美联社感谢文卡特山教授、新加坡国立大学纳米和纳米技术倡议和副校长办公室 (R-398-000-084-646) 的支持。CLD 承认新加坡卫生部国家医学研究理事会在其临床科学家资助计划 NMRC/CSA/035/2012 和新加坡国立大学的支持。资助者在研究设计、数据收集和分析、决定出版或准备原稿方面没有作用。

1. 传感器的制造和数据的获取
<ol>
	<li>镍模的制备
	<ol>
		<li>将一层220纳米厚的负电子束阻挡在600µm 厚的4的硅片上。使用电子束光刻系统编写该晶片上设计的纳米阵列。<ol>
			<li>为了加速电子束写入, 请为每个300µm 字段大小 (a) (即,在每个300µm2区域上映射4亿点, 并将每个点都由电子束或不显示, 以低 dotmap (N) 为20k 写入模式。, 具体取决于图案设计)。设置抗电子束抗照射剂量为抵抗到110µC cm2并且写在当前 (I) 800 pA。 注意: 在电子束写入时, 曝光剂量 (D) 由每个点 (T点) 的曝光时间控制, 由计算. <img alt="Equation 1" src="/files/ftp_upload/55597/55597eq1.jpg"> </img>对于暴露剂量在110µC cm2, 电子束的居住时间在每个暴露的点是0.5 µs12。由于数组捕获的区域为 1.8 mm2, 因此总共有36块 300-µm2字段区域缝合在一起, 形成一个大的金纳米阵列。</li>
		</ol>
		</li>
		<li>开发的抵抗, 浸泡4英寸硅片在开发商的解决方案, 十年代, 让硅片干燥在空气中。</li>
		<li>在硅片上沉积金属的种子层, 如镍、铜或铝。</li>
		<li>在镀镍氨基镀液中电镀晶片。两步进行电镀。在第一步, 持续95分钟, 使用电流密度为0.7 的 dm2;这完全填补了 nanopatterns 与镍。在第二步, 持续125分钟, 使用 12 dm2达到300µm 作为最终的镍模具厚度 (20 nm)。确保 pH 值在 3.5-3.8, 温度在 52-54 ° c。</li>
		<li>使用柔和的机械力将镍模与硅基体分离。将镍模浸泡在约100毫升的正光刻胶去除剂中过夜, 清洗掉电子束的残留物。</li>
		<li>将镍模放入烤箱中, 在100° c 的3小时内将其晾干. 在等离子蚀刻系统中, 用 O2气体在 10 sccm 和 100 W 处清洁它, 3 分钟。</li>
	</ol>
	</li>
	<li>金纳米结构的制备
	<ol>
		<li>在80° c 的自组装单层 (SAM) 涂层机上, 在镍模上涂150µL heptadecafluoro-1,1,2,2-tetrahydrodecyl 硅 (FDTS)。 注: 这将形成一个粘层, 这将使模具分离的光刻胶 ("脱模") 后完成的 nanoimprinting 步骤。气化时间应 180s, 反应时间应900s。</li>
		<li>印记的 nanopatterns 4 在玻璃晶片已被涂上300纳米厚的光固化无抵抗使用纳米印刷机在压力的 10 bar 和温度40° c 为10分钟。</li>
		<li>将模具、光刻胶和玻璃晶片转换为 uv 光固化系统, 并在三十年代使用 75 mW cm-2的 uv 曝光 photocure。 注: 如果所有步骤都被正确地遵循, 镍模具应该很容易 demolded 从光刻胶。</li>
		<li>在反应离子蚀刻系统中, 对玻璃基板上的光刻胶进行一个空白蚀刻, 用 O2气体流量为 10 sccm, 在 50 W 处为 2 s, 在缩进区域中暴露玻璃。</li>
		<li>在电子束沉积机的玻璃晶片上沉积一层5纳米厚的铬 (Cr), 用于金属附着力和100纳米金 (Au) 的浆传感器。为 Au 使用1Å s1的沉积速率, 用于 Cr 和2Å的− 1 。</li>
		<li>用 O2等离子蚀刻进行3分钟的光刻, 然后在丙酮中进行15秒的可步骤。</li>
		<li>将样品切成5毫米 x 5 毫米的筹码。该纳米阵列将占据中央2毫米 x 2 毫米的芯片。</li>
	</ol>
	</li>
	<li>数据的承购
	<ol>
		<li>设置该仪器, 使光学测量, 使得通过发射器光纤的一端的白光光束是准直的, 是入射到传感器表面 (纳米阵列) 在90°。 注意: 光通过整个纳米阵列传输。</li>
		<li>收集传输信号与接收器光纤和记录它与紫外可见光谱仪操作在范围300到1000毫微米之内。</li>
		<li>将每个帧的获取时间设置为20毫秒平均100帧, 以获得最终频谱以降低测量中的噪音。</li>
		<li>使用绘图软件根据先前确定的传输峰值 (使用 Lorentz-based 方法) 分析数据。</li>
	</ol>
	</li>
</ol>
2. 传感器体积灵敏度测试
<ol>
	<li>将标准 ri 液体存入液体细胞, ri 的变化从1.31 到1.39。</li>
	<li>将传感器芯片浸入标准 RI 液体中, 并将其与白光光束对齐。获得传输频谱。</li>
	<li>在每次测量后用 surface-active 清洗剂清洗传感器芯片, 并用氮气将其烘干。</li>
</ol>
3. 传感器表面改性
<ol>
	<li>在任何化学修饰之前, 通过依次浸入异丙醇、丙酮和去离子水来清洗传感器芯片。在干燥的氮气流中室温干燥。</li>
	<li>在室温下, 在 0.4 mm 10-羧基-1-癸和 1.6 mm 1-硫的乙醇溶液中孵育传感器芯片。 注: 这将形成一个胺反应自组装单层 (SAM)。</li>
	<li>使用乙醇在室温下彻底冲洗和干燥。</li>
	<li>做一个75毫米磺 n-羟基琥珀酰亚胺 (磺-NHS) 和15毫米 1-乙基 3-(3-dimethylaminopropyl) 二的混合物。将此混合物中的筹码浸泡15分钟。 注意: 这将激活 SAM 的羧酸组。</li>
	<li>斑点50µL 的200µg/毫升 anti-troponin 抗体溶液在 pH 值4.5 醋酸酯缓冲液中的传感器表面和孵化30分钟。</li>
	<li>将传感器芯片浸入1米乙醇胺-HCl 溶液中15分钟, 使未酯钝化。</li>
	<li>用去离子水冲洗芯片, 在室温下在干氮气的气流中干燥。</li>
</ol>
4. cTnI 测定
<ol>
	<li>通过在表面上发现100µL 1% 牛血清白蛋白 (BSA) 溶液, 阻止任何非特异性结合。</li></ol>孵育15分钟。
	<li>用磷酸盐缓冲盐水 (PBS) 溶液冲洗传感器芯片三次。将芯片插入测量单元以记录传输频谱。 注: 这是参考光谱。</li>
	<li>斑点50µL 的 cTnI 标准到芯片表面, 在潮湿的环境中孵育30分钟。</li>
	<li>在 PBS 溶液中冲洗三次传感器芯片, 并将其插入测量单元以记录传输频谱。 注: 这是 after-binding 频谱。</li>
	<li>将芯片浸入 50 mM 甘氨酸盐酸盐 (pH 2) 中1分钟, 然后在 PBS 溶液中冲洗三次以再生晶片表面。测量 PBS 的透射光谱, 验证再生步骤是否成功。</li>

5. 表面等离子体共振 (SPR) 测量
<ol>
	<li>用 PBS t 缓冲器在 spr 系统上运行复用 spr 传感器芯片。 注: PBS t 缓冲液的组成为20毫米钠磷酸盐, 150 毫米氯化钠, 0.05% 吐温20。pH 值为7.4。</li>
	<li>使用 cTnI 标准和抗体, 如步骤4所述。</li>
	<li>激活3出6可用通道与混合的0.2 米和磺-NHS (0.05 米) 为5分钟. 执行5分钟注射50µg/毫升抗体560和5分钟注射1米乙醇胺盐酸溶液。</li>
	<li>将传感器芯片旋转90度, 并在不同浓度 (75、30、7.5 和 2.5 ng/毫升) 中注入 cTnI 标准。</li>
	<li>通过 SPR 读出, 观察芯片上的相互作用点上的抗体的共轭。</li>
	<li>通过注射50毫米甘氨酸盐酸盐 (pH 2) 来再生芯片, 1 分钟。</li>
</ol>

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作者没有相互竞争的金融利益。

通过模拟入射光和纳米结构之间的相互作用, 可以识别出合适的峰值 (在透射谱中), 其位移可记录为分析物浓度的函数。重要的是要注意, 对传感器结构的波段的本地化是至关重要的选择正确的波段, 其转移可以跟踪, 以感知分析物。可视化可以通过仿真实现。这也是关键的设计一个优化结构, 使传感的分析。正如这里所看到的, 第一和第三波段是在玻璃金色的界面上定位的, 因此对传感是无用的。在...

基于纳米阵列的化学传感器一直是许多调查的主题, 因为在 1998年1中, Ebbesen et al.发布了第一份关于特殊光传输 (EOT) 的报告。当光撞击波长维纳米结构的周期阵列时, 增强的透射在特定波长上发生。当入射光与极化 (BW) 和/或局部表面等离子体 (LSP)2时发生。
当与这样的周期数组传感时, 所利用的底层物理原理很简单。分子在金属界面上的吸附, 改变了介质在接触金属时的介电常数, 反过来又转移了光谱中传输波段的位置。频谱本身可以通过纳米工程来调整形状、大小和分离距离3,4,5。根据设计, 基于 EOT 的传感器在其光谱中具有特征波段, 在分子结合事件的调查过程中, 它能促进特定的赋值6、7、8 。这是一个关键的优势比商业可用表面等离子共振 (SPR) 平台。
使用 EOT 的传感器通常涉及光源的光学对准, 使准直光束在传感表面发生入射。生成大型纳米表面的技术 (如共聚模板和干涉和纳米光刻) 的重现性较差,9。由于这些限制在精确地制造出显示 EOT 现象的大表面, 需要光学显微镜来正确定位光源和探测器。为了简化技术, 采用了高质量的 nanoimprinting 光刻 (零)10 。这使得大的传感器表面区域的生产11 (毫米级), 消除了需要显微镜来寻找传感表面上的芯片。相反, 这种传感器可以很容易地与标准的光缆连接。
由于此纳米阵列的传输峰值包含在可见光近红外区域 (近红外线), 因此它非常适合于在水环境中感知生物分子的结合事件。模拟了纳米阵的期望光学行为。通过对标准折射率 (RI) 液体的研究, 验证了实验结果的正确性。该阵列用于测定心肌肌钙蛋白 i (cTnI) 在人血清复杂背景下的浓度。cTnI 是诊断急性心肌梗死的临床金标准。
使用该传感器, 可以检测和量化的 cTnI 在人血清中的检测限度 (LOD) 的 0.55 ng/毫升, 这是临床相关的。该检测比最常用的技术在这个领域, 酶联免疫吸附试验 (ELISA) 快得多。此外, 传感表面可以很容易地再生, 因此可重复使用。因此, 这项工作表明, 纳米阵列的承诺, 作为一个可行的点 (POC) 技术的传感在复杂的 biofluids。

<table><tbody><tr><td>Electron Beam Lithography setup</td><td>Elionix ELS 700</td><td></td><td></td></tr><tr><td>o-Xylene</td><td>Sigma Aldrich</td><td>95662</td><td></td></tr><tr><td>EB resist</td><td>Sumitomo</td><td>NEB-22A2</td><td></td></tr><tr><td>Developer reagent</td><td>Shipley Company</td><td>Microposit MF 321</td><td></td></tr><tr><td>Electroplating machine</td><td>Technotrans AG RD 50</td><td></td><td></td></tr><tr><td>Photoresist stripper&amp;nbsp;</td><td>Rohm and Haas Electronic Materials&amp;nbsp;LLC</td><td>Microposit Remover 1165</td><td></td></tr><tr><td>Etching System</td><td>Trion Phantom</td><td></td><td></td></tr><tr><td>Heptadecafluoro-1,1,2,2-tetrahydrodecyl)trichlorosilane&amp;nbsp;</td><td>Gelest (PA, USA)</td><td>78560-44-8</td><td></td></tr><tr><td>SAM coater&amp;nbsp;</td><td>Sorona Inc.</td><td>AVC 150M</td><td></td></tr><tr><td>Photo-curable NIL resist</td><td>micro resist technology GmbH</td><td>mr-UVCur21-300nm</td><td></td></tr><tr><td>Light Curing System</td><td>Dymax&amp;nbsp;</td><td>Model 2000 Flood</td><td></td></tr><tr><td>E-beam deposition machine</td><td>Denton Explorer</td><td></td><td></td></tr><tr><td>UV-visible spectrometer&amp;nbsp;</td><td>Ocean optic HR2000+ (Dunedin, FL, USA)</td><td></td><td></td></tr><tr><td>Standard refractive index liquids&amp;nbsp;</td><td>Cargill Inc (Cedar Grove, USA)</td><td>18032</td><td></td></tr><tr><td>Plotting software</td><td>Origin</td><td>Origin Pro 9</td><td></td></tr><tr><td>10-carboxy-1-decanethiol&amp;nbsp;</td><td>Dojindo Laboratories (Japan)</td><td>C385-10</td><td></td></tr><tr><td>1-octanethiol&amp;nbsp;</td><td>Sigma-Aldrich, MO, USA</td><td>471386</td><td></td></tr><tr><td>Sulfo-N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide&amp;nbsp;</td><td>BioRad (CA, USA)</td><td>1762410</td><td></td></tr><tr><td>Anti-troponin antibody 560</td><td>Hytest (Finland)</td><td>4T21</td><td></td></tr><tr><td>Ethanolamine-HCl solution</td><td>BioRad (CA, USA)</td><td>1762450</td><td></td></tr><tr><td>Surface Plasmon Resonance setup</td><td>BioRad XPR36 (Haifa, Israel)</td><td></td><td></td></tr><tr><td>Multiplexed SPR chip</td><td>BioRad</td><td>GLC</td><td></td></tr><tr><td>Human cTnI standard</td><td>Phoenix Pharmaceuticals</td><td>EK -311-05</td><td></td></tr><tr><td>Glycine-HCl</td><td>BioRad (CA, USA)</td><td>1762221</td><td></td></tr></tbody></table>

using extraordinary optical transmission to quantify cardiac biomarkers in human serum

对于一个传感平台具有临床相关性的点 (POC) 的设置, 化验灵敏度, 重现性, 并能够可靠地监测分析的背景下, 人血清是至关重要的。
Nanoimprinting 光刻 (零) 被用来制造, 在低成本, 传感面积高达1.5 毫米 x 1.5 毫米。传感面是由高保真阵列的 nanoholes, 每一个面积约 140 nm2。零的巨大再现性使得在12个单独制造的表面上使用一个单芯片的单测量策略成为可能, 最小的芯片变化。这些 nanoimprinted 的局部表面等离子体共振 (LSPR) 芯片被广泛测试的能力, 以可靠地测量 bioanalyte 在浓度变化, 从2.5 到 75 ng/毫升在一个复杂的背景 biofluid-in 这种情况下, 人血清。零的高保真度使产生大的传感区, 这反过来又消除了对显微镜的需要, 因为这种生物传感器可以很容易地与一个普通的实验室光源接口。这些生物传感器可以检测高灵敏度的血清心肌肌钙蛋白, 在 0.55 ng/毫升的检测限度 (LOD), 这是临床相关的。它们也显示出低的芯片方差 (由于制造过程的高质量)。结果是相称与广泛使用的酶联免疫吸附试验 (ELISA) 的基础上, 但该技术保留了 LSPR-based 传感平台的优势 (即,可的小型化和多路复用, 使其更适合于 POC 应用程序)。

用于测量的光学设置显示在图 1A中。在图 1B中给出了实际纳米数组的图像。为了了解物理驱动传感过程, 利用 COMSOL 仿真软件模拟了浆场在水环境中的分布。模拟结果与实测值有关。先前发布的研究包含了所做假设的详细信息和模拟1113中使用的参数。用于模拟纳米阵列的浆场的物理尺...

Watch this Scientific Journal Video about Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum at JoVE.com

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

这项工作描述了 nanoimprinting 光刻方法, 制造高质量的传感阵列的工作原理, 非凡的光传输。该生物传感器成本低廉, 健壮, 易于使用, 并能检测血清心肌肌钙蛋白 i 在临床相关浓度 (99th百分比截止∼10-400 pg/毫升, 取决于化验)。

用超常光透射法定量测定人血清中的心脏标志物

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

using-extraordinary-optical-transmission-to-quantify-cardiac

Research

JoVE Journal

Bioengineering

6.2K 次观看.  National University of Singapore. 这项工作描述了 nanoimprinting 光刻方法, 制造高质量的传感阵列的工作原理, 非凡的光传输。该生物传感器成本低廉, 健壮, 易于使用, 并能检测血清心肌肌钙蛋白 i 在临床相关浓度 (99th百分比截止∼10-400 pg/毫升, 取决于化验)。

视频: Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

生成和动脉病变的小鼠的三维定量使用光学投影层析成像

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

科研

医学

Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spots meet this need, and can be collected in the field with high response rates. These elements are particularly important in longitudinal study designs including interventions where attrition is critical to avoid, and high response rates improve the interpretation of results. Dried Blood Spot sample collection is simple, quick, relatively painless, less invasive then venipuncture, and requires minimal field storage requirements (i.e.&#160;samples do not need to be immediately frozen and can be stored for a long period of time in a stable freezer environment before assay). The samples can be analyzed for a variety of different analytes, including cholesterol, C-reactive protein, glycosylated hemoglobin, numerous cytokines, and other analytes, as well as provide genetic material. DBS collection is depicted as employed in several recent studies.

Biomarkers are directly-measured biological indicators of disease or health. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spot (DBS) collection meets this need, can be collected in the field with high response rates&#160;and analyzed for a variety of biomarkers.

干生生物标志物的血斑收藏最大限度地参与人口研究

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

心脏内侧燃光导管,用于监测细胞代谢,使用渗透哺乳动物心脏的透射吸收光谱

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

生物工程

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

3D全心脏心肌组织分析

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

同时布莱特菲尔德， 荧光， 和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

数字 PCR 在急性心肌梗死和心血管疾病中量化循环 MicroRNAs 的临床观察

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

电化学发光免疫法测定血清心肌肌球蛋白结合蛋白-C敏感而特异的定量方法

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

生物学

Coronary Progenitor Cells and Soluble Biomarkers in Cardiovascular Prognosis after Coronary Angioplasty

Major adverse cardiovascular events (MACEs) negatively impact the cardiovascular prognosis of patients undergoing coronary angioplasty due to coronary ischemic injury. The extent of coronary damage and the mechanisms of vascular repair are factors influencing the future development of MACEs. Intrinsic vascular features like the plaque characteristics and coronary artery complexity have demonstrated prognostic information for MACEs. However, the use of intracoronary circulating biomarkers has been postulated as a convenient method for the early identification and prognosis of MACEs, as they more closely reflect dynamic mechanisms involving coronary damage and repair. Determination of coronary circulating biomarkers during angioplasty, such as the number of subpopulations of mononuclear progenitor cells (MPCs) as well as the concentration of soluble molecules reflecting inflammation, cell adhesion, and repair, allows for assessment of future developments and the prognosis of MACEs 6 months post coronary angioplasty. This method is highlighted by its translational nature and better performance than peripheral blood circulating biomarkers regarding prediction of MACEs and its effect on the cardiovascular prognosis, which may be applied for risk stratification of patients with coronary artery disease undergoing angioplasty.

Development of major adverse cardiovascular events, which impact cardiovascular prognosis after coronary angioplasty, are influenced by the extent of coronary damage and vascular repair. The use of novel coronary cellular and soluble biomarkers, reactive to vascular damage and repair, are useful to predict the development of MACEs and prognosis.

冠状血管化后心血管预后冠状动脉祖细胞和可溶性生物标志物

Major adverse cardiovascular events (MACEs) negatively impact the cardiovascular prognosis of patients undergoing coronary angioplasty due to coronary ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Mesoscopic Optical Imaging of Whole Mouse Heart

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical dysfunctions and, eventually, lead to arrhythmia. Current predictive models of these functional alterations are based on non-integrated and low-resolution structural information. Placing this framework on a different order of magnitude is challenging due to the inefficacy of standard imaging methods in performing high-resolution imaging in massive tissue. In this work, we describe a methodological framework that allows imaging of whole mouse hearts with micrometric resolution. The achievement of this goal has required a technological effort where advances in tissue transformation and imaging methods have been combined. First, we describe an optimized CLARITY protocol capable of transforming an intact heart into a nanoporous, hydrogel-hybridized, lipid-free form that allows high transparency and deep staining. Then, a fluorescence light-sheet microscope able to rapidly acquire images of a mesoscopic field of view (mm-scale) with the micron-scale resolution is described. Following the mesoSPIM project, the conceived microscope allows the reconstruction of the whole mouse heart with micrometric resolution in a single tomographic scan. We believe that this methodological framework will allow clarifying the involvement of the cytoarchitecture disarray in the electrical dysfunctions and pave the way for a comprehensive model that considers both the functional and structural data, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.

We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope.

全小鼠心脏的介观光学成像

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition ...

用超常光透射法定量测定人血清中的心脏标志物

摘要

探索更多视频

此视频中的章节

生成和动脉病变的小鼠的三维定量使用光学投影层析成像

干生生物标志物的血斑收藏最大限度地参与人口研究

心脏内侧燃光导管,用于监测细胞代谢,使用渗透哺乳动物心脏的透射吸收光谱

3D全心脏心肌组织分析

同时布莱特菲尔德，荧光，和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

数字 PCR 在急性心肌梗死和心血管疾病中量化循环 MicroRNAs 的临床观察

电化学发光免疫法测定血清心肌肌球蛋白结合蛋白-C敏感而特异的定量方法

冠状血管化后心血管预后冠状动脉祖细胞和可溶性生物标志物

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

全小鼠心脏的介观光学成像

用超常光透射法定量测定人血清中的心脏标志物

摘要

探索更多视频

此视频中的章节

生成和动脉病变的小鼠的三维定量使用光学投影层析成像

干生生物标志物的血斑收藏最大限度地参与人口研究

心脏内侧燃光导管,用于监测细胞代谢,使用渗透哺乳动物心脏的透射吸收光谱

3D全心脏心肌组织分析

同时布莱特菲尔德， 荧光， 和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

数字 PCR 在急性心肌梗死和心血管疾病中量化循环 MicroRNAs 的临床观察

电化学发光免疫法测定血清心肌肌球蛋白结合蛋白-C敏感而特异的定量方法

冠状血管化后心血管预后冠状动脉祖细胞和可溶性生物标志物

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

全小鼠心脏的介观光学成像

同时布莱特菲尔德，荧光，和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo