作者承认戈登和贝蒂摩尔基金会授予加州理工学院4037和4038资助这项工作。

1. 细菌的生长和计数 注意: 这适用于在适当培养基中生长的几乎任何细菌菌株 36 。在我们的例子中, 我们使用三菌株: 沙雷菌 作为一个常见的、容易识别的实验室菌株;和一个较小的, 高度能动的环境应变, 希瓦 oneidensis MR-1。为了比较检测的运动与 non-motile 细胞, non-motile 希瓦 突变体, 和 #916; FlgM, 也用于比较 37 。所有菌株都生长在性肉汤 (LB). 
<ol> <li> 准备无菌 LB 培养基 (每公升蒸馏水:10 克 bacto 胰, 5 克 bacto 酵母, 10 克氯化钠; 过滤器或高压釜)。准备标准的100毫米直径 LB 琼脂板 (相同的配方, 中等加 15 g 琼脂每升; 蒸压釜 (121, #176; C, 20 分钟), 以杀菌和倒入时, 冷却足够处理)。在冰箱中存放介质和盘子. </li>
	<li> 试验前的前一天 5-6 毫升的 #34; 硕士和 #34; 培养基从新鲜细菌菌落, 使用正确的无菌和生物安全技术。在30和 #176 的振动器上孵育 (120 rpm); C 为 ~ 12 h. 潜伏期将取决于应变和生长速率。在我们的实验中, 希瓦 菌株孵育12小时;但是, 沙雷 在8小时后收获, 以确保文化将显示 mid-logarithmic 的增长. </li>
	<li> 实验的当天, 采取分光光度读数 (OD 600 ) 的细菌的主文化, 这是预计的范围0.6 至0.7。它应该在对数增长阶段 (如先前所确定)。如果不是, 亚文化100和 #181; L 到5毫升培养基, 并允许细胞生长到 mid-log 阶段. </li>
	<li> 采用主区域性样本, 并使用 Petroff-豪塞尔计数室直接计数单元格。这将枚举活的和死的细胞. 
	<ol> <li> 提取10和 #181; 未稀释的文化的样品与微和转移入房间. </li>
		<li> 图像下的 high-dry 物镜显微镜 (40X 和 #160; 或 63X, NA 0.7-0.8) 使用相衬. </li>
		<li> 统计至少20平方和平均值的细菌数. 
		注: 只计算那些完全在一个正方形内的细菌, 只有那些越过顶部和左边的边界 (如果你愿意的话)。如果正方形由几行分隔, 请选择一个作为边界. </li>
		<li> 计算浓度为20平方 x 稀释因子的平均值。注意, 直接计数对浓度和 #60 不起作用; 10 7 /mL. </li>
	</ol> </li> <li> 用于对菌落形成单元 (CFU) 计数的区域性进行串行稀释。这将仅枚举活动单元格. 
	<ol> <li> 将每个选定的细菌样本与无菌0.9% 盐水溶液进行连续稀释。转移20和 #181; l 的细菌溶液和稀释它与180和 #181; l 盐水。重复直到最低浓度为 ~ 10 3 单元格/mL. </li>
		<li> 采取100和 #181; L 从至少两个稀释建议是 10 3 和 10 4 /mL 和 #160; #8212; 在适当的固体介质板上板。与无菌摊铺机。执行每次稀释的至少3复制. </li>
		<li> 在适当的温度下孵育细菌过夜或直到菌落生长. </li>
		<li> 计数殖民地和计算殖民地形成单位 (CFU) 根据 (# 的殖民地 x 稀释因子)/体积镀 = CFU/毫升。对复制的平均 CFU. </li>
	</ol> </li> </ol> 2。DHM 的高稀释样品的制备 <ol> <li> 在 LB 介质板上为 DHM 和后 DHM 计数的菌落形成单元稀释的主区域性的序列 (见 1.4.1-1.4.4)。执行这双盲, 使录音的人不知道每个测量样本的浓度. 
	<ol> <li> 将细菌稀释到 10-15 毫升的最小培养基中, 这将鼓励运动 (视情况而定), 但抑制细胞分裂, 以便在实验过程中细胞的浓度不会有明显变化。这可能是0.9% 盐水或更具体的运动介质。例如, 如果使用 大肠杆菌 , 则运动介质必须包含 EDTA 38 。对于这些例子, 我们使用0.9% 盐水. </li>
	</ol> </li> </ol> 3。录制 DHM 视频 <ol> <li> 使用无菌注射器, 抽取约10毫升的稀释利息. </li>
	<li> 使用无菌管件和油管将注射器连接到 DHM 样品室. 
	注: custom-made 微流控室是为了使样品的一致流动通过仪器的光学通路而建造的。有关详细信息, 请参见 结果 部分。在实验之前, 样品室的所有组分应该由灭菌消毒. </li>
	<li> 从注射器通过样品室源源不断地使用注射器泵. 
	注: 适当的流速取决于射流通道的尺寸、所需的吞吐量以及数据获取的限制. </li>
	<li> 当样品流经样品室时, 以适当的帧速率连续获取全息图。将创建一个时间文件, 记录每个图像捕获的时间, 以便以后在数据分析期间使用 (协议4节)。获取所有全息图在环境温度 (23 和 #176; C)。通过执行由照相机制造商提供的预先 c++ 可执行文件来记录全息图. 
	注: 适当的帧速率根据选择的流速而变化。对于这个实验, 建议使用一个帧速率, 这样细菌就会被成像和 #62; 2 倍, 因为它横跨仪器和 #39; s 的视野. </li>
	<li> 允许足够的时间让整个10毫升的样品流经 DHM. </li>
	<li> 为了确保在实验过程中细菌不会生长或死亡, 用100和 #181 对培养基中的富集进行接种; DHM 图像捕获。用无菌的吊具铺上, 并在适当温度下孵育24小时;计数殖民地存在. </li>
</ol> 4。计算细胞密度和检测限值 <ol> <li> 已获得的全息图视频, 分析数据中是否存在细菌. 
	<ol> <li> 计算全息图的时间序列的中值像素值, 然后从每个各自的像素中减去此中间数值, 以消除全息图像中的固定工件。这可以通过执行以下步骤在 ImageJ 中完成: <ol> <li> 转换为32位 (Image-Type-32 位) </li> <li> 计算中值 (图像堆栈-Zproject-中值) </li> <li> 从堆栈的其余部分减去中间值 (进程-ImageCalculator 减去) </li> </ol> </li> <li> 计算可见的通风环和焦点单元格的数目手动.在 ImageJ 中, 这是通过用点工具指向对象来完成的. </li>
		<li> 每系列全息图将附有一个时间文件 (在全息图像采集时记录)。使用时间文件计算在捕获图像时抽取的样本总量. </li>
	</ol> </li> <li> 通过将检测到的总体积的单元格总数除以图像的总数来计算单元格的密度。平均 5-10 帧用于精确统计. </li>
</ol> 5。图像重构到振幅和相位 <ol> <li> 选择每帧 10-200 单元格的代表性视频。装载原始 (不是中位减去) 全息图入重建软件 (例子: ImageJ 数值传播 39 ;洗发水 [GitHub]). 
	<ol> <li> 使用 ImageJ, 转到 OD-数值传播-数值衍射. </li>
		<li> 在提示时, 绘制一个傅里叶掩码以选择真实或虚拟图像, 并消除任何正弦噪声特征. </li>
		<li> 通过进入重建距离, 在适当的 z 步重建振幅和相位. </li>
	</ol> </li> <li> 中值减去振幅数据以减少噪音, 如4.1.1 中所示. </li>
	<li> 将数据绘制为3D 多维数据集或投影。转到插件和 #8212; 卷查看器. </li>
</ol>

<ol>
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K., Ramphal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+functional+characterization+of+flgM,+a+gene+encoding+the+anti-sigma+28+factor+in+Pseudomonas+aeruginosa.">Identification and functional characterization of flgM, a gene encoding the anti-sigma 28 factor in Pseudomonas aeruginosa.</a> J Bacteriol. 184, 1514-1521 (2002).</li><li>Adler, J., Templeton, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+environmental+conditions+on+the+motility+of+Escherichia+coli.">The effect of environmental conditions on the motility of Escherichia coli.</a> Journal Gen Microbiol. 46, 175-184 (1967).</li><li>Piedrahita-Quintero, P., Castaneda, R., Garcia-Sucerquia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numerical+wave+propagation+in+Image+J.">Numerical wave propagation in Image J.</a> Appl Opt. 54, 6410-6415 (2015).</li><li>Schnars, U., J&#252;ptner, W. 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作者没有什么可透露的。

全息图的数值重建: 用于全息图的数值重建, 采用了角谱法 (ASM)。这涉及到全息图的卷积与格林函数的 DHM。利用傅里叶卷积定理可以计算出在特定焦平面上的复杂波前, 如下所示:
<img alt="Equation 2" src="/files/ftp_upload/56343/56343eq2.jpg">(1)
其中, 是傅里叶变换运算符, 是全息图矩阵, 是 DHM 的绿色函数, 定义为: <img alt="Equation 3" src="/files/ftp_upload/56343/56343eq3.jpg"> <img alt="Equation 4" src="/files/ftp_upload/56343/56343eq4.jpg"> <img alt="Equation 5" src="/files/ftp_upload/56343/56343eq5.jpg">
<img alt="Equation 6" src="/files/ftp_upload/56343/56343eq6.jpg">(2)
其中, 是要重建的所需焦平面的距离 (沿光轴), 是光照波长、和分别是 x 和 y 方向的像素数, 以及和<img alt="Equation 7" src="/files/ftp_upload/56343/56343eq7.jpg"> <img alt="Equation 8" src="/files/ftp_upload/56343/56343eq8.jpg"> <img alt="Equation 9" src="/files/ftp_upload/56343/56343eq9.jpg"> <img alt="Equation 10" src="/files/ftp_upload/56343/56343eq10.jpg"> <img alt="Equation 11" src="/files/ftp_upload/56343/56343eq11.jpg"> <img alt="Equation 12" src="/files/ftp_upload/56343/56343eq12.jpg">分别是 x 和 y 方向的像素大小 (在探测器平面上)40。
从复波前, 强度可以通过的幅度平方来计算, 并且可以通过反正的虚和实部分之间的商的不确定来获得相位. <img alt="Equation 13" src="/files/ftp_upload/56343/56343eq13.jpg"> </img> <img alt="Equation 13" src="/files/ftp_upload/56343/56343eq13.jpg"> </img>
修改和故障排除 涉及稀疏细菌浓度计数的实验非常容易受到污染、误报和假阴性的影响。因此, 细菌的生长和计数, 以及 DHM 的高稀释样品的制备是本实验的关键步骤。在这个实验中, 必须谨慎地控制细菌生长和列举的环境, 以避免污染。适当的杀菌技术, 包括灭菌, 有助于防止污染, 以及假阳性。为了进一步防止误报, 细菌被选作这个实验, 表现出相对独特的特征 (例如, 菌在培养时具有非常鲜明的红色)。为了防止假阴性, 选择这个实验的细菌选择这样, 他们将能够生长在丰富的文化板块, 以及有特征的形状和/或运动模式, 通过 DHM 识别。通过电镀样品和通过平板计数对细菌进行定量, 定期检测污染和/或意外细胞生长是很重要的。
在进行此实验时, 预计每1毫升样本的数据大小大约为 2.4 GB。当然, 这个数字取决于所使用的相机和文件格式。报告的数据大小是使用 4 Mpx 检测器和标准 TIFF 文件格式。数据的后处理需要每1毫升样品大约6分钟的计算。在使用 3.5 GHz 和 32 GB RAM 的英特尔 i7 8 核处理器时, 观察到了处理时间。这些计算可以并行到 GPU, 从而大大缩短了处理时间, 但在这里没有完成。
技术的局限 虽然使用 DHM 检测极低浓度的细菌是可能的, 这种方法是非特异性的。单独的形态学不是鉴定细菌菌株的决定性手段。由于非目标细胞和聚合体的存在, 诊断应用程序将面临特定的挑战。然而, 双光束仪器的优势在于, 它能够在相对混浊的样品中成像细菌。在临床样品中检测细菌的能力, 以及所需样品的预处理程度仍有待确定。
协议中的关键步骤 
步骤 2:获取精确的稀释是定量枚举的关键。任何稀释做的都应该用板数来备份。步骤 3: 必须仔细选择流速和相应的帧速率, 以确保所有的电池都能被看到。在进行检测试验的极限之前, 应仔细调整泵的参数。步骤 4: 对于非常低的细菌浓度, 捕获足够的数据以确信检测是至关重要的。可能有必要重建一些数据, 以确定细胞, 而不是碎片, 正在看到。在极端情况下, 可能需要超过10毫升的解决方案。非能动菌株比运动菌株更容易发生歧义。步骤 5: 如果数据有任何歧义, 重建可以帮助证明全息图代表的是细胞而不是碎片。高分辨率的重建将显示经典的细胞形态, 和运动可以清楚地看到, 在轨道上的能动菌株。

在非常稀的样品中准确的细菌计数的测定在许多应用中是至关重要的: 几个例子是水和食品质量分析1,2,3;检测血液、脑脊液或痰液中的病原体4,5;生产医药产品, 包括无菌水6;和环境群落分析在营养环境中如开阔的海洋和沉积物7,8,9。在木星和土星的冰冷卫星上, 特别是木卫二10、11和土卫二12、13、 14, 已知有地下液态海洋。由于维京人在1978年试图在另一个星球上寻找现存的生命, 因此没有任何任务, 因此在空间飞行任务的过程中, 细菌识别和计数的技术和仪器的开发是有限的15。
传统的平板计数方法只发现培养细胞, 它能在环境菌株中代表少数物种, 有时和 #60; 1%16。盘子需要几天或几周的孵化来获得最大的成功, 这取决于应变。荧光显微镜在很大程度上取代了板数作为快速准确的微生物计数的金标准。核酸标记荧光染料, 如 4, 6-diamidino-2-吲盐酸盐 (DAPI), SYBR 绿色, 或吖啶橙绑定到核酸是典型的染料使用17,18,19, 虽然许多研究使用了克标志的荧光指标20,21,22,23,24。使用这些没有预步骤的方法, 每个 mL 会导致检测 (检测) 105单元格的限制。改进的 LoD 是可能的使用过滤。液体样品是真空过滤到膜上, 通常是聚碳酸酯和理想的黑色, 以减少背景。低背景染料, 如上面提到的 DNA 污渍, 可以直接应用于过滤器25。为了准确地按眼睛计数, 每个过滤器需要 105单元格, 这意味着对于每个 mL 的样本比 105单元更稀, 必须收集和筛选大量的样本卷。为了系统地探索过滤器的所有区域, 并因此减少了计数所需的单元数, 将检测范围推到每 mL26102单元格, 从而开发了激光扫描设备。然而, 这些在大多数实验室是不可用的, 并且需要先进的硬件和软件, 允许专家确认观察到的粒子是细菌而不是碎片。
作为参考, 成人脓毒症通常开始显示的症状和 #60; 100 细胞/毫升的血液, 和婴儿在和 #60; 10 细胞/毫升。从一个成年人的血液提取需要10毫升, 从一个婴儿, 1 毫升。pcr 方法被存在人和致病植物群脱氧核糖核酸和由 PCR 抑制的组分抑制在血液27,28。尽管有各种新兴技术, 文化仍然是诊断血流感染的金标准, 特别是在更多的农村地区或发展中国家。为了探测其他行星上的生命, 热力学计算可以估计生命的能量预算, 从而预测可能的生物量。1-100 细胞/毫升预计将热力合理的欧罗巴29。从这些数字可以很容易地看出, 大量的水溶液中的少量细胞的检测是一个重要的未决问题。
在本文中, 我们演示了检测沙雷菌和希瓦 oneidensis (野生类型和 non-motile 变种) 的浓度为 105, 104, 103, 和100细胞/毫升使用离轴数字全息显微镜 (DHM)。DHM 优于传统光学显微镜的关键优势是在高-的同时成像厚样本体积, 该实现, 样品室是0.8 毫米厚。这些样品室由烷 (soft-lithography) 由精密机械加工的铝模制成, 公差为±50µm。这代表大约100倍的改善在领域的深度在大功率光学显微镜。DHM 还提供了定量的相位信息, 允许测量光路长度 (折射率和厚度的乘积)。DHM 和类似的技术已用于监测细菌和酵母细胞周期和细菌干质量的计算30,31,32;散射差异甚至可以用来区分细菌菌株33。
我们使用的仪器是 custom-built 专门用于微生物, 如以前发布的34,35, 它的设计和构造是演示和讨论的。通过注射器泵连续向0.25 µL 容积样品室提供水溶液;流率是由相机帧率确定的, 以确保整个样本量的成像。统计计算预测的样本量必须被成像, 以便检测到一个相当数量的细胞在一定浓度。
对于细胞检测应用, 不需要将全息图重建为振幅和相位图像;对原始全息图进行了分析。这节省了大量的计算资源和磁盘空间: 重建时, 500 Mb 的全息图视频将是 1-2 Tb。然而, 我们确实讨论了通过样品的深度重建, 以确认全息图代表了所需的物种。DHM 的一个重要特点是它能够监测图像的强度和相位。在强度上几乎是透明的生物体 (如大多数生物细胞) 在相时清晰地显现出来。由于它是一种无标签的技术, 没有使用染料。这是一个dvantage 为可能的太空飞行应用, 因为染料可能不生存使命的条件, 并且-更重要地-不能假设与外星有机体一起使用, 可能不用脱氧核糖核酸或 RNA 为编码。这也是在诸如北极和南极等极端环境中工作的一个优势, 在那里, 染料可能难以带到偏远的地方, 并可能在储存时降解。图像的相位和振幅重建是使用一个开源软件包, 我们已经提供了 GitHub (洗发水) 或使用 ImageJ。

<table>
	<tbody>
		<tr>
			<td>Bacto Yeast</td>
			<td>BD Biosciences</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Tryptone</td>
			<td>BD Biosciences</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7710</td>
			<td>Many options for purchase</td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD Biosciences</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Petri dishes</td>
			<td>VWR</td>
			<td>10053-704</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL culture tubes</td>
			<td>Falcon (Corning Life Sciences)</td>
			<td>352002</td>
			<td>Loose-capped</td>
		</tr>
		<tr>
			<td>Petroff-Hauser chamber</td>
			<td>Electron Microscopy Sciences</td>
			<td>3920</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL syringes</td>
			<td>BD Biosciences</td>
			<td>309604</td>
			<td>Luer-Lok tip not necessary</td>
		</tr>
		<tr>
			<td>Male Luer to 1/16&#8221; barbed fitting</td>
			<td>McMaster-Carr</td>
			<td>51525K291</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclavable 1/16&#8221; ID PVC tubing for flow</td>
			<td>Nalgene</td>
			<td>8000-0004</td>
			<td>Sold by length, purchase accordingly</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>PHD 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample Chamber</td>
			<td>Custom</td>
			<td>n/a</td>
			<td>See Materials Section</td>
		</tr>
		<tr>
			<td>Holographic Microscope</td>
			<td>Custom</td>
			<td>n/a</td>
			<td>See Wallace et al.</td>
		</tr>
		<tr>
			<td>Open-source software</td>
			<td>Custom</td>
			<td>https://github.com/bmorris3/shampoo</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying microorganisms at low concentrations using digital holographic microscopy (dhm)

准确地检测和计数稀疏细菌样品在食品、饮料和制药加工业、医疗诊断和机器人任务到太阳系其他行星和卫星的生命检测中有许多应用。目前, 稀疏细菌样品通过培养电镀或荧光显微镜进行计数。培养皿需要很长的孵育时间 (天到周), 荧光显微镜需要广泛的染色和样品的浓度。在这里, 我们演示如何使用离轴数字全息显微镜 (DHM) 在非常稀的文化中列举细菌 (100-104细胞/毫升)。首先, 讨论了定制 DHM 的构建, 并详细说明了如何构建低成本的仪器。讨论了全息术的原理, 根据仪器的光学性能特点和细菌溶液浓度 (表 2), 利用统计模型估计视频检测细胞的时间长度。.视频检测的细胞在 105, 104, 103, 和100细胞/毫升是实时演示使用 un-reconstructed 全息图。利用开源软件包对振幅和相位图像进行重建。

结果应表明, DHM 能在极低的水平上检测活细菌和死菌。计数的细菌数量应与使用 Petroff-豪瑟计数室和钢板计数所得的结果一致。标准统计方法提供了各种细菌浓度不同检测方法的准确性信息。
图 1显示了 Petroff-豪塞尔计数室, 以及用于直接枚举单元格的计数室的显微结构。图 2A显示了一个典型的富集文化板块, 允许有足够的时间为S. 菌生长菌落 (室温16小时)。图 2B显示了所使用的所有菌株的生长曲线。当精确分光光度计的测量值不同时, OD600与菌落计数之间的关系应在分光光度计的可靠范围内线性化;我们测量〜 OD600 = 0.1 为 ~ 108/毫升。直接 Petroff-豪塞尔计数和菌落计数应同意在10% 内, 直到细胞开始死亡, 由营业额在增长曲线看到。
图 3显示了自定义采样室的设计。本实验采用了自定义微流控腔, 使样品在全息显微镜的视场中连续流动。在样品室的建造中采用了标准的微流控技术和材料。在微流体装置中插入带有雄性口的钝尖不锈钢针, 为样品室提供入口和出口端口。微流体通道几何是由烷软光刻法建立的, 采用了机械加工的铝制主模, 通过铝框架在两个光学质量玻璃窗之间进行压缩。该公司建立了流动通道的几何形状, 并确保皮下注射针头, 提供流体输送到和从流动通道。这些样品室仅用于建立流道几何, 从而在仪器的光学路径中不含任何的硅橡胶。请注意, 由于离轴全息显微镜的光学性质, 两个微通道在样品室中相互平行成型。这是为了匹配干涉仪的两臂之间的光路长度 (称为 "标本" 和 "参考" 武器)。参考通道应只包含无菌介质, 不应受流量的限制。
图 4显示了数字全息显微镜在实验室实施过程中的示意图和图像。它由照相机提供的软件操作。图 5显示了用于细菌计数的具有代表性的 DHM 数据及其与每个视场中的细胞预测数的对应关系。从原始全息图, 没有重建, 可以看到和计数的细胞中位减法和手动跟踪。这大大降低了计算负担与重建所有 z 平面, 因为原始全息图显示的细胞在整个会议厅的深度。指出了不同浓度的文化形态及其与板数和 Petroff 数的对应关系。总的细胞计数是通过平均每个样本体积中所看到的细胞总数来确定的。检测的限制是由映像的样本卷总数决定的。在这个实验中, 我们对这个值设定了一个最大的限制, 最大总计为10毫升的样品成像0.25 µL 增量 (总共4万图像每个样本)。如果在20帧的每个帧中清晰地观察到单元格, 则在达到最大总音量之前停止录制。在前一篇论文中, 我们报告了一个公式, 用于估计检测细菌的数量, 在50% 的浓度从 1-105细胞/毫升 (表 1)。根据这一估计, 对所有4万帧的检查应允许检测 1/毫升的细胞, 虽然这种计算是密集的。
图 6显示了重建软件 (斐济)39的使用情况。一个单一的全息图打开, 软件 "OD-数值传播-数值衍射" 运行。在对话框中给出了成像参数;振幅、强度和相位都可以重建。可以选择单个 z 平面, 也可以使用批重建重建整个深度。
图 7显示了选定示例中不同深度的振幅和相位图像的重建。用于生成重建的傅里叶掩模, 连同中值减去强度图像、相位图像、通过相栈的最大投影以及强度叠加的体积投影。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56343/56343fig1.jpg"> 
图 1: 细菌细胞的直接计数.在细胞/毫升的细菌浓度计算计数的细菌总数计数室和结垢的数字核算的事实, Petroff-豪瑟室只包含 20 nL 样本。(A) Petroff-豪塞尔计数室。(B) 在10X 相位对比度下计数室显微结构的图像, 显示不同大小的盒, 对枚举不同大小的单元格有用。(C) 在计数室最小的框内出现S. 菌;40X 相衬。(D) 用于计数的方法, 它排除掉在上、右边缘 (N) 上的单元格, 而不是下边缘和左边的边 (Y)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56343/56343fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56343/56343fig2.jpg"> 
图 2: 按菌落生长和光学密度计算细胞数.(A) S. 菌在室温下孵育16小时。在正确的稀释, 文化板材应该有 20-200 殖民地准确计数和统计。显示的板块有点过于拥挤, 无法可靠计数。(B) 用于3菌株的生长曲线。光学密度0.1 对应于 ~ 108单元/mL, 但精确值因仪器而异。直接 Petroff-豪瑟计数同意板块计数在10% 以内的线性增长范围。误差线表示5独立样本的标准偏差。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56343/56343fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56343/56343fig3.jpg"> 
图 3: 样品室设计.(A) 微流体原理图 (样本和参考微标记)。(B) 完全组装的样品箱。(C) 分解 CAD 视图, 显示样品室的组成元素。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56343/56343fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56343/56343fig4.jpg"> 
图 4: 数字全息显微镜的示意图和图像.(A) 示意图显示四主要元素: 来源, 样品 (标本路径被标记为规范. 和参考路径被标记 Ref.), 显微镜, 和传感器。(B) 硬件的实体模型。纤维馈源组件位于底部, 而成像相机位于顶部。显微镜光学-由两个非球面透镜和继电器透镜组成-包含在300毫米长的透镜管之内。三轴阶段之间的来源显微镜光学提供简单的手工操作的标本正在研究中。(C) 实验室中仪器的照片。图像下半部分的刻度线表示1英寸。示意图是重新从华莱士et al.34.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56343/56343fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56343/56343fig5.jpg"> 
图 5: 增长曲线和具有代表性的 DHM 数据.(A) 根据365µm x 365 µm x 1 mm 的样本量预测每个视场的单元格。DHM 枚举的最佳范围显示在矩形中, 实际的示例表示为 (B) 和 (C)。(B) DHM 8 x 10 5单元/ml 的全息图, 由 Petroff-豪瑟和 7.7 x 105单元/ml 测量, 按盘数测量;这个图像显示110细胞, 非常适合预测。(C) DHM 2.0 0.1 x 10 5单元/ml 的全息图, 由 Petroff-豪瑟和 2.0 0.3 x 105单元/ml 测量, 按盘数测量;此图像显示27单元格。缩放栏 = 35 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56343/56343fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56343/56343fig6.jpg"> 
图 6: 执行重建.(a) 在斐济打开全息图。(B) 运行数值传播并输入波长和图像大小。选择接近0的 z 距离开始。(C) 在提示选择真实图像或虚拟映像时绘制一个傅立叶掩码。(D) 浴处理允许在整个卷中进行重建。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56343/56343fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56343/56343fig7.jpg"> 
图 7: 重建的外观.沙雷区域性的图像在 ~ 106单元格/mL。(A) 原始全息图。(B) 傅立叶变换。圆圈内的区域是要使用的区域;其余的都是蒙面的(C) 在单 z 平面上进行振幅重构。(D) 在单 z 平面上进行相位重建;刻度线显示相移的度数。(E) 通过相位堆栈中的所有 z 平面进行最大强度投影。(F) 全振幅堆栈 (365 x 365 x 800 µm) 的音量视图。缩放栏 = 35 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56343/56343fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>浓度 [每毫升细胞]</td>
			<td>需要的帧</td>
		</tr>
		<tr>
			<td>1.00E+00</td>
			<td>6686</td>
		</tr>
		<tr>
			<td>1.00E+01</td>
			<td>669</td>
		</tr>
		<tr>
			<td>1.00E+02</td>
			<td>67</td>
		</tr>
		<tr>
			<td>1.00E+03</td>
			<td>7</td>
		</tr>
		<tr>
			<td>1.00E+04</td>
			<td>1</td>
		</tr>
		<tr>
			<td>1.00E+05</td>
			<td>1</td>
		</tr>
	</tbody>
</table>
表 1:DHM 检测细菌所需样本量的最小数量 (检测置信度为 50%).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>属性</td>
			<td>值</td>
			<td>单位</td>
		</tr>
		<tr>
			<td>工作波长</td>
			<td>405</td>
			<td>nm</td>
		</tr>
		<tr>
			<td>物镜数值孔径</td>
			<td>0。3</td>
			<td>-</td>
		</tr>
		<tr>
			<td>系统放大倍数</td>
			<td>20</td>
			<td>-</td>
		</tr>
		<tr>
			<td>横向分辨率</td>
			<td>0。7</td>
			<td>μ</td>
		</tr>
		<tr>
			<td>样品成像量</td>
			<td>360 x 360 x 800</td>
			<td>μ m x μ m</td>
		</tr>
		<tr>
			<td>仪器长度</td>
			<td>400</td>
			<td>毫米</td>
		</tr>
	</tbody>
</table>
表 2:数字全息显微镜的光学和性能规范

Watch this Scientific Journal Video about Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM at JoVE.com

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

数字全息显微镜 (DHM) 是一种体积技术, 允许成像样品50-100X 厚比明显微镜在可比的分辨率, 与重点进行后处理。DHM 用于识别、计数和跟踪非常低密度的微生物, 并与光学密度测量、平板计数和直接计数相比较。

用数字全息显微镜 (DHM) 定量化低浓度微生物

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in ...

quantifying-microorganisms-at-low-concentrations-using-digital

Nanyang Technological University

Research

JoVE Journal

Bioengineering

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

10.2K 次观看.  California Institute of Technology. 数字全息显微镜 (DHM) 是一种体积技术, 允许成像样品50-100X 厚比明显微镜在可比的分辨率, 与重点进行后处理。DHM 用于识别、计数和跟踪非常低密度的微生物, 并与光学密度测量、平板计数和直接计数相比较。

视频: Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

使用介电泳分离珠和多通道微流控芯片细胞和层流

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

科研

生物工程

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

低成本的低温光学显微镜舞台制作相关的光/电子显微镜

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with &alpha;5&beta;1-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration1,2. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes.
The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal3, causes integrin redistribution1, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions4, and thereby induces directional migration3. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement5. In this protocol we describe the method by which 30 mW/cm2, 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair6. The method uses recombinant fibronectin fragments to engage &alpha;5&beta;1-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts., The positive effect of ultrasound on repair mechanisms is well documented7,8, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.

This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.

诱导粘附依赖使用低强度超声波信号

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from ...

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

量化的内皮细胞糖复合物的力学性能，用原子力显微镜

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

设计和人工细胞外基质的构建（AECM）蛋白质<em&gt;大肠杆菌</em&gt;对皮肤组织工程

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.

This article provides an in depth guide for the assembly and operation of a structured illumination microscope operating with total internal reflection fluorescence illumination (TIRF-SIM) to image dynamic biological processes with optical super-resolution in multiple colors.

指南结构照明显微镜TIRF在高速，多种颜色

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations at the point of contact. It can be achieved by complementing traditional rigid grippers with soft robotic pneumatic gripper devices. This manuscript describes a rod-based approach that combined both 3D-printing and a modified soft lithography technique to fabricate the soft pneumatic gripper. In brief, the pneumatic featureless mold with chamber component is 3D-printed and the rods were used to create the pneumatic channels that connect to the chamber. This protocol eliminates the risk of channels occluding during the sealing process and the need for external air source or related control circuit. The soft gripper consists of a chamber filled with air, and one or more gripper arms with a pneumatic channel in each arm connected to the chamber. The pneumatic channel is positioned close to the outer wall to create different stiffness in the gripper arm. Upon compression of the chamber which generates pressure on the pneumatic channel, the gripper arm will bend inward to form a close grip posture because the outer wall area is more compliant. The soft gripper can be inserted into a 3D-printed handling tool with two different control modes for chamber compression: manual gripper mode with a movable piston, and robotic gripper mode with a linear actuator. The double-arm gripper with two actuatable arms was able to pick up objects of sizes up to 2 mm and yet generate lower compressive forces as compared to elastomer-coated and non-coated rigid grippers. The feasibility of having other designs, such as single-arm or hook gripper, was also demonstrated, which further highlighted the customizability of the soft gripper device, and it's potential to be used in delicate surgical manipulation to reduce the risk of tissue grip damage.

This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels.

娇嫩的组织操纵定制的软机器人气动设备手爪基于杆制造

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations ...

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 ...

用数字全息显微镜 (DHM) 定量化低浓度微生物

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