Name: quantifying microorganisms at low concentrations using digital holographic microscopy (dhm)
Uploaded: 2017-11-01T15:00:00
Description: Watch this Scientific Journal Video about Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM at JoVE.com

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...

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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/56343/56343e...

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?...

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 ...

Research

JoVE Journal

Bioengineering

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

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

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

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

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

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

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

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

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

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

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

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 ...