作者感谢安娜 Labernadie, 纪尧姆 Charrière 和帕特里克 Delobelle 为这项工作的最初贡献, 并马修桑切斯和弗朗索瓦斯·汉普森 Viala 为他们的帮助, 视频拍摄和编辑。这项工作得到了研究所 (ANR14-CE11-0020-02)、la 基金会研究所 Médicale (FRM DEQ2016 0334894)、INSERM 计划癌症、图卢兹癌症和人类前沿科学计划 (RGP0035/2016) 的支持。

1. Formvar 涂层网格的制备
<ol><li>清洁电子显微镜网格与纯丙酮和干燥他们在滤纸上。然后用纯乙醇清洁显微镜滑梯, 用透镜纸擦拭, 用鼓风机清除灰尘。</li>
	<li>将乙醇清洗过的玻璃垂直放在薄膜浇铸装置的漏斗上, 其中含有下部 Formvar 的溶液。覆盖漏斗顶部。</li>
	<li>将100毫升的 Formvar 溶液 (二氯化乙烯中的 0.5%) 与雾化灯泡泵浦, 直到水平达到滑动的两个以上。</li>
	<li>在解决方案中保留幻灯片1分钟。</li>
	<li>打开设备的阀门以在稳定的流中排出 Formvar 溶液。流速为10至15毫升/秒将产生一个 Formvar 膜 30-80 nm。薄膜的厚度是由 Formvar 溶液的浓度和排水速率决定的。 注: 通过将阀门慢慢打开, 通过排气阀排出压力, 可以控制排水速率。排水越快, 胶片越厚。在实践中, 由于难以预测薄膜厚度从 Formvar 流速, 我们准备了几批 Formvar 薄膜和控制其厚度的 AFM (见步骤 2)。</li>
	<li>从腔室取出滑块, 并在滤纸上微妙地晾干, 以除去任何液体 Formvar。</li>
	<li>用剃刀刀片从 Formvar 胶片上剪下一个20毫米 x 50 毫米的长方形。</li>
	<li>用干净的蒸馏水填满烧杯, 慢慢地将幻灯片垂直地浸入水中漂浮在胶片上: 胶片应该漂浮在水面上。</li>
	<li>将干丙酮清洗的网格放在胶片上, 面朝上发亮。</li>
	<li>将圆形玻璃盖玻片 (直径12毫米) 放到几个网格上。此盖玻片将用于测量胶片厚度 (见步骤 2)。</li>
	<li>覆盖一个显微镜幻灯片与一个白色的标签调整大小, 以适应它。将幻灯片垂直倾斜在浮动 Formvar 胶片的边框上, 直到整个胶片粘附在幻灯片上。用滤纸把多余的水从滑梯上取出, 让它在室温下一夜之间晾干。</li>
</ol>2. 薄膜厚度的测量
<ol><li>用镊子把盖玻片周围的 Formvar, 从滑梯上分离出来。</li>
	<li>用钢笔在盖玻片下标记网格的位置。</li>
	<li>在标记的网格周围剪切 Formvar, 将其与盖玻片分离。因此, 在剩余的 Formvar 板上会有一个洞, 这将允许测量胶片的厚度。用 PBS 覆盖盖玻片, 并将其放在显微镜上的玻璃滑梯上。</li>
	<li>在 AFM 玻璃块上安装氮化硅悬臂, 确保金色条纹不被弹簧的尖端覆盖。 注意: 悬臂的灵敏度和弹簧常数应该以前在 PBS 中校准过。</li>
	<li>将玻璃块安装到 AFM 模块上, 然后将该模块放到显微镜上。</li>
	<li>将 Formvar 涂层的盖玻片放在观察室上, 并盖上500µL PBS。</li>
	<li>用 AFM 在接触模式和 0.5 nN 力设定点扫描 Formvar 板孔的边界。</li>
	<li>使用玻璃盖玻片表面作为高度测量的参考, 并评估 Formvar 厚度为剖面的高度 (每 Formvar 批次至少执行10次测量)。</li>
</ol>3. 在网格上播种细胞
<ol><li>在无菌罩下, 在盖玻片上放置一个带 (12 毫米 x 3 毫米) 的双面胶带。</li>
	<li>用镊子剪掉一个 Formvar 的网格, 把它倒在胶条上 (只有网格的边缘需要附着在胶带上)。Formvar 电影需要面对盖玻片。</li>
	<li>把这个设备放在一个文化中。</li>
	<li>放置一个10µL 滴 RPMI 没有 FCS 含有 104巨噬细胞分化为人单核细胞16。 注意: 确保不要用吸管尖接触网格, 以防止损坏 Formvar 涂层。</li>
	<li>孵育30分钟 (37 °c, 5% CO2) 让细胞坚持。</li>
	<li>用2毫升的含10% 的 RPMI 填充井。</li>
	<li>在37摄氏度和 5% CO2上孵化设备2小时, 让细胞在 AFM 观察之前坚持。</li>
</ol>4. Podosome 诱发变形的地形测量
<ol><li>将两条双面胶带贴在玻璃底培养皿上。 注: 两条线之间的距离应小于网格直径。</li>
	<li>小心地从胶带上分离出与巨噬细胞电镀的格子。</li>
	<li>将网格倒置, 使单元格面对玻璃, 并将栅格粘在两条粘合带之间。</li>
	<li>固定网格与两个新的双面胶条: 网格将因此被夹在两个胶条之间, 使网格的中心区域可进入 AFM 尖端。 注意: 确保网格是固定的, 而不是扭曲的;否则, AFM 悬臂可能无法接近 Formvar 表面。</li>
	<li>用2毫升预加热的37°c 培养基 (RPMI 和 10% FCS) 填充培养皿, 辅以10毫米 HEPES (pH 7.4)。</li>
	<li>将养殖盘放在37摄氏度的盘子加热器上。</li>
	<li>在 AFM 阶段安装碟式加热器。</li>
	<li>让系统稳定在37摄氏度。</li>
	<li>在接触模式与 0.5 nN 力量, 使第一个全局图像定位 podosomes, 然后扫描 Formvar 地形在3赫兹与大约 20 nm 大像素。 注意: 避免在网格边缘附近进行扫描。</li>
</ol>

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没有宣布利益冲突。

材料性能
可变形膜材料的选择, 在我们的案例中 Formvar, 需要满足一些要求。该材料必须是透明的可见光和展示有限的自动荧光, 以允许观察在明亮的领域和荧光显微镜。薄膜的粗糙度必须低于10纳米, 以避免任何地形对细胞黏附的影响, 并允许通过 AFM 成像清晰地观察细胞诱发的突起。最后, 根据杨氏模量和材料厚度的不同, 膜的刚度需要足够高, 以诱导 podosomes 的形...

动物细胞与基质和构成其环境的其他细胞在物理上相互作用1。这是需要他们迁移, 内化机构, 获取外部信息, 或区分。在这种过程中, 细胞必须产生机械力, 正如近年来许多研究表明的那样, 细胞产生力量和探测其环境的能力会影响其生物学行为, 例如扩散或分化2,3。反过来, 细胞力的测量是研究力量生成规律的主要辅助因素, 并了解它在细胞行为和组织命运4,5中的意义。
最近几年目睹了许多技术的发展, 以测量细胞在其环境中所能发挥的力量6。大多数这些都有助于揭示细胞在移动探针或变形基板上施加的牵引力。然而, 在细胞外环境中所涉及的机械力缺乏测量技术, 至今尚无良好的特征。
为了克服这一限制, 我们提出了一种测量施加正交到基体上的力的方法。它包括在一个薄的弹性薄板上的电镀活细胞, 可以在正交方向上变形, 使其能够测量基底变形的细胞和推断所涉及的力量。用原子力显微镜测量基片的形貌, 用纳米尺度的分辨率测定, 变形力的评估依赖于突出细胞结构的几何知识7,8,9。
在这里, 我们描述的设置和它的应用, 以测量 podosomes 产生的力量, 突出黏附结构形成的巨噬细胞为他们的间充质迁移在三维环境10,11, 12,13,14,15,16,17。我们相信, 这一技术将促进理解的力量产生和它的调节, 在许多细胞过程涉及突出。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">200 mesh nickel grids</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">G200-Ni</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">Filter paper</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">1001-055</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">Microscope slides</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">10235612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">White stickers 26 x 70 mm</td>
			<td style="width:71px;">Avery</td>
			<td style="width:119px;">DP033-100</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Film casting device with valve in its outlet</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">71305-01</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Razorblades</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">72000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">1.08543.0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetone</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">20066.321</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Formvar 0.5% solution in ethylene dichloride</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">12 mm coverslips</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">631-0666</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted microscope</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;">Axiovert 200</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Atomic Force Microscope</td>
			<td style="width:71px;">JPK Instruments</td>
			<td style="width:119px;">NanoWizard III</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Temperature-controlled sample holder&#160;</td>
			<td style="width:71px;">JPK Instruments</td>
			<td style="width:119px;">BioCell</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Silicon nitride cantilever with a nominal spring constant of 0.01 N/m</td>
			<td style="width:71px;">Veeco Instruments</td>
			<td style="width:119px;">MLCT-AUHW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Double-sided adhesive tape</td>
			<td style="width:71px;">APLI AGIPA</td>
			<td style="width:119px;">118100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">31870-025</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">FCS</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F7524</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">HEPES&#160;</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm glass-bottom Petri dishes</td>
			<td style="width:71px;">WPI</td>
			<td style="width:119px;">FD35-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

protrusion force microscopy: a method to quantify forces developed by cell protrusions

在许多生物学环境中, 动物细胞需要通过培养机械力来与环境相互作用。其中, 牵引力具有良好的特征, 但缺乏技术允许测量正交细胞所施加的凸出力。我们设计了一个实验装置来测量附着细胞在基板上施加的凸出力。在兼容的 Formvar 板上镀的细胞变形了这种基底, 由此产生的地形被原子力显微镜 (AFM) 在纳米尺度上映射。然后从基于突出细胞结构几何的变形剖面分析中提取力值。因此, 一个活细胞的个别凸出单位施加的作用力可以随着时间的推移而测量。这一技术将使研究的力量产生和它的调节, 在许多细胞过程涉及突出。在这里, 我们描述了它的应用, 以测量由人巨噬细胞形成的 podosomes 产生的突出力。

上述协议描述了如何准备实验设置, 以量化在 Formvar 基板上应用巨噬细胞 podosomes 的突力。这是使用 AFM 实现的, 如图 1所示。
利用 JPK 数据处理软件对 podosomes 下凸起的地形图像进行分析时, 应独立地从每条扫描线中减去三度多项式拟合。可以通过在图像侧面添加 z 色刻度来评估 podosome 引起的颠簸的最大高...

Watch this Scientific Journal Video about Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions at JoVE.com

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

在这里, 我们详细介绍了用于评估 podosomes 在兼容薄膜上应用的突出力的实验技术, 从薄膜的制备到地形图像的自动分析。

凸出力显微镜: 一种量化细胞突起力的方法

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...

protrusion-force-microscopy-method-to-quantify-forces-developed-cell

Research

JoVE Journal

Biology

5.5K 次观看.  IPBS, Université de Toulouse, CNRS, UPS. 在这里, 我们详细介绍了用于评估 podosomes 在兼容薄膜上应用的突出力的实验技术, 从薄膜的制备到地形图像的自动分析。

视频: Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

微阵列后探测器（mPADs）：机械力的方法来隔离

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

科研

生物学

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3&#39;UTR记者构造的microRNA识别目标在贴壁细胞

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

适用于比色金纳米粒子检测方法的选择结构切换适配方法

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

设计和适体 - 金纳米粒子基于比色法的开发在这领域的应用

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

电穿孔对小鼠膀胱 Urothelium 质粒 DNA 传递的新方法

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

时空映射和粒子分析技术在细胞内 ca2 +原位信令定量中的应用

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high throughput. In general, the acquisition of single-cell time courses from cultured cells is hampered by cell motility and diversity of cell shapes. Adhesive micro-arrays standardize single-cell conditions and facilitate image analysis. LISCA combines single-cell microarrays with scanning time-lapse microscopy and automated image processing. Here, we describe the experimental steps of taking single-cell fluorescence time courses in a LISCA format. We transfect cells adherent to a micropatterned array using mRNA encoding for enhanced green fluorescent protein (eGFP) and monitor the eGFP expression kinetics of hundreds of cells in parallel via scanning time-lapse microscopy. The image data stacks are automatically processed by newly developed software that integrates fluorescence intensity over selected cell contours to generate single-cell fluorescence time courses. We demonstrate that eGFP expression time courses after mRNA transfection are well described by a simple kinetic translation model that reveals expression and degradation rates of mRNA. Further applications of LISCA for event time correlations of multiple markers in the context of signaling apoptosis are discussed.

Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site.

单细胞阵列的活细胞成像 （LISCA） - 量化细胞动力学的多功能技术

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...