (12:0) lipid</td>
			<td style="width:183px;">Avanti Polar Lipids</td>
			<td style="width:245px;">850335P</td>
			<td style="width:167px;">Powder form</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Volumetric flasks&#160;</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">20-812A; 20-812C</td>
			<td style="width:167px;">1 ml; 5 ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Hamilton Syringes</td>
			<td style="width:183px;">Hamilton Co.</td>
			<td style="width:245px;">80300, 80400</td>
			<td style="width:167px;">1-10 &#956;l; 1-25 &#956;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Whatman #1 filter paper</td>
			<td style="width:183px;">Whatman</td>
			<td style="width:245px;">1001 090</td>
			<td style="width:167px;">100 pieces, 90 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass Petri dishes</td>
			<td style="width:183px;">Corning&#160;</td>
			<td style="width:245px;">70165-101</td>
			<td style="width:167px;">100 mm x 15 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass Pasteur pipettes</td>
			<td style="width:183px;">VWR</td>
			<td style="width:245px;">14673-010</td>
			<td style="width:167px;">14673-010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass culture tubes</td>
			<td style="width:183px;">VWR</td>
			<td style="width:245px;">47729-566</td>
			<td style="width:167px;">6 mm x 50 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Acetone</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">&#160;A11-1</td>
			<td style="width:167px;">1 L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Methanol</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">&#160;A412-1</td>
			<td style="width:167px;">1 L</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">Chloroform&#160;</td>
			<td style="width:183px;">Electron Microcopy Sciences</td>
			<td style="width:245px;">12550</td>
			<td style="width:167px;">100 ml&#160;</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">His-tagged Protein A</td>
			<td style="width:183px;">Abcam, Inc.</td>
			<td style="width:245px;">ab52953</td>
			<td style="width:167px;">10 mg</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">Milli-Q water system</td>
			<td style="width:183px;">EMD Millipore Corp.</td>
			<td style="width:245px;">Z00QSV001</td>
			<td style="width:167px;">Ultrapure Water</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">HEPES</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">BP310-500</td>
			<td style="width:167px;">500 g</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Equipment&#160;</td>
			<td style="width:183px;"></td>
			<td style="width:245px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Poseidon In situ specimen holder</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;"></td>
			<td>&#160;FEI compatible</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">FEI Spirit BioTwin TEM</td>
			<td style="width:183px;">FEI Co.</td>
			<td style="width:245px;"></td>
			<td>120 kV</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Eagle 2k HS CCD camera</td>
			<td style="width:183px;">FEI Co.</td>
			<td style="width:245px;"></td>
			<td>10&#160;&#197;/pixel sampling at 30,000X</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:237px;">Gatan 655 Dry pump station</td>
			<td style="width:183px;">Gatan, Inc.</td>
			<td style="width:245px;"></td>
			<td>Pump holder tip to 10-6&#160;range</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:237px;">PELCO easiGlow, glow discharge unit</td>
			<td style="width:183px;">Ted Pella, Inc.</td>
			<td style="width:245px;"></td>
			<td>&#160;Negative polarity mode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Isotemp heated stir plate</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;"></td>
			<td>Heat to 150 &#186;C for 1.5 hr</td>
		</tr>
	</tbody>
</table>

in situ tem of biological assemblies in liquid

研究人员定期使用透电电子显微镜（TEM）来检查生物实体和评估新材料。在这里，我们描述了这些仪器的附加应用 - 在液体环境中查看病毒组件。这种令人激动和新颖的生物结构可视化方法利用了最近开发的基于微流体的标本支架。我们的视频文章演示了如何组装和使用微流体支架在 TEM 内对液体标本进行成像。特别是，我们使用模拟轮状病毒双层颗粒 （DLP） 作为我们的模型系统。我们还描述了用亲和力生物膜覆盖液体室表面的步骤，这些生物膜将 DLP 与观看窗口绑在一起。这使我们能够以适合 3D 结构确定的方式对组件进行成像。因此，我们首次在原生液体环境中看到亚病毒粒子。

使用发光释放的电子芯片（图 3A）在液体中使用 DDLP 的代表性图像显示，与在亲和力捕获设备上浓缩的 DDLP相比，在给定观看区域（大概是由于扩散）中的 DLP 较少。在成像室中加入尿素用于增强标本的对比度，从而提高溶液中单个 DLP 的可见性（图 3C，顶部面板）。更好的对比度允许下游图像处理，如粒子平均（图3C，底部面板）和3D重建例程（<...

Watch this Scientific Journal Video about In situ TEM of Biological Assemblies in Liquid at JoVE.com

In situ TEM of Biological Assemblies in Liquid

在这里，我们描述了一个程序，用传输电子显微镜以纳米分辨率对液体中的病毒复合物进行成像。

就地 液体生物组件的 TEM

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

in-situ-tem-of-biological-assemblies-in-liquid

Research

JoVE Journal

Bioengineering

In situ TEM of Biological Assemblies in Liquid

9.9K 次观看.  Protochips, Inc.. 在这里，我们描述了一个程序，用传输电子显微镜以纳米分辨率对液体中的病毒复合物进行成像。

视频: In situ TEM of Biological Assemblies in Liquid

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

科研

生物工程

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

可扩展的，金属的高宽比纳米复合材料在生物液体中产生

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

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 ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

交变磁场敏感的混合明胶微凝胶药物控释

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

在生物环境的新技术的产生和观测化学发光

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

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; ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

生物相容的液晶弹性体泡沫的合成中用作细胞支架的三维空间细胞培养

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

用于刺激生物组织的电动和磁场设备

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

就地 液体生物组件的 TEM

就地液体生物组件的 TEM