资金：美国陆军医学研究和装备司令部（USAMRMC）及远程医疗与先进技术研究中心管理。顺溪籍提供如这里所描述的注射器的修改。

1，系统设置了该系统由配有单级混合器，在线高剪切均化器，辊子泵来移动HCT和均化器之间的流体，和一个热交换器（图1）的保持和浓缩罐（HCT）的。 <ol><li>放置消毒，广口4升的玻璃集装上2底座端口和3个侧端口的单级混频器下方容器。降低混频器头在容器的口部，并确保气密接头用橡胶密封件或带（为了防止环境空气污染的头部空间）。 </li><li>适合的基地港口之一，用无菌3/8的HCT（图1，端口＃1）“（ID）的透明管，约10”长，配备了3路阀在尖端收集浓缩乳液。 </li><li>配合第二基本端口（图1，口＃2）用无菌3/8“（ID）的油管，approximatelŸ36“的长度。通过滚压泵送入这个管道。适合高剪切均质机的入口有一个T形件包括两个端口，并连接如下：从端口＃2连接管，通过滚轮泵和连接到T形件的侧端口。使用低流量的氧气流量计连接的另一端口，氧气罐。 </li><li>高剪切均化器的出口端连接至保持在4℃下对在线热交换器的入口所述热交换器的出口连接到HCT的返回口（图1，端口＃3），产生一个闭环系统。 </li><li>附加氧气罐（经由流量计）的HCT（图1，端口＃4）。附加气体成分监测，是开放的对HCT的顶部端口（图1，口＃5）气氛 ​​。 </li><li>如果不育性是需要的，每次使用由高压釜之前消毒的玻璃和金属部件。消毒管道元件和塑胶有限公司nnectors由环氧乙烷每次使用前。这是特别重要的，如果产品是在体内进行测试。 </li></ol> 2，LOM制造<ol><li>放置20克GMP的1,2 -二硬脂酰-sn-甘油-3 -磷酸胆碱（DSPC）和10克胆固醇在HCT的基部的。添加1L的等离子体莱特A向HCT和手搅拌1分钟，尽可能多的脂质尽可能集成到水相中。 </li><li>降低单级混频器进入水相，从而确保了整个混合器头部分在水相中。确保HCT的顶部是气密性（参见上面的步骤1.1），且没有打开的侧口。打开连接到端口＃4气源，然后等待，直到HCT顶部空间的氧气分数达到&gt; 95％。在10升/分钟（LPM），这应该采取〜10分钟。 </li><li>使用单级混合器中，混合所述前体乳液5分钟，在5,000 rpm。将所得的混合物应该出现淡白色的，不包含任何可见的梨皮ð团块。一旦混合后，未使用的脂质 - 水混合物可以储存在4℃为单次使用前最多30天。 </li><li>素的整个闭环系统通过接通滚筒泵在1.3 LPM的前体乳液。一旦系统催芽，保持泵的1.3 LPM。 </li><li>开始制造的LOM的，打开的行高剪切均化器以7500转每分钟。紧随其后，打开氧气流至均化0.5 LPM的入口部分。保持单级混频器（在HCT）上在3,500 rpm。的LOM形成在在线均化器（图2）内的转子叶片和emulsor画面的界面。几分钟之内，流体应该成为明显更粘稠。更严格的方法是确定的粘度作为时间的函数，这可以通过在制造过程中从端口1取出等分试样并用粘度计分析来完成。 注意：如果可见气泡存在于管排出的混合器，氧流向在线均化器是太高了。滴定向下气流，直至流体是不透明和不含有可见的气泡。 </li><li>运行系统15分钟，然后关闭高剪切均质机和氧气入口吧。继续在HCT运行的单级混合器直至乳液被去除;这减轻了相分离，并保持产品的HCT内相对统一。 注意：所述气体填充的乳浊液的体积应在串行浓度相增加约2-3倍。如果没有，请检查以确保氧气流入高剪切均质机和前体乳液脂质浓度是正确的。生产效率降低脂质浓度降低。 </li></ol> 3，收集，浓缩，评估，和的LOM的存储<ol><li>附上一个无菌，修改140毫升鲁尔锁注射器连接到基座端口＃1的收集容器的活塞。制定100毫升液。盖紧瓶盖注射器和重复，直到所有液体已被删除。 <ol><li>通过抽出百毫升空气进入注射器，然后锯切多余柱塞和注射器材料上面的140毫升标记修改注射器。填写并利用齿镊制定柱塞空的注射器。这一修改使得更容易离心。 </li></ol></li><li>离心机注射器与封顶年底在冷藏（4℃）离心桶在225×g离心10分钟朝下。 </li><li>离心后三层材料就会出现。排出体内多余的混浊水相的底层和丢弃。第二层是明亮的白色和含有浓缩的LOM。转浓泡沫使用三路活塞，以防止环境气体污染不透气注射器。放弃最后一层，其中含有游离氧的气体从破裂的LOM。 </li><li>泡沫质量可以通过达到浓泡沫的≥90％的气体进行评估。演算吃气体浓度如下： 体积％的气体= [（泡沫重量/体积泡沫） - 1]×100 <ol><li>作为第二质量控制，微气泡大小以减光，以确定是否粒径是所期望的范围内。但应注意的是，在均化时间或制剂的改变可能改变气泡的大小。 </li></ol></li><li>盖紧瓶盖与鲁尔锁接头的玻璃注射器。浓缩的LOM可与等离子莱特一个在使用时被摊薄。注射器可存放在22，4，或-20℃;较低温度下可提供增强的保质期稳定性7。 </li></ol>

<ol>
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作者宣称，他们有没有竞争的财务权益。

最重要的步骤，以建立集中的，高度氧化的LOM包括：1）确保HCT内的顶部空间保持完全氧化; 2）确保所述脂质赋形剂的纯度为最佳（包括储存条件和使用GMP的产品）; 3）确保粉状脂质一起吸系统之前水相混匀;和4）密切关注的HCT内增加的气体馏分，以确保气体的体积分数不超过70％。 我们在这里描述的方法，利用高剪切均质创建空心微。两亲性脂质悬浮的水相，它是用于承载脂...

微泡的蛋白质，聚合物和脂质壳的组成已经被开发作为药物递送载体，基因治疗，和超声造影剂1-5。因为这些治疗用途需要血管内微泡的持久性，这样的微泡通常填充有惰性的，高分子量的气体如全氟化碳6，它具有低的溶解度在血液和稳定的气泡3,4。 最近，基于脂质的氧气微泡（的LOM）被设计用于提供治疗剂量的氧，这可能保留终末器官的氧输送和防止在气道阻塞或低氧血症7期血流动力学不稳定。设计用于静脉内气体输送乳剂需要比那些用于超声造影剂或靶向药物递送不同的设计特点。首先，因为身体消耗大量的氧气（〜200毫升/分钟）的，的LOM必须生产和注射大规模。这需要在制造过程是有效的。第二，在制造过程中应该是闭环，以避免氮气污染过的LOM的曝光（它应该充满100％的氧气）到环境空气中。第三，因为的LOM的目的是静脉内气体输送，LOM中的气体馏分应最大化，认识到通过乳液粘度7所施加的限制。最后，与任何静脉注射，精确地控制粒度分布是必要的，避免微血管梗阻8。 有用于制造微泡几个成熟的方法。超声处理采用强度高，低频超声应用于乳液，它包括表面活性剂，如两亲性的磷脂，以气体的顶部空间，以产生微泡7,9的存在下的空气-液体界面。这个过程是可控的，通过改变超声音的频率，功率和脉冲持续时间，并将得到的粒度分布可以根据产生特定尺寸分布的微泡，虽然超声处理在临床上使用的微泡的制备很少使用。合并是表面活性剂和气体的封闭系统，该系统也难于扩大，以适应大容量的2的强烈机械搅拌。液滴型微流控使微气泡大小分布10-13的精确控制。虽然传统上难以扩大规模，多通道，高速微流体已经描述了增加微泡的生产效率13。使用上述任何一种方法制造的微泡可能需要制造后尺寸减小过程中，如离心分馏14,15和微泡浮选16,17。 另一种既定方法的高度稳定微泡的制造是剪切homogeniz振动性6，这可能会导致对微泡的表面18上的稳定六边形磷脂图案。在此基础上的概念，我们描述了在线高剪切均质机来创建自组装的LOM 19日注册成立。在这个过程中，利用匀化器快速旋转接近双细网眼emulsor屏幕叶片，产生高的机械和液压剪用于创建微泡。脂质乳剂通过本系统的串行浓度产生越来越浓的气体馏分，可进一步通过离心浓缩。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="42">
			<td height="42" style="height:43px;width:239px;">Name of Material/ Equipment</td>
			<td style="width:141px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:405px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)</td>
			<td style="width:141px;">Avanti Polar Lipids</td>
			<td style="width:119px;">770365</td>
			<td>Alternate product: non-GMP from NOF America (Coatsome MC-8080)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Cholesterol</td>
			<td style="width:141px;">Sigma Aldrich</td>
			<td style="width:119px;">C75209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Plasma-Lyte A</td>
			<td style="width:141px;">VWR</td>
			<td style="width:119px;">80089-818</td>
			<td>Alternatively can use NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Glass collection vessel</td>
			<td style="width:141px;">Specialty Glass, Inc.</td>
			<td style="width:119px;">Custom</td>
			<td>Contact: Pam Zurbrick - 281-595-2210</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Gas composition (oxygen) monitor</td>
			<td style="width:141px;">Precision Medical</td>
			<td style="width:119px;">PM5900L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Sarns 8000 roller pump</td>
			<td style="width:141px;">Calicut Medical</td>
			<td style="width:119px;">16407</td>
			<td>Part of a modular perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">BIOtherm Heat Exchanger</td>
			<td style="width:141px;">Medtronic</td>
			<td style="width:119px;">ECMOtherm-II</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Verso laboratory in-line mixer</td>
			<td style="width:141px;">Silverson Machines, Inc</td>
			<td style="width:119px;">TH-IL-102-VERSO</td>
			<td>Use multistage workheads and front-end extension with T piece</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">T-piece for Silverson Verso inlet port</td>
			<td style="width:141px;">Process Innovations</td>
			<td style="width:119px;">Custom</td>
			<td>Contact: Brian Leavitt - 508-423-2266</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">L5M-A laboratory mixer</td>
			<td style="width:141px;">Silverson Machines, Inc</td>
			<td style="width:119px;">NC0136483</td>
			<td>Use mesh emulsor screen (fine)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Rochester-Ochsner toothed forceps</td>
			<td style="width:141px;">Fisher Scientific</td>
			<td style="width:119px;">13-812-18</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">140 mL syringe</td>
			<td style="width:141px;">Kendall Healthcare Monoject&#160;</td>
			<td style="width:119px;">8881114030</td>
			<td>Ensure there is a luer lock.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">IX71 Inverted light microscope</td>
			<td style="width:141px;">Olympus</td>
			<td style="width:119px;">IX71</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Retiga-2000R microscope camera</td>
			<td style="width:141px;">QImaging</td>
			<td style="width:119px;">RET-2000R-F-M-12</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:239px;">Accusizer 780A Autodilution</td>
			<td style="width:141px;">PSS-NICOMP Particle Sizing Systems</td>
			<td style="width:119px;">Out of production</td>
			<td></td>
		</tr>
	</tbody>
</table>

manufacture of concentrated, lipid-based oxygen microbubble emulsions by high shear homogenization and serial concentration

充气微泡已经开发作为超声造影和药物递送剂。微泡可以通过使用超声处理，机械搅拌，微流体装置，或均化处理的表面活性剂来制备。最近，基于脂质的氧气微泡（的LOM）已经被设计在医疗紧急静脉输送氧气，扭转危及生命的低氧血症，防止后续的脏器损伤，心脏骤停而死亡。我们提出使用闭环高剪切均化器的方法按比例扩大生产的高度氧化的微泡。该过程可产生2升浓缩的LOM（90体积％）中90分钟。产生气泡的平均直径为〜2μm时，以及流变更新与该血液时，稀释至60％（体积）是一致的。该技术生产的高容量和高纯度氧的LOM，这表明该技术可用于转化研究实验室有用。

高剪切均质使效率（ 即在一个下午）生产足够的LOM对动物的研究，也不需要专业技术。一旦熟练，高达2升浓缩的LOM的可以在90分钟来制造。 微气泡的大小和形态，光镜和光遮蔽评估。时的LOM的10微升样品是可视化，球形的LOM中所指出的，以及脂质碎片的相对贫乏（图3A）。这一点尤其是当GMP产物被使用。时的LOM的相同的样品，通过减光评估的平均粒径为2.62...

Watch this Scientific Journal Video about Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration at JoVE.com

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

我们描述了大容量的基于脂质的氧气微气泡（的LOM），使用高剪切均化和串行浓度设计用于静脉内的氧气输送的制造方法。

制造浓缩，脂质为基础的氧气微泡的乳液由高剪切均质化和串行集中

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using ...

manufacture-concentrated-lipid-based-oxygen-microbubble-emulsions

Research

JoVE Journal

Bioengineering

14.9K 次观看.  Harvard Medical School. 我们描述了大容量的基于脂质的氧气微气泡（的LOM），使用高剪切均化和串行浓度设计用于静脉内的氧气输送的制造方法。 

视频: Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents7-9. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region10. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.

Ultrasound Targeted Microbubble Destruction (UTMD) can be used to direct site-specific delivery of bioactive molecules, including therapeutic genes, to target organs accessible to ultrasound, such as the heart and liver1-6.

有针对性的超声微泡销毁技术简介

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid ...

科研

生物工程

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.&#160;low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 &#181;m biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions.

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

的剪切粘合地图使用SynVivo合成微血管网络代

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

在脉冲激光激发金纳米粒子集成光电响应脂质体及其微泡空化的测量的合成

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

纳米丝绸制造和药物输送应用

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Quantifying Intermembrane Distances with Serial Image Dilations

A recently-described extracellular nanodomain, termed the perinexus, has been implicated in ephaptic coupling, which is an alternative mechanism for electrical conduction between cardiomyocytes. The current method for quantifying this space by manual segmentation is slow and has low spatial resolution.We developed an algorithm that uses serial image dilations of a binary outline to count the number of pixels between two opposing 2 dimensional edges.This algorithm requires fewer man hours and has a higher spatial resolution than the manual method while preserving the reproducibility of the manual process.In fact, experienced and novice investigators were able to recapitulate the results of a previous study with this new algorithm.The algorithm is limited by the human input needed to manually outline the perinexus and computational power mainly encumbered by a pre-existing pathfinding algorithm.However, the algorithm&#39;s high-throughput capabilities, high spatial resolution and reproducibility make it a versatile and robust measurement tool for use across a variety of applications requiring the measurement of the distance between any 2-dimensional (2D) edges.

The purpose of this algorithm is to continuously measure the distance between two 2-dimensional edges using serial image dilations and pathfinding. This algorithm can be applied to a variety of fields such as cardiac structural biology, vascular biology, and civil engineering.

用序列图像量化膜间隙距离伸缩

A recently-described extracellular nanodomain, termed the perinexus, has been implicated in ephaptic coupling, which is an alternative mechanism for ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

阴离子氧化铈纳米粒子催化清除体内植物活性氧的研究

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to nanomolar ranges, several orders of magnitude less than many physiological ligand concentrations. Optical nanostructures called zero mode waveguides (ZMWs), which are 100&#8722;200 nm in diameter apertures fabricated in a thin conducting metal such as aluminum or gold, allow imaging of individual molecules at micromolar concentrations of fluorophores by confining visible light excitation to zeptoliter effective volumes. However, the need for expensive and specialized nanofabrication equipment has precluded the widespread use of ZMWs. Typically, nanostructures such as ZMWs are obtained by direct writing using electron beam lithography, which is sequential and slow. Here, colloidal, or nanosphere, lithography is used as an alternative strategy to create nanometer-scale masks for waveguide fabrication. This report describes the approach in detail, with practical considerations for each phase. The method allows thousands of aluminum or gold ZMWs to be made in parallel, with final waveguide diameters and depths of 100&#8722;200 nm. Only common lab equipment and a thermal evaporator for metal deposition are required. By making ZMWs more accessible to the biochemical community, this method can facilitate the study of molecular processes at cellular concentrations and rates.

Described here is a nanosphere lithography method for parallel fabrication of zero mode waveguides, which are arrays of nanoapertures in a metal-clad glass microscopy coverslip for single molecule imaging at nano- to micromolar concentrations of fluorophores. The method takes advantage of colloidal crystal self-assembly to create a waveguide template.

为高浓度单分子显微镜制造零模式波导

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to ...