1。影像设定很多，可以在一个紧凑和轻巧便携领域的架构8组装，或者optofluidic显微镜截面成像能力。9在这份报告中，但是，我们会向静态描述断层成像为台式实施的基本设置样品。 <ol><li> 照明模块：地段，部分相干光源，如发光二极管（LED）可以利用。实验的灵活性，我们使用了氙气灯的单色（T260基石，新港公司）。单色仪的调整，以提供一个〜1-10纳米围绕一个中心， 如 450-650纳米波长的光谱宽度的输出。那么，这部分相干输出耦合到多模光纤的纤维（Thorlabs AFS105/125Y）提供部分相干光系统。 光纤安装在一个电动的旋转舞台（ThorlabsPRM - 1Z8驱动Thorlabs TDC001控制器）来改变照明角度。机动阶段，光源，安装在一个两维的直线的XY阶段（新港ILS50CC由新港外交部的PPD控制器驱动），这是用来实现在一个特定的角度，光源平面的变化。 </li><li> 检测：Lensfree光断层扫描是一种可扩展的探测器阵列可以根据应用要求选择没有显着变化的图像采集步骤或数据处理技术等。在这份报告中，我们采用的2.2微米像素大小（IDS成像，UI-1485LE-M）的500万像素，CMOS传感器阵列。探测器是用来记录宽领域的视图（ 如 24毫米2）全息图像样本，其中直接放置在顶部的探测器的有效面积。如果双轴断层扫描设置是实施7，探测器应莱索托被安装在水平（光表）旋转阶段（ 如 Thorlabs RP-01）在正常视神经轴的平面旋转的样品（连同探测器）7。 </li></ol> 2。样品制备虽然lensfree光学断层扫描图像的各种对象，如细 ​​胞和微生物，我们将说明执行的C.立体显微镜的基本原则线虫样本。 <ol><li>使用手术刀或压舌板，采取的Petri包含了 C盘的琼脂小块线虫文化。一个立方米一块几毫米，沿着每个维度将包含数百线虫。 </li><li>聚丙烯小瓶含1 mL去离子（DI）水将琼脂小块。 </li><li>轻轻涡0.5-1分钟，等待10-15分钟，直至蠕虫爬行到DI水的琼脂块。 </li><li>以暂时固定蠕虫，加1毫升5-10毫米左旋咪唑溶液（Sigma-Aldrich公司），并等待10分钟。 </li><li>吸取5-10微升样品小瓶底部，两盖玻片和三明治。这个范例中，含有大量的临时固定的蠕虫（ 如 50-100蠕虫），可放置在探测器上启动数据采集。 </li></ol> 3。数据采集在这里，我们总结为一个典型的大量实验，这是由自动使用一个自定义开发的LabVIEW接口的图像采集步骤。 <ol><li>调整旋转舞台的初始角度-50°，0°对应的垂直位置的光源。 </li><li>调整的XY阶段的初始位置为（0,0），这是起始位置。 </li><li>调整探测器的曝光时间，以最好地利用其动态范围，这样的形象是没有任何S尽可能明亮aturated像素。 </li><li>在不改变角度的旋转舞台，捕捉9张图像。每幅图像，移动XY工作台在一个3x3的方格，比前一个大约有四分之一像素，每个图像转移到新的位置。在各个角度获取更多的图像， 例如在6μ6电网，可能会提高分辨率取决于对象的类型和信号噪声比（SNR）10。 </li><li>调整的XY阶段的初始位置为（0,0），这是起始位置。 </li><li>增加2°递增，直至达到50°角度的旋转舞台。每个增量后， 即在每一个新的角度，重复步骤3-5。角增量可以更细或更粗，根据收购时间与成像分辨率的优化。 </li><li> （可选步骤，如果一个双轴断层计划是利用）旋转90°安装探测器的水平阶段。然后，重复步骤1-6。 </li></ol> 4。数据分析继步骤1-6在第3节，收购了459图像集，其中包含9个子像素转向照明的51个不同的角度的图像。首先，每套9张图像数字化处理，使用像素超高分辨率算法10，获得一个高分辨率（HR）投影每个角的全息图。应当指出的像素的超高分辨率，是指克服像素大小的限制，而不是衍射极限lensfree图像的空间分辨率。像素的超级解决全息图，然后以数字7-10获得51投影图像重建。这套51投影图像，然后背投影使用TomoJ，插件ImageJ的（一个开源的图像分析软件）11背投影操作。输出样本（tomograms）一个三维图像。虽然这里不落实，双轴断层计划也可以利用在文献中所述。 7。在这个方案中，第二组的tomograms方面的第一个旋转轴，它提供了更多的空间信息有关的样本，并提高轴向分辨率的正交轴沿旋转光源。 5。代表结果大视场的视角（FOV）lensfree光断层扫描显示在图1。作为样品被直接放置在探测器阵列的顶部，物体的全息图像可以记录超过24个视野2毫米，可进一步增加新兴探测器阵列用较大的活跃的地区。 虽然探测器阵列的像素大小限制记录的全息图像的分辨率，像素的超高分辨率技术缓解这一问题。 图2显示像素的超级解决的全息图沿提供三个不同的角度照明亚微米的空间分辨率，图像重建（ 即投影图像）。 投影图像可以合并使用断层图像重建技术（ 如滤波反投影）计算试样tomograms（ 即三维图像）。 图3显示了在xy平面的切片图像通过蠕虫的前三个，其中咽管是可见的，只有在通过Z = 8微米，预计从这个〜5微米外径大致圆柱形结构切片。此外，在xz平面的截面图像（ 图3顶部面板中插图）清楚地表明了该蠕虫的边界和咽管内（箭头指向），展示咽成功的三维成像。 <img src="/files/ftp_upload/4161/4161fig1.jpg" alt="图1" /> <strONG&gt;图1显示了一个完整的现场视图（24毫米2）全息图像记录使用的lensfree光断层扫描（地段）设置垂直照度。由于地段的成像面积大，许多蠕虫可以同时成像与一个单一的数据采集步骤。 <img src="/files/ftp_upload/4161/4161fig2.jpg" alt="图2" /> 图2显示像素为C。超分辨全息图（左图）和数字重建投影图像（右图） 线虫蠕虫在三个不同光照角度（从一个大场的视图裁剪）。对于不同的视角，使通过滤波反投影操作的三维结构计算每个投影图像中包含的信息。 <img src="/files/ftp_upload/4161/4161fig3.jpg" alt="图3" /> 图3显示的C.计算tomograms 线虫蠕虫ØF 图2。 （上排）显示了整个蠕虫在Z = 3微米的切片图像。插图显示了从蠕虫前横断面图像。插图比例尺为50μm。 （下排）显示通过蠕虫的前三个切片图像，展示lensfree光断层扫描光学切片能力。整个图中的箭头指示蠕虫的咽管的相同点。还提供了一个显微镜图像（X40，0.65-NA）的视觉比较。

<ol>
	<li>Schmitt, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+(OCT):+a+review,+J.+Sel.+Top.">Optical coherence tomography (OCT): a review, J. Sel. Top.</a> Quant. Elect. 5, 1205-1215 (1999).</li><li>Keller, P. J., Schmidt, A. D., Wittbrodt, J., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+zebrafish+early+embryonic+development+by+scanned+light+sheet+microscopy.">Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy.</a> Science. 322, 1065-1069 (2008).</li><li>Sharpe, J., Ahlgren, U., Perry, P., Hill, B., Ross, A., Hecksher-S&#248;rensen, J., Baldock, R., Davidson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Projection+Tomography+as+a+Tool+for+3D+Microscopy+and+Gene+Expression+Studies.">Optical Projection Tomography as a Tool for 3D Microscopy and Gene Expression Studies.</a> Science. 296, 541-545 (2002).</li><li>Sung, Y., Choi, W., Fang-Yen, C., Badizadegan, K., Dasari, R. R., Feld, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+diffraction+tomography+for+high+resolution+live+cell+imaging.">Optical diffraction tomography for high resolution live cell imaging.</a> Opt. Exp. 17, 266-277 (2009).</li><li>Debailleul, M., Simon, B., Georges, V., Haeberle, O., Lauer, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Holographic+microscopy+and+diffractive+microtomography+of+transparent+samples.">Holographic microscopy and diffractive microtomography of transparent samples.</a> Meas. Sci. Technol. 19, 074009 (2008).</li><li>Charri&#232;re, F., Pavillon, N., Colomb, T., Depeursinge, C., Heger, T. J., Mitchell, E. A. D., Marquet, P., Rappaz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+specimen+tomography+by+digital+holographic+microscopy:+Morphometry+of+testate+amoeba.">Living specimen tomography by digital holographic microscopy: Morphometry of testate amoeba.</a> Opt. Exp. 14, 7005-7013 (2006).</li><li>Isikman, S. O., Bishara, W., Mavandadi, S., Yu, S. W., Feng, S., Lau, R., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lens-free+optical+tomographic+microscope+with+a+large+imaging+volume+on+a+chip.">Lens-free optical tomographic microscope with a large imaging volume on a chip.</a> Proc. Nat. Acad. Sci. 108, 7296-7301 (2011).</li><li>Isikman, S. O., Bishara, W., Sikora, U., Yaglidere, O., Yeah, J., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field-portable+Lensfree+Tomographic+Microscope.">Field-portable Lensfree Tomographic Microscope.</a> Lab Chip. 11, 2222-2230 (2011).</li><li>Isikman, S. O., Bishara, W., Zhu, H., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optofluidic+tomography+on+a+chip.">Optofluidic tomography on a chip.</a> App. Phys. Lett. 98, 161109 (2011).</li><li>Bishara, W., Su, T. W., Coskun, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lensfree+on-chip+microscopy+over+a+wide+field-of-view+using+pixel+super-resolution.">Lensfree on-chip microscopy over a wide field-of-view using pixel super-resolution.</a> Opt. Exp. 18, 11181-11191 (2010).</li><li>Messaoudi, C., Boudier, T., Sorzano, C. O. S., Marco, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TomoJ:+tomography+software+for+three-dimensional+reconstruction+in+transmission+electron+microscopy.">TomoJ: tomography software for three-dimensional reconstruction in transmission electron microscopy.</a> BMC Bioinformatics. 8, 288 (2007).</li><li>Heng, X., Erickson, D., Baugh, L. R., Yaqoob, Z., Sternberg, P. W., Psaltis, D., Yang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optofluidic+Microscopy:+A+Method+for+Implementing+High+Resolution+Optical+Microscope+On+A+Chip.">Optofluidic Microscopy: A Method for Implementing High Resolution Optical Microscope On A Chip.</a> Lab on a Chip. 6, 1274-1276 (2006).</li></ol>

没有利益冲突的声明。

重要的是要强调的是独特的几何lensfree片上的全息显微实现像素的超高分辨率层析成像的关键推动者。由于录制的影像不认为是投影图像在语无伦次接触成像接近12，但投影全息图 ，透射光衍射，直到它撞击探测器可以数字全息重建纠正。因此，在样品到传感器的距离变化可占。此外，由于样品到传感器的距离通常~~ 0.5-5毫米，而光源放置在〜4-10厘米的距离从样品，实现亚像素的变...

<table border="1"><tbody><tr><td>名称</td><td>品牌授权</td><td>产品编号</td><td>评论</td></tr><tr><td>线性的XY阶段</td><td>新港公司</td><td>外交部聚丙烯</td><td>微型直线阶段</td></tr><tr><td>电动旋转舞台</td><td> thorlabs </td><td> PRM1Z8 </td><td>电动精密旋转摩</td></tr><tr><td>多模光纤</td><td> thorlabs </td><td> AFS105/125Y </td><td>多模光纤</td></tr><tr><td>光源</td><td>新港公司</td><td> 6255 </td><td>臭氧免费氙气灯</td></tr><tr><td>单色</td><td>新港公司</td><td> 74100 </td><td>基石260 1/4中号单色</td></tr><tr><td> CMOS传感器阵列</td><td> Aptina的公司</td><td> MT9P031STC </td><td> 500万像素CMOS传感器</td></tr><tr><td> 线虫样本</td><td>卡罗来纳州Biosupply </td><td> 173500 </td><td>野生型C.线虫 </td></tr><tr><td>左旋咪唑</td><td> Sigma Aldrich公司</td><td> L9756-5G </td><td>四咪唑盐酸盐</td></tr></tbody></table>

lensfree on-chip tomographic microscopy employing multi-angle illumination and pixel super-resolution

层析成像已在医学上广泛使用的工具，因为它可以提供三维（3D）结构信息，对于大小不同尺度的对象。在微米和毫米尺度，光学显微镜的方式发现，由于越来越多地使用非电离性的可见光，并提供一套丰富的照明光源（如激光器和发光二极管）和检测元件（如大幅面CCD和CMOS探测器阵列）。在最近开发的光学层析显微镜的方式，可以包括光学相干断层扫描，衍 ​​射光断层扫描，光学投影层析成像和光表镜。1-6这些平台提供的细胞，微生物和动物模型如 C截面成像线虫 ，斑马鱼和小鼠胚胎。 现有的3D光学成像一般都比较笨重和复杂的结构，限制日é可用性，这些设备先进的实验室，并妨碍他们的实验室在一个芯片平台和微流控芯片的集成。提供一种替代的断层显微镜，我们最近开发的一个高通量，结构紧凑，成本效益的光学断层扫描方式lensfree光断层扫描（很多）。7拍镜头和笨重的光学元件的使用将被丢弃，而不是依靠多角度照明和数字计算，实现了一个大的成像量深度解决微型物体的成像。很多形象在&lt;1微米的空间分辨率X &lt;1微米x &lt;3微米，在X，Y和Z尺寸，分别超过了15-100毫米3大成像容积，并就特别有用的生物标本实验室在一个芯片平台。

Watch this Scientific Journal Video about Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution at JoVE.com

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

lensfree光学断层扫描是一个立体显微镜技术，提供了一个&lt;1微米的空间分辨率×&lt;1微米×&lt;3微米，在X，Y和Z尺寸，分别超过15-100毫米大成像容积<sup&gt; 3</sup&gt;，这可能与实验室的单芯片平台集成特别有用。

lensfree芯片断层用人多角度的光照和像素超高分辨率的显微镜

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

lensfree-on-chip-tomographic-microscopy-employing-multi-angle

Research

JoVE Journal

Bioengineering

11.4K 次观看.  University of California, Los Angeles. lensfree光学断层扫描是一个立体显微镜技术，提供了一个

视频: Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

生物分子检测的干涉反射成像传感器（IRIS）

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

科研

生物工程

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

对芯片等速电泳离子的分离和纯化核酸

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

片上内皮炎症表型

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

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 ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

干膜 Photoresist-based 电化学微流控生物传感器平台: 器件制备、片上检测制备及系统运行

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

用集成电极直接量化 Transendothelial 电阻的 Organ-on-chip 系统的制作与验证

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab and industrial scale. However, due to its toxicity and flammability, there is a desire to avoid methanol while maintaining the high productivity of P. pastoris. Small scale bioreactor cultivations (0.5-5 L working volume) are commonly used to evaluate a strain and its protein production characteristics since microscale cultivation in deep well plates can be hardly controlled or relies on expensive equipment. Furthermore, traditional protocols for the cultivation and induction of P. pastoris were established for constitutive expression or methanol induction and so far, no reliable protocols were described to screen P. pastoris expression strains with derepressible promoters in (controlled and monitored) parallel cultivations. To simplify such initial cultivations to characterize and compare new protein production strains, we established a simple shake flask cultivation system for methanol free expression that simulates bioreactor conditions including a constant slow glycerol feed and online monitoring, thereby coming closer to the real conditions in bioreactors compared to mostly applied small scale batch cultivations. To drive recombinant protein expression in P. pastoris, the carbon source repressed promoters PDC and PDF were applied. Polymer discs with embedded carbon source, releasing a constant amount of glycerol, assured a feed rate delivering the necessary energy for maintaining the promoters active while keeping the biomass generation low.

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

This method for the first time describes reliable experimental procedures for efficient inducible methanol-free recombinant protein production in Pichia pastoris (Komagataella phaffii), employing the carbon source regulated promoters PDCand PDF in small scale experiments while cultivation parameters can be monitored online, and a constant glycerol feed is applied.

多夏巴氏杆菌自主表达采用除抑制技术

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab ...

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S.&#160;cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

The CRISPR/Cas12a system in combination with a single crRNA array enables efficient multiplex editing of the S.&#160;cerevisiae genome at multiple loci simultaneously. This is demonstrated by constructing carotenoid producing yeast strains which are subsequently used to create yeast pixel art.

CRISPR/Cas12a 多路复用基因组编辑糖母细胞和酵母像素艺术的创作

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

开源微孔板兼容照明面板的应用

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...

Building a Simple and Versatile Illumination System for Optogenetic Experiments

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical control allows for an unprecedented ability to dissect function and holds the potential for enabling novel genetic therapies. However, optogenetic experiments require adequate light sources with spatial, temporal, or intensity control, often a bottleneck for researchers. Here we detail how to build a low-cost and versatile LED illumination system that is easily customizable for different available optogenetic tools. This system is configurable for manual or computer control with adjustable LED intensity. We provide an illustrated step-by-step guide for building the circuit, making it computer-controlled, and constructing the LEDs. To facilitate the assembly of this device, we also discuss some basic soldering techniques and explain the circuitry used to control the LEDs. Using our open-source user interface, users can automate precise timing and pulsing of light on a personal computer (PC) or an inexpensive tablet. This automation makes the system useful for experiments that use LEDs to control genes, signaling pathways, and other cellular activities that span large time scales. For this protocol, no prior expertise in electronics is required to build all the parts needed or to use the illumination system to perform optogenetic experiments.

This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer.

为光遗传学实验构建简单通用的照明系统

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical ...