This research was supported by the research programs of the National Research Foundation of Korea (NRF) (NRF-2015R1A1A1A05001548 and NRF-2015R1A5A1037668).

与血液样本实验是在遵守有关法律和机构指导进行。的样品已被获取并在该机构的临床试验处理剩余血样。 1. PT-AS光学装置注意:一个可以利用一个空微毛细管用于初始的PT-AS的设置。 <ol><li>装入一个空的微毛细管具有分别为200和330微米，内径和外径，和大于约5厘米的毛细管夹具的长度。市售的纤维夹具可被用作管夹具。 </li><li>安全锚650nm的激光指示器， 即探测光源，照亮毛细管。探测光束应该比毛细管大。将毛细管垂帘听政（ 如白皮书），观察的角度周期性模式。 </li><li>对于检测部分，删除任何镜头摄像头可以直接捕捉scatt化工e圈格局。在相对于探测光束方向25-35度的一个角度的毛细管后面的网络摄像头位置。确保由毛细管产生的角度周期性图案可与检测器（ 图1）来测量。观察的角度周期性图案在图像传感器的中间时，图像传感器被正确地定位。 </li><li>一个532纳米PT激发光源来照亮毛细管位置。在PT光源在任何角度，只要该PT激励光与毛细管探测光束重叠，不直接到达探测器的位置。采用高光功率通常血样PT激发改善对PT-AS的灵敏度，因为它会导致在对RI更大的变化。 <ol><li>使用采用PT激发光源的最高光功率。此外，确保在PT激励光重叠在毛细管的探测光。使用PT激发光的光束尺寸的至少两倍的探测光来加热整个探头体积。 </li></ol></li><li>放置一个长通滤波器在探测器的前面以阻止532纳米的光，并测量仅650纳米探测光。 </li><li>照明毛细管之前安装在PT激发光的路径的光学菜刀。光学斩波器被用来调节该PT激发光强度。 </li></ol> 2.血液样品制备<ol><li>绘制的6ml新鲜全血中贫血状况入乙二胺四乙酸血液采样管，并充分混合样品井。不需要其他处理。 </li><li>使用提取的24小时内PT-AS传感器，以防止凝血测量血液样本。 </li></ol> 3. PT-AS测量协议<ol><li>加载一个微毛细管与血液样品以测量。通过将管进入血液个饱通过毛细作用血液毛细管充足。测定所需的最小样品体积由毛细管的内径和探测光束大小决定。 <ol><li>采用管的200微米的内径。探测光束尺寸为2mm中的代表性结果，这表明该测量可以与&gt; 63 NL的样本量来进行。 </li></ol></li><li>装入毛细管在夹具的指定位置。 </li><li>打开650纳米探针激光照射血装载微毛细管。角周期性图案应与摄像头观察到。 </li><li>打开532纳米的PT激发激光照亮管。 </li><li>运行的光斩波器调制以2Hz该PT激发光的强度。 注:此操作条件的选择的理由讨论和Kim 等人说明。 12。 <ol><li>在光学斩波器的马达头组件安装斩光轮系统。 </li><li>打开斩波控制箱，并使用该控制旋钮在控制台设定调制频率。 </li><li>通过运行控制旋钮菜刀。 </li></ol></li><li>记录通过在MPEG-4（MP4）格式的摄像头，持续5秒的波动散射图案。 </li></ol> 4.信号处理注:PT-AS的信号处理被使用实验室开发的MATLAB代码执行。 <ol><li>加载视频文件中提取图像。对于每个图像[参见图2（a）为一代表图像]，由沿垂直方向的计算的像素值的平均值获得平均散射图案[ 图2（B，C）。 </li><li>评估的傅立叶变换平均散射图案，并且在峰值空间频率计算的相位。所有录制的图像的所有帧执行这些操作。 </li><li>利用所有图像获得的相位值，绘制时间相位波动〔 如图2（d）]。注意，相位波动在该PT调制频率。取傅立叶在调制频率变换在时域的相位变动的，并且得到的幅度。此信号被称为对PT-AS信号〔 如图2（E）]。 </li><li>由它的PT-AS信号转换成测量血液样品中的[血红蛋白]使用在协议5中获得的校正曲线相应的[血红蛋白。 </li></ol> 5. PT-AS校准<ol><li>制备血液样品中，具有[血红蛋白]被均匀地分布在PT-AS传感器的检测范围内的值（ 例如 ，0 - 29克/分升）。 </li><li>校准之前，量化使用参考血液分析仪的样品的[血红蛋白]值。测量样品的PT-AS信号。 </li><li>通过派生进行线性最小二乘法拟合有关[血红蛋白]将PT-AS信号的校正曲线，[血红蛋白] = A [PT-AS信号] + B，该experimenta的升的结果。对于表1中指定的操作条件，间[血红蛋白]和PT-AS信号的关系被认为是[血红蛋白] = 5.13 [PT-AS信号] - 0.09。利用MATLAB代码来执行的线性拟合。 </li></ol>

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No conflict of interest is declared.

在PT-AS传感器表示能够未处理的血液样本的直接[血红蛋白]测量的全光学方法。的方法，使用在红细胞的血红蛋白分子的内在的PT响应量化[血红蛋白]血液中。下通过532纳米的光照射，血红蛋白分子吸收的光的能量，并产生热量。所得温升改变血样的RI。 BSI的高灵敏度RI被利用来测量血液中这种RI变化。先前，我们表明，在PT-AS传感器使[血红蛋白]测量0.12克/分升以上0.35-17.9克/分升，这与在市场上的商...

验血通常以评估整体的人类健康和检测与某些疾病生物标志物。例如，在血液中的胆固醇浓度用作用于高脂血症的标准，这是密切相关的心血管疾病和胰腺炎。血糖含量应经常测量，如葡萄糖水平与并发症如糖尿病酮症酸中毒和高血糖高渗综合征。严重的疾病，如疟疾，人类免疫缺陷病毒和获得性免疫缺陷综合征是由血液检查确诊，和血液成分，包括红细胞，血小板的量化和白细胞使胰腺和肾脏疾病筛查。 血红蛋白，血的关键组成部分，构成了红细胞的约96％，并输送氧气到人体器官。其质量浓度显著改变（[血红蛋白]）可能表明我tabolic变化，肝胆疾病，和神经，心血管及内分泌紊乱1。 [血红蛋白]因此在常规验血测量。尤其是贫血患者，透析患者和孕妇强烈建议监察[血红蛋白]作为一个重要的任务2。 各种[血红蛋白]检测方法就这样被开发。血红蛋白氰化法，对于[血红蛋白]量化最常用的技术之一，使用氰化钾（KCN）摧毁红细胞3的脂质双层。通过在540nm附近的化学展品高吸收产生的氰化物血红蛋白;因此，[血红蛋白]测量值可以通过比色分析进行。这种方法由于其简单性广泛采用，但所采用的化学品（ 如 ，KCN和dimethyllaurylamine氧化物）是有毒的对人体和环境。血细胞比容方案测量与总血液体积的红血细胞的体积比乌梅通过离心分离;然而，它需要一个相对大的血液量（50-100微升）4。分光光度法测量方法[血红蛋白]正是没有任何的化学物质，但在多个波长和大血容量测量需要5,6。同样，用于测量[血红蛋白]几种光学方法已经提出了包括基于光散射的检测方法，但其测量精度上的理论血模型的准确度在很大程度上取决于。 为了克服这些限制，基于血红蛋白的光热（PT）作用[血红蛋白]检测方法近来已经提出7。血红蛋白，它是由主要是铁的氧化物，在532纳米吸收光的光能量转换成热8-10。这个PT温度增加可以光学地通过测量血液样品的折光指数（RI）的变化来检测。 Yim 等。就业谱域光学相干reflectometry以测量在含有血液室11在PT光路长度变化。虽然该方法能够化学 - 自由和直接[血红蛋白]测定，使用分光计和干涉装置可阻碍其小型化。我们最近提出一种替代[血红蛋白]检测方法，称为光热角光散射（PT-AS）的传感器，它更适合于设备的小型化12。在PT-AS传感器利用了背散射干涉（BSI）的高RI灵敏度测量毛细管内的血液样品的RI PT变化。 BSI已用来测量各种解决方案13-15中RI和监测在自由溶液16生物化学相互作用。该PT-AS传感器采用类似的光学结构为BSI，但结合光热激发的设置来测量血液样本中的RI PT增加。在BSI和PT-AS传感器的工作原理进行了详细的描述别处<sup&gt; 12,15。 PT-AS传感器表现出高灵敏度[血红蛋白]测量在很宽的检测范围（0.35-17.9克/分升），并能够与&lt;100 NL的样品体积运行。血液试样的无预处理是必需的，并且在测量时间仅为〜5秒。在这里，实验装置和详细的测量协议进行说明。使用血液样品从贫血患者提供代表的PT-AS的结果，其结果对那些从血液分析仪比较，以评估对PT-AS传感器的精度。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>650nm laser pointer</td>
			<td>LASMAC</td>
			<td>LED-1</td>
			<td>Probe light</td>
		</tr>
		<tr>
			<td>Hollow round glass capillaries</td>
			<td>Cm Scientific</td>
			<td>CV2033</td>
			<td>Blood sample container</td>
		</tr>
		<tr>
			<td>Webcam</td>
			<td>Logitech</td>
			<td>C525</td>
			<td>CMOS optical sensor</td>
		</tr>
		<tr>
			<td>532-nm DPSS laser</td>
			<td>CNI Laser</td>
			<td>MGL-&#8546;-532</td>
			<td>Photothermal light source</td>
		</tr>
		<tr>
			<td>Optical chopper system</td>
			<td>Thorlabs</td>
			<td>MC2000-EC</td>
			<td>Optical chopper</td>
		</tr>
		<tr>
			<td>Plastic long-pass filter</td>
			<td>Edmund Optics</td>
			<td>#43-942</td>
			<td>To reject 532-nm PT excitation light</td>
		</tr>
		<tr>
			<td>Fiber clamp</td>
			<td>Thorlabs</td>
			<td>SM1F1-250</td>
			<td>Capillary tube fixture</td>
		</tr>
		<tr>
			<td>EDTA coated blood sampling tube</td>
			<td>Greiner Bio-One</td>
			<td>VACUETTE 454217</td>
			<td>Blood sampling &#38; anticoagulating</td>
		</tr>
		<tr>
			<td>Hematology analyzer</td>
			<td>Siemens AG</td>
			<td>ADVIA 2120i</td>
			<td>Reference hematology analyzer</td>
		</tr>
	</tbody>
</table>

a rapid and chemical-free hemoglobin assay with photothermal angular light scattering

光热角光散射（PT-AS）是用于测量血液样本的血红蛋白浓度（[血红蛋白]）一种新颖的光学方法。对血红蛋白分子的固有光热响应的基础上，该传感器可实现高灵敏度，[血红蛋白]的无化学计量。 [血红蛋白] 0.12克/分升以上0.35的范围内的极限检测能力 - 17.9克/ dl的先前已证实。该方法可容易地使用便宜的消费电子设备，诸如激光指示器和网络摄像头实现。作为血液容器使用微毛细管还使用纳升规模的血容量和低运行成本的血红蛋白测定。这里，对于对PT-AS光学设置和信号处理程序的详细说明介绍。实验方案和用于贫血的条件血样代表性的结果（[血红蛋白] = 5.3，7.5，和9.9克/分升）也提供，并测量与这些往复相比马血液分析仪。其在实施和操作简单应使其广泛应用在临床实验室和资源有限的环境。

使用PT-AS传感器被执行的血红蛋白测定法，其测量结果与那些从血细胞分析仪进行了比较。实验用1.4瓦/厘米2，2赫兹的PT调制频率，并测量5秒的时间的一个PT激励光强度进行的。 表1总结了实验条件。探针和PT激发光的光束尺寸分别为5.5和2毫米。摄像头以30 fps的帧速率录制的影像。测量中，采用了三种不同的Hb浓度的贫血的血液样本。在PT-AS测量之前，?...

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A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

一种快速和无农药血红蛋白检测法与光热角光散射

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...

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Research

JoVE Journal

Bioengineering

9.6K 次观看.  Yonsei University. A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

视频: A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

活细胞层的快速消减带图案微流控探头

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

科研

生物工程

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

仿生纳米羟基磷灰石的快速混合制备生物医学应用

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

用于动态生长测试的温度，光照和pH监测的台式藻类光合生物反应器的构建和设置

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa.

Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

一种基于变形虫的快速图像细菌毒力检测方法

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

Rapid Characterization of Genetic Parts with Cell-Free Systems

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a rapid and cost-effective method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.

Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.

使用无细胞系统快速表征遗传部分

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

用于微生物化学和蛋白质生产的光控发酵

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

体内 使用双光子荧光和受激拉曼散射显微镜对生物组织进行成像

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

A Nonsequencing Approach for the Rapid Detection of RNA Editing

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger sequencing and RNA sequencing techniques. However, these methods can be costly and time-consuming. In this protocol, a portable microtemperature gradient gel electrophoresis (&#181;TGGE) system is used as a nonsequencing approach for the rapid detection of RNA editing. The process is based on the principle of electrophoresis, which uses high temperatures to denature nucleic acid samples as they move across a polyacrylamide gel. Across a range of temperatures, a DNA fragment forms a gradient of fully double-stranded DNA to partially separated strands and then to entirely separated single-stranded DNA. RNA-edited sites with distinct nucleotide bases produce different melting profiles in &#181;TGGE analyses. We used the &#181;TGGE-based approach to characterize the differences between the melting profiles of four edited RNA fragments and their corresponding nonedited (wild-type) fragments. Pattern Similarity Scores (PaSSs) were calculated by comparing the band patterns produced by the edited and nonedited RNAs and were used to assess the reproducibility of the method. Overall, the platform described here enables the detection of even single base mutations in RNAs in a straightforward, simple, and cost-effective manner. It is anticipated that this analysis tool will aid new molecular biology findings.

Rapid detection and reliable quantification of RNA editing events at a genomic scale remain challenging and currently rely on direct RNA sequencing methods. The protocol described here uses microtemperature gradient gel electrophoresis (&#181;TGGE) as a simple, quick, and portable method of detecting RNA editing.

一种快速检测RNA编辑的无序列方法

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger ...