作者承认资助NIH / NIDCR R00DE018212（SPH），NIH/NIDCR-R01DE022032（SPH），美国国立卫生研究院/ NIDCR T32 DE07306（AJ，JDL），美国国立卫生研究院/ NCRR S10RR026645，（SPH），以及预防和恢复牙科学系和颜面部科学，加州大学旧金山分校。此外，作者承认Xradia公司研究生奖学金（AJ），Xradia公司公司，加利福尼亚州Pleasanton。 作者感谢凯瑟琳枫，加州大学旧金山分校博士为她与数据后处理援助;博士。斯蒂芬·韦纳和吉利Naveh，科学，雷霍沃特，以色列魏兹曼研究所;罗恩沙哈尔博士，耶路撒冷希伯来大学，以色列针对其特定的原位装载设备有见地的讨论。作者还要感谢生物材料与生物工程微型电脑成像设备在加州大学旧金山分校的使用微XCT和原位装载设备。

动物住房和安乐死:在本演示中使用的所有动物均按照该机构动物护理和使用委员会（IACUC）和美国国立卫生研究院（NIH）的指引，安置无病原体条件下。 提供动物与标准硬颗粒大鼠饲料和水的随机应变 。通过二氧化碳窒息，双侧开胸的两步法按照UCSF的标准协议安乐死动物经IACUC。 24小时动物献祭的范围内进行生物力学测试，以避免组织退化。 1。制备及大鼠下颌骨或上颌骨的解剖<ol><li>通过轻轻地切割膜组织和肌肉组织的附件，同时保持整个下颌骨，包括冠突和髁过程（ 图1）15除去大鼠下颌骨。 </li><li>独立hemimandibles车efully切割下颌联合纤维组织带有手术刀。 注:冠状动脉及髁流程，以及下颌骨升支（ 图1）应该被删除，如果他们身体挡住第二磨牙的生物力学测试。 </li><li>切门牙不暴露牙髓腔不妨碍加载磨牙。 </li></ol> 2。 原位压极限试样制备的（图2） <ol><li>通过使用比实验标本显著较硬之前加载它在原位装载装置（ 图2A）的材料固定在一个钢存根试样。 注:聚甲基丙烯酸甲酯（PMMA）是用于固定在这项研究中与过量的试样，如果有的话，用牙科探针除去。 </li><li>配合使用在两个直边的原子力显微镜的金属试样圆盘息平行的摩尔数的咬合面平面（ 即近远中和颊舌）。 </li><li>建立与周边磨牙钝刀一个低谷。 注意:这个空间应该作为一个"护城河"包含多余的液体，并在原位负载维持组织水化。 </li><li>制备在牙齿表面使用牙科用复合建立为同心（ 图2B）或偏心轮（ 图2C）加载。蚀刻所关心的牙齿表面以15秒的牙合面的35％磷酸凝胶。 </li><li>用去离子水彻底冲洗，蚀刻剂和使用空气/水注射器或压缩空气罐干燥的表面上。随着探险家，分布一滴粘接剂成公开尖在薄薄的一层。固化复合材料牙科光固化机。 注:应该没有直接的光从灯泡进行所有涉及复合材料的步骤。这样的条件下会不希望地促进聚合过程中，并且不需经过ð防止复合材料的妥善安置。房间里的灯是可以接受的。 </li><li>从邻牙去除多余的粘接剂用细手术刀或刀片。 </li><li>把可流动的牙科用复合材料的表面上的表面的制备方法以下，并用牙科探针铺到感兴趣的摩尔数的凹槽。 </li><li>暴露复合牙科光固化机，持续30秒。 </li><li>用牙科用复合树脂成形为约3-4毫米的咬合堆积，从感兴趣的摩尔数和光固化30秒的咬合平面。 </li><li>降低复合堆积的顶部表面平行的平面通过使用直边和一个高速手持件，以使一致的装载方案在所有标本。 注意:在生物力学测试，其他标本应存放在三-磷酸盐缓冲溶液（TBS）与50毫克/毫升的青霉素，链霉素和15。 </li></ol> 3。加载装置和漂移刚度，材料属性辨能力， 原位纤维联合的装载<ol><li>使试样与在加载阶段的砧座的复合材料形成，并且测试为均匀的载荷， 如图2B所示 。 </li><li>放置在复合材料的表面上形成的咬合纸随后装入试样，以一个有限的负载，以检查同心或偏心载荷（ 图2B和2C）。 </li><li>周围放置试样TBS浸泡过的Kimwipe，以确保样品水化。使周围的样品槽和用TBS填充它来保持成像过程中的水分的器官。 </li><li>输入峰值负载和位移速率到德夯软件来压缩的摩尔到下面的单侧的下颚骨的固定化所需的峰值负荷位移速度。 注意:典型的读数应该包括反动载荷作为材料被压缩，随着时间的推移（负荷传感器灵敏度= 0.1N）。从负荷-时间和位移-时间，用于压缩材料的负荷-位移曲线应获得16-18。使用从加载周期所收集的数据，关节的各种性能，也可测定。关节的刚度应采取的直线部分的载荷与位移曲线19的加载阶段（约最后30个数据的％）的斜率来计算。 </li></ol> 4。染色软组织中，PDL，用磷钨酸（PTA） 注意:为了提高X射线衰减的对比中，PDL应染色，用5％的PTA溶液20。 <ol><li>回填PTA染色溶液倒入一个干净的1.8毫升玻璃安瓿，并把装安瓿到注射器中。 </li><li>注入溶液缓慢（5分钟/卡普耳）到相邻牙齿的PDL-空间，以防止周围的兴趣磨牙牙周组织结构损伤。 注:以上步骤B应该要ë重复，直到溶液约5全carpules（9毫升）中被注入并允许流入周围的组织。该prepped标本也可以在剩余的PTA溶液（8小时）浸泡过夜。 </li></ol> 5。推荐μ-XCT扫描设置进行M-XCT具有下列扫描设置: <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td>物镜放大倍数</td><td> 4X，10X </td></tr><tr><td></td><td> 1,800的图像</td></tr><tr><td> X射线管电压</td><td> 75 kVp的（50 kVp的对PTA染色样品） </td></tr><tr><td></td><td> 8 W </td></tr><tr><td>曝光时间</td><td> 〜8-25秒* </td></tr><tr><td></td><td> 〜4微米（4X物镜），〜2微米（10X物镜）** </td></tr></tbody></table> *曝光时间可以根据几何形状和试样和X射线管的画外音的光密度变化ltage。 *基于源，样品和检测器的结构的实际像素分辨率将略有不同。

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在建立这个协议的第一个步骤涉及评估的装载架的使用刚性体的刚性。根据结果​​，刚度是显著更高使得能够使用的装载装置的标本与显著较低的硬度值进一步测试。第二步强调了仪器的使用两个阶段的装载 - 卸载曲线通过使用刚性体，不同的交联密度的PDMS材料和纤维连接生成区分不同硬度值的能力。从在间隙相的加载阶段和回送的刚度被用来确定该材料的装载和材料的回收的阻力以下卸载（...

几个实验方法继续被用来研究diarthrodial和纤维状关节生物力学。具体到牙齿器官生物力学的方法包括使用应变计1-3，光弹性方法4，5，云纹干涉6，7，电子散斑干涉8，和数字图像相关（DIC）9-14。在这项研究中，创新的方法包括非侵入性的成像使用的X射线暴露的纤维接头的内部结构（矿化组织和它们的接口由较软的区域，和接口组织如韧带）在负载相当于在体内条件。耦合到微透视显微镜原位装载装置将被使用。负载时间和负载 - 位移曲线将被收集感兴趣的新鲜收获的大鼠半侧下颌骨内装载的摩尔。该米在本研究中提出的方法的AIN的目标是通过在比较条件下，以强调牙骨的三维形貌的影响:1）无负载时加载，并且当2）同心和偏心加载。省去了切标本，并在潮湿条件下的整体完好的器官进行实验将允许三维应力状态的最大保护。这将打开调查，在各种载荷情况下理解复杂的动态过程的一个新领域。 在这项研究中，方法为只SD大鼠一个完整的纤维关节内测试PDL生物力学，被视为最佳的生物工程模型系统联合进行详细说明。实验将包括以突出关节的三个重要特点，因为它们涉及到器官水平的生物力学仿真水合条件下咀嚼负荷。这三点将包括:1）反动力与位移:肺泡插座内齿的位移和其装载反动响应，2）的三维（3D）空间构型和形态测量:用肺泡插座牙齿的几何关系，以及3）在读出器1和2中，由于在一个变化的变化加载轴， 即从同心偏心负荷。所提出的技术的三个基本读数可以应用于调查接头的脊椎动物中任一所述自适应性质变化引起的功能性要求，和/或疾病。可用于改变上述读数，特别是在不同的加载速率反动负载之间的相关性与位移，并导致反动的加载时间和载荷 - 位移曲线，突出联合生物力学整体变化。提出的协议的有效性可以通过耦合力学测试读数为3D形态测量和关节的生物力学整体评估。

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			<td height="21" style="height:21px;width:169px;">Bard Parker Blade</td>
			<td style="width:101px;">BD</td>
			<td style="width:123px;">MEDC-001054</td>
			<td style="width:259px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:169px;">AFM metal disk</td>
			<td style="width:101px;">Ted Pella</td>
			<td style="width:123px;">16218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Polymethyl methacrylate&#160;</td>
			<td style="width:101px;">GC America</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Uni-Etch</td>
			<td style="width:101px;">Bisco</td>
			<td style="width:123px;">E5502EBM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Optibond Solo Plus</td>
			<td style="width:101px;">Kerr Corp</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Filtek Flow</td>
			<td style="width:101px;">3M</td>
			<td style="width:123px;">N/A</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Hurculite Ultra</td>
			<td style="width:101px;">Kerr</td>
			<td style="width:123px;">34346</td>
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			<td height="21" style="height:21px;width:169px;">Tris buffer</td>
			<td style="width:101px;">Mediatech Inc.</td>
			<td style="width:123px;">N/A</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Articulating paper</td>
			<td style="width:101px;"></td>
			<td style="width:123px;"></td>
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			<td height="21" style="height:21px;width:169px;">Phosphotungstic Acid</td>
			<td style="width:101px;">Sigma Aldrich</td>
			<td style="width:123px;">HT152</td>
			<td></td>
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in situ compressive loading and correlative noninvasive imaging of the bone-periodontal ligament-tooth fibrous joint

这项研究表明，一种新型的生物力学测试协议。此协议的优点包括在耦合到高分辨率X-射线显微镜原位装载装置使用的，从而使模拟生理负荷和潮湿条件下的内部结构元件的可视化。实验样品将包括完整的骨牙周膜（PDL）齿纤维连接。结果将说明该协议的三个重要特点，因为它们可以应用到器官水平的生物力学:1）反动力与位移:位移牙牙槽窝内，其装载，2），三维（3D）空间配置反动响应和形态测量:用肺泡插座牙齿的几何关系，以及3）在读出器1和2中，由于在加载轴的变化， 即从同心偏心载荷的变化。提出的协议的有效性可以通过机械耦合TE进行评估蜇读数为3D形态测量和关节的生物力学整体。此外，这种技术将强调需要平衡的实验条件下，前获取纤维连接的断层图像特别反动负载。应当注意，该协议被限制为检测离体条件下的试样，并且使用造影剂进行可视化的软组织机械响应可能导致对组织和器官水平的生物力学错误的结论。

的装载装置"齿隙"恒定负荷下，"推回"，刚度，和系统漂移估计 差:循环加载和卸载部分之间，存在3秒的暂停期间，反向齿轮电机内卸真正开始前， 即作为试样拉离顶下颚（ 图3）。这一时期被称为系统，它代表一个时间段，当系统试图从闭到开的夹爪切换的反弹。但应注意的是，所有的负载周期都将包含一个类似的齿隙响应无论...

Watch this Scientific Journal Video about In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint at JoVE.com

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

在这项研究中， 在加上微X射线计算机断层摄影术为纤维状关节生物力学原位装载装置使用一个将被讨论。识别与关节生物力学的整体改变的实验读数将包括:1）反动力与位移，在牙槽窝内，即牙齿位移及其装载反动响应，2）三维（3D）空间构型和形态测量， 即几何齿与齿槽插座，和3）中读出1和2的变化是由于在加载轴， 即同心或偏心载荷的变化关系。

原位压极限和骨牙周韧带齿纤维联合的相关性无创成像

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

in-situ-compressive-loading-correlative-noninvasive-imaging-bone

Research

JoVE Journal

Bioengineering

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

13.3K 次观看.  University of California San Francisco. 在这项研究中， 在加上微X射线计算机断层摄影术为纤维状关节生物力学原位装载装置使用一个将被讨论。识别与关节生物力学的整体改变的实验读数将包括:1）反动力与位移，在牙槽窝内，即牙齿位移及其装载反动响应，2）三维（3D）空间构型和形态测量， 即几何齿与齿槽插座，和3）中读出1和2的变化是由于在加载轴， 即?...

视频: In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

高分辨率光纤内窥镜为原位细胞成像

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

科研

生物工程

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but important bioinstruments capable of testing the force dependence of structural features in proteins. Scale has been a limiting parameter on how accurately researchers can peer into the nanomechanical world of molecules, such as nucleic acids, enzymes, and motor proteins that perform life-sustaining work. Atomic force microscopy (AFM) is well tuned to determine native structures of fibrous proteins with a distance resolution on par with electron microscopy. However, in AFM force studies, the forces are typically much higher than a single molecule might experience 1, 2. Optical traps (OT) are very good at determining the relative distance between the trapped beads and they can impart very small forces 3. However, they do not yield accurate absolute lengths of the molecules under study. Molecular simulations provide supportive information to such experiments, but are limited in the ability to handle the same large molecular sizes, long time frames, and convince some researchers in the absence of other supporting evidence2, 4.
The gravitational force spectrometer (GFS) fills a critical niche in the arsenal of an investigator by providing a unique combination of abilities. This instrument is capable of generating forces typically with 98% or better accuracy from the femtonewton range to the nanonewton range. The distance measurements currently are capable of resolving the absolute molecular length down to five nanometers, and relative bead pair separation distances with a precision similar to an optical trap. Also, the GFS can determine stretching or uncoiling where the force is near equilibrium, or provide a graded force to juxtapose against any measured structural changes. It is even possible to determine how many amino acid residues are involved in uncoiling events under physiological force loads 2. Unlike in other methods where there is extensive force calibration that must precede any assay, the GFS requires no such force calibration 5. By complementing the strengths of other methods, the GFS will bridge gaps in understanding the nanomechanics of vital proteins and other macromolecules.

This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.

展示新型引力光谱仪的使用拉伸和测量纤维蛋白

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but ...

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 ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

组织工程生物反应器的双轴机械装载设计

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

FIBS功能的无创性代谢轮廓

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

研究表皮生长因子受体的完整细胞使用相关显微镜的化学计量

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

表征细胞迁移在三维体外伤口环境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

用原位滞后测量进行磁畴成像的优化设置和协议

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

纳米传感器检测体内蛋白酶活性的无创诊断

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...