Name: sivq-lcm protocol for the arcturusxt instrument
Uploaded: 2014-07-23T14:00:00.000+00:00
Duration: 7 min 37 s
Description: Watch this Scientific Journal Video about SIVQ-LCM Protocol for the ArcturusXT Instrument at JoVE.com

这项研究是由美国国家癌症研究所癌症研究中心院内研究计划美国国立卫生研究院，部分支持。

所描述的协议是采用按照关于使用人体组织样本的美国国立卫生研究院的规则。 1，组织准备<ol><li>前年初，根据机构审查委员会（IRB）的协议得到人体组织标本。 </li><li>选择组织/细胞块的类型和相应的处理方法[福尔马林固定石蜡包埋（FFPE），冷冻，或乙醇固定，石蜡包埋（EFPE）]。福尔马林固定提供了最佳的组​​织学，随后用乙醇固定，并快速冷冻。然而，固定和组织处理方法可影响DNA，RNA和蛋白质的数量和质量的下游分子分析，并应予以考虑。 </li><li>切割组织/细胞块切片上选择的滑动型（玻璃，膜玻璃，或金属框架的膜）。 SIVQ分析同样适用于所有三种类型的幻灯片。请注意，（如下所述）用二甲苯和乙伪盖玻片方法醇不能在金属框架膜幻灯片进行由于滑动，必须在舞台上被颠倒。 </li><li>选择一个化学或免疫组织化学为基础的组织染色，以确定所关注的细胞的背景。需要注意的是染色方法也可影响DNA，RNA和蛋白质的质量和组织的数量。在进行染色与协议前评估生物分子的基准质量测试后的组织中。免疫组织化学（IHC）与民建联15，免疫荧光，快红， 从头红，甲苯胺蓝，苏木精伊红（H＆E）（未发表资料）:SIVQ-LCM已对组织/细胞学载玻片沾满了被执行。 </li></ol> 2。标本成像<ol><li>负载滑动到显微切割工具的机动阶段，并启动相关的软件。选中相应的复选框来指定加载幻灯片的位置，并确保所拍摄的图像文件将被保存为JPEG格式。 </li><li>优化IM通过调整图像的亮度和聚焦在屏幕上，并使用本手动对焦轮或通过解剖仪器的软件年龄质量。 <ol><li>使用解剖仪器的软件中的图像工具箱，适当地将灯泡的亮度和摄像机的增益。示例值有亮度= 60和增益= 220，与扩散器。 </li><li>手动或使用该软件中的自动对焦功能对焦。 </li></ol></li><li>捕捉幻灯片提供一个路线图解剖过程的缩略图概览图像。 <ol><li>对于一个uncoverslipped滑动的最佳的图像质量，利用仪器上的冷凝器下方的扩散器，或者，添加乙醇或二甲苯少量（约30微升）以提高耐火指数（伪盖玻片）。当使用二甲苯，要知道他们必须用有毒的和适当的安全措施，包括使用通风橱和防护实验室外套，护目镜和手套。 </li><li> 请勿在投影片上的LCM帽，直到乙醇或二甲苯溶液中完全清除或盖上的聚合物会被扭曲。 </li></ol></li><li>导航在10X，20X，40X或放大要解剖方面的幻灯片，拍摄的图像。如果有必要，加强与软件，如自动更正（在Microsoft Office图片管理器）图像如前所述22。图像必须被捕获JPEG格式，让他们重新导入到自动选型软件。 </li></ol> 3，算法的图像分析<ol><li>转让从捕获显微切割仪图像到SIVQ文件夹。安装并打开ArcturusXT，自动扫描和SIVQ软件包安装到解剖仪器的电脑上。用于访问SIVQ软件，请联系尤利西斯巴里斯博士（ulysses@med.umich.edu）。 </li><li>开SIVQ并加载所捕获的图像的兴趣（JPEG）。&lt;/ LI&gt; <li>导航到感兴趣的区域和/或调节的显示窗口（视口5＆6）的大小。在SIVQ软件，视口5示出了预处理的图像和视口6示出了后处理的图像16。 </li><li>选择环矢量和环要使用的数字的大小。 </li><li>选择要通过右击视口6抓获其上的谓词图像特征。 </li><li>点击"扫描"来分析图像。 </li><li>采用两个滑动杆调节图像匹配的统计概率。上部杆调整整体矢量特异性，并用于从初始扫描（具有灵敏度由选定的"统计"变量的定义），以排除区域可表示包含过多的区域。相反，较低的滑块被用于增加灵敏度，之后进行的扫描，与被分类为是匹配增加面积的意图。这两个滑块控件的UTI丽泽的"统计"变量作为初始基线灵敏度阈值。 </li><li>保存图像，点击"保存为JPEG"（图像已保存在C :/ vq_test文件夹/图片）。 </li><li>分析与算法的图像。算法分析的输出需要产生一个带注释的图像为它在SIVQ-LCM中使用。目前，SIVQ核心引擎的当前版本是beta版测试，并期望在满负荷生产版本（2014年第一季度上市），将包括一个完整的软件开发工具包（SDK）和应用程序编程接口（API），用于简化集成用户生成的空间滤波器，并与核心环匹配引擎的下游流程的数据处理步骤。该SDK将分发了一套完整的文档。 <ol><li>确保SIVQ热图改变为均匀的红色。 </li></ol></li><li>导出图像。至关重要的是，在p重新嵌入的位置坐标通过使用一个十六进制编辑器从原来的解剖仪器的图像文件头粘贴OST分析JPEG图像。相应的数据可能会"启动图像"标记（0xFF的，为0xD8）和第一"定义量化表"标记（0xFF的，为0xDB）之间发现。 </li></ol> 4。显微切割<ol><li>放置在LCM盖在感兴趣的区域，其中所述图像捕获为SIVQ分析的中心。 </li><li>校准和质量控制（QC）的紫外/红外激光器和优化参数，包括功率，持续时间，激光的位置，和UV切割速度（如制造商推荐的）。之前重新导入所分析的图像进行这些校准。 </li><li>打开AutoScanXT（自动选型软件），并从C :/ vq_test文件夹/图片导入所分析的图像。 </li><li>列车自动选择软件来识别SIVQ注释和创建解剖图。 <ol><li>要创建一个寨宁文件，对"红色漆"的SIVQ分析图像选择感兴趣的四个区域（标记为"蓝色圆圈"）。 </li><li>选择的背景区域（标记为"红方块"），它们不被解剖。 </li><li>单击"分析"按钮，生成培训文件，该文件可以保存供日后使用。 </li></ol></li><li>使用适当的红外（IR）和/或紫外（UV）激光器进行显微解剖。 <ol><li>选定区域复制到"活"的形象。 </li><li>在"microdissect"工具箱中，选择适当的红外捕捉或UV切割按钮。 </li></ol></li><li>剥离完成后，将LCM盖到QC站和捕捉解剖组织/细胞的图像。另一种方法是把帽上之前，将其移动到QC站幻灯片的空白区域，这允许用户在拍摄照片以不同的放大倍率。 </li><li>捕获感兴趣的组织区域的图像显微切割后，进一步评估起重效率。 </li><li>如果所需的细胞已被成功地解剖，点击在ArcturusXT软件的"现阶段"按钮，删除该LCM帽发起的分子提取程序用于下游分析。 </li></ol>

<ol>
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迈克尔·埃默特 - 巴克是美国国立卫生研究院持有的专利，涵盖激光捕获显微切割技术的发明者，并通过美国国家卫生研究院技术转让项目获得特许权为基础的支付。

我们提出了一个协议，用于SIVQ-LCM的应用，以microdissect从FFPE人体乳腺组织免疫染色上皮细胞。使用图像分析算法，如SIVQ，降低了实际操作所需的显微切割过程的时间的量。这对于该领域的潜在的重要进步，因为操作者的时间和精力，通常为所关注的细胞的精确解剖中的限速步骤。在本协议中，我们特别适合我们的程序到ArcturusXT仪器，虽然它很可能将有可能适应SIVQ等算法其他市售的显微仪器，以?...

最初开发于90年代中期，激光捕获显微切割（LCM）使用户通过微观可视化1，2，精确捕捉从组织学组织切片特定的细胞或细胞区域。比较LCM的分子分析与组织擦伤许多研究说明了该方法3-12的值。此外，也有关于可用于观看13,14的技术3视频协议的出版物。然而，尽管它被证明价值，LCM可以是繁琐和费力的，当感兴趣的目标是一个分散的细胞群体中的异质组织切片时，或者当需要进行特定的下游应用，如蛋白质组学大量的细胞。放置在操作人员的负担，导致我们结合一个强大的图像分析算法，以指导对LCM进程15制定LCM半自动夹层的方法。 &lt;P类="j​​ove_content"&gt;在与密歇根大学合作，我们的实验室在NIH扩展先前制定和报告的空间不变的矢量量化（SIVQ）算法的方式，使其到半自动化的组织选择过程中固有的引导显微切割，从而使可用在头脑里的病理学家或生命科学家的工具。空间不变的矢量量化（SIVQ）是一种算法，允许用户简单地"点击"感兴趣的组织学特征来创建一个环载体（谓词图像特征），可以用来搜索整个组织学图像，调整阈值的统计根据需要16-21。由此产生的热图显示匹配的质量初始谓词图像特征，并随后被转换成一个单一的颜色，可以导入到LCM仪器（红色）标注地图。自动选型软件，AutoScanXT，然后用于绘制地图的基础上SIVQ的注解引导从组织样品中的靶细胞的捕获。下面详细的协议描述的实施SIVQ进入显微切割工作流程。

<table><tbody><tr><td>Positive Charged Glass Slides</td><td>Thermo Scientific</td><td>4951Plus-001</td><td></td></tr><tr><td>Xylenes, ACS reagent, &amp;ge;98.5% xylenes + ethylbenzene basis&amp;nbsp;</td><td>Sigma Aldrich</td><td>247642</td><td>CAUTION: PLEASE USE PROPER SAFETY PROCEDURES.</td></tr><tr><td>Ethyl Alcohol, U.S.P. 200 Proof, Anhydrous</td><td>The Warner-Graham Company</td><td>6.505E+12</td><td>CAUTION: PLEASE USE PROPER SAFETY PROCEDURES.</td></tr><tr><td>Arcturus CapSure Macro LCM Caps</td><td>Life Technologies</td><td>LCM0211</td><td></td></tr><tr><td>ArcturusXT Laser Microdissection Instrument</td><td>Life Technologies</td><td>ARCTURUSXT</td><td></td></tr><tr><td>AutoScanXT Software</td><td>Life Technologies</td><td></td><td>An optional image analysis program for the ArcturusXT Laser Microdissection Device. This is software is required for SIVQ-LCM.</td></tr><tr><td>Spatially Invariant Vector Quantization (SIVQ)</td><td>University of Michigan</td><td></td><td>This tool suite is publicly available for academic collaborations. For access to the SIVQ algorithm, please contact Dr. Ulysses Balis [Ulysses@med.umich.edu]</td></tr></tbody></table>

sivq-lcm protocol for the arcturusxt instrument

SIVQ-LCM是一种新的方法，它可以自动和简化了较为传统的，依赖于用户的激光剥离过程。它的目的是建立一个先进的，快速定制的激光剥离技术平台。在这份报告中，我们描述了图像分析软件空间不变的矢量量化（SIVQ）整合到ArcturusXT仪器。该ArcturusXT系统包含一个红外（IR）和紫外（UV）激光，允许特定的细胞或大面积解剖。主要目标是提高速度，精度，激光剥离，以提高样品通量的重现性。这种新方法有利于动物和在研究和临床工作流程人体组织的显微切割。

一个FFPE人体乳腺组织切片的免疫染色用标准IHC协议23细胞角蛋白AE1/AE3。染色后，将组织切片放置在ArcturusXT阶段，如上述的SIVQ-LCM协议发起。自组织不能盖玻片为显微切割的IHC +染色的细胞可以是难以辨别视觉（ 图1A）。因此，为了提供更好的折射率匹配和改进的图像，二甲苯加入到该组织部分来创建一个伪盖玻片15（ 图1B）。 JPEG图像，然后拍摄的伪盖片?...

Watch this Scientific Journal Video about SIVQ-LCM Protocol for the ArcturusXT Instrument at JoVE.com

SIVQ-LCM Protocol for the ArcturusXT Instrument

SIVQ-LCM是一种创新的方法，运用计算机算法，空间不变的矢量量化（SIVQ），驱动激光捕获显微切割（LCM）的过程。该SIVQ-LCM流程大大提高显微切割的速度和精度，以在这两个研究和临床应用中的设置。

为ArcturusXT仪器SIVQ-LCM协议

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an ...

sivq-lcm-protocol-for-the-arcturusxt-instrument

Research

JoVE Journal

Biology

8.6K 次观看.  National Institutes of Health.  SIVQ-LCM是一种创新的方法，运用计算机算法，空间不变的矢量量化（SIVQ），驱动激光捕获显微切割（LCM）的过程。该SIVQ-LCM流程大大提高显微切割的速度和精度，以在这两个研究和临床应用中的设置。 

视频: SIVQ-LCM Protocol for the ArcturusXT Instrument

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC- and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.

The determination of the solution structure of a protein by small angle X-ray scattering (SAXS) requires monodisperse samples. Here, we present two possibilities to ensure minimal delays between sample preparation and data acquisition: online size-exclusion chromatography (SEC) and online ion-exchange chromatography (IEC).

在线体积排阻和离子交换色谱法SAXS光束线

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, ...

科研

生物化学

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). SM is a common sphingolipid composed of a phosphorylcholine and a ceramide as the hydrophilic and hydrophobic component, respectively. A number of SM species are present in mammalian cells due to a variety in the sphingoid long chain base (LCB) and an N-acyl moiety in the ceramide. In this report, we show a method of estimating the number of carbon and double bonds in a LCB and an N-acyl moiety based on their corresponding product ions in MS/MS/MS (MS3) experiments. In addition, we present a quantitative analysis method for SM using two stable isotopically labeled SM species, which facilitates determining the range used in SM quantitation. The present method will be useful in characterizing a variety of SM species in biological samples and industrial products such as cosmetics.

Here, we present a protocol to quantify and qualify each sphingomyelin species using multiple reaction monitoring and MS/MS/MS mode, respectively.

用两个稳定 Isotopically 标记鞘磷脂种鞘磷脂的定量和定性方法

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

Applying X-ray Imaging Crystal Spectroscopy for Use as a High Temperature Plasma Diagnostic

X-ray spectra provide a wealth of information on high temperature plasmas; for example electron temperature and density can be inferred from line intensity ratios. By using a Johann spectrometer viewing the plasma, it is possible to construct profiles of plasma parameters such as density, temperature, and velocity with good spatial and time resolution. However, benchmarking atomic code modeling of X-ray spectra obtained from well-diagnosed laboratory plasmas is important to justify use of such spectra to determine plasma parameters when other independent diagnostics are not available. This manuscript presents the operation of the High Resolution X-ray Crystal Imaging Spectrometer with Spatial Resolution (HIREXSR), a high wavelength resolution spatially imaging X-ray spectrometer used to view hydrogen- and helium-like ions of medium atomic number elements in a tokamak plasma. In addition, this manuscript covers a laser blow-off system that can introduce such ions to the plasma with precise timing to allow for perturbative studies of transport in the plasma.

X-ray spectra provide a wealth of information on high temperature plasmas. This manuscript presents the operation of a high wavelength resolution spatially imaging X-ray spectrometer used to view hydrogen- and helium-like ions of medium atomic number elements in a tokamak plasma.

运用X射线成像光谱水晶的使用为高温等离子体诊断

X-ray spectra provide a wealth of information on high temperature plasmas; for example electron temperature and density can be inferred from line ...

工程学

Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the results of femtosecond X-ray free electron laser (XFEL) experiments. An increasing number of experimental observations have shown that although nuclear motion can be negligible, given a short enough incident pulse duration, electronic motion cannot be ignored. The current and widely accepted models assume that although electrons undergo dynamics driven by interaction with the pulse, their motion could largely be considered &#39;random&#39;. This would then allow the supposedly incoherent contribution from the electronic motion to be treated as a continuous background signal and thus ignored. The original aim of our experiment was to precisely measure the change in intensity of individual Bragg peaks, due to X-ray induced electronic damage in a model system, crystalline C60. Contrary to this expectation, we observed that at the highest X-ray intensities, the electron dynamics in C60 were in fact highly correlated, and over sufficiently long distances that the positions of the Bragg reflections are significantly altered. This paper describes in detail the methods and protocols used for these experiments, which were conducted both at the Linac Coherent Light Source (LCLS) and the Australian Synchrotron (AS) as well as the crystallographic approaches used to analyse the data.

We describe an experiment designed to probe the electronic damage induced in nanocrystals of Buckminsterfullerene (C60) by intense, femtosecond pulses of X-rays. The experiment found that, surprisingly, rather than being stochastic, the X-ray induced electron dynamics in C60 are highly correlated, extending over hundreds of unit cells within the crystals1.

在纳米富勒的飞秒衍射实验的长程电子关联的测量

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the ...

化学

High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis

The goal of this protocol is to build a parallel high-extinction and high-resolution optical Brillouin spectrometer. Brillouin spectroscopy is a non-contact measurement method that can be used to obtain direct readouts of viscoelastic material properties. It has been a useful tool in material characterization, structural monitoring and environmental sensing. In the past, Brillouin spectroscopy has usually employed scanning Fabry-Perot etalons to perform spectral analysis. This process requires high illumination power and long acquisition times, making the technique unsuitable for biomedical applications. A recently introduced novel spectrometer overcomes this challenge by employing two VIPAs in a cross-axis configuration. This innovation enables sub-Gigahertz (GHz) resolution spectral analysis with sub-second acquisition time and illumination power within the safety limits of biological tissue. The multiple new applications facilitated by this improvement are currently being explored in biological research and clinical application.

Here we present a protocol to build a rapid Brillouin spectrometer. Cascading virtually imaged phase array (VIPA) etalons achieve a measurement speed more than 1,000 times faster than traditional scanning Fabry-Perot spectrometers. This improvement provides the means for Brillouin analysis of tissue and biomaterials at low power levels in vivo.

高速的sub-GHz光谱仪激布里渊散射分析

The goal of this protocol is to build a parallel high-extinction and high-resolution optical Brillouin spectrometer. Brillouin spectroscopy is a ...

生物工程

X-ray Beam Induced Current Measurements for Multi-Modal X-ray Microscopy of Solar Cells

X-ray beam induced current (XBIC) measurements allow mapping of&#160;the nanoscale performance of electronic devices such as solar cells. Ideally, XBIC is employed simultaneously with other techniques within a multi-modal X-ray microscopy approach. An example is given herein combining XBIC with X-ray fluorescence to enable point-by-point correlations of the electrical performance with chemical composition. For the highest signal-to-noise ratio in XBIC measurements, lock-in amplification plays a crucial role. By this approach, the X-ray beam is modulated by an optical chopper upstream of the sample. The modulated X-ray beam induced electrical signal is amplified and demodulated to the chopper frequency using a lock-in amplifier. By optimizing low-pass filter settings, modulation frequency, and amplification amplitudes, noise can efficiently be suppressed for the extraction of a clear XBIC signal. A similar setup can be used to measure the X-ray beam induced voltage (XBIV). Beyond standard XBIC/XBIV measurements, XBIC can be measured with bias light or bias voltage applied such that outdoor working conditions of solar cells can be reproduced during in-situ and operando measurements. Ultimately, the multi-modal and multi-dimensional evaluation of electronic devices at the nanoscale enables new insights into the complex dependencies between composition, structure, and performance, which is an important step towards solving the materials&#39; paradigm.

A setup for X-ray beam induced current measurements at synchrotron beamlines is described. It unveils the nanoscale performance of solar cells and extends the suite of techniques for multi-modal X-ray microscopy. From wiring to signal-to-noise optimization, it is shown how to perform state-of-the-art XBIC measurements at a hard X-ray microprobe.

太阳能电池多模态X射线显微镜的X射线光束诱导电流测量

X-ray beam induced current (XBIC) measurements allow mapping of&#160;the nanoscale performance of electronic devices such as solar cells. Ideally, XBIC is ...

Sample Preparation and Transfer Protocol for In-Vacuum Long-Wavelength Crystallography on Beamline I23 at Diamond Light Source

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, chlorine, or calcium, that are often natively present in macromolecules. This enables the direct structure solution of proteins and nucleic acids via experimental phasing without the need of additional labelling. To eliminate the significant air absorption of X-rays in this wavelength regime, these experiments are performed in a vacuum environment. Beamline I23 at Diamond Light Source, UK, is the first synchrotron instrument of its kind, designed and optimized for MX experiments in the long wavelength range towards 5 &#197;. 
To make this possible, a large vacuum vessel encloses all endstation components of the sample environment. The necessity to maintain samples at cryogenic temperatures during storage and data collection in vacuum requires the use of thermally conductive sample holders. This facilitates efficient heat removal to ensure sample cooling to approximately 50 K. The current protocol describes the procedures used for sample preparation and transfer of samples into vacuum on beamline I23. Ensuring uniformity in practices and methods already established within the macromolecular crystallography community, sample cooling to liquid nitrogen temperature can be performed in any laboratory setting equipped with standard MX tools. 
Cryogenic storage and transport of samples only require standard commercially available equipment. Specialized equipment is required for the transfer of cryogenically cooled crystals from liquid nitrogen into the vacuum endstation. Bespoke sample handling tools and a dedicated Cryogenic Transfer System (CTS) have been developed in house. Diffraction data collected on samples prepared using this protocol show excellent merging statistics, indicating that the quality of samples is unaltered during the procedure. This opens unique opportunities for in-vacuum MX in a wavelength range beyond standard synchrotron beamlines.

Here, we present a protocol for cryogenic sample preparation and transfer of crystals into the vacuum endstation on beamline I23 at Diamond Light Source, for long-wavelength macromolecular X-ray crystallography experiments.

金刚石光源下光束线I23真空长波长晶体学的样品制备和转移实验方案

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, ...

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 ...

Quantitative Atomic-Site Analysis of Functional Dopants/Point Defects in Crystalline Materials by Electron-Channeling-Enhanced Microanalysis

A novel elemental and chemical analysis scheme based on electron-channeling phenomena in crystalline materials is introduced, where the incident high-energy electron beam is rocked with the submicrometric pivot point fixed on a specimen. This method enables us to quantitatively derive the site occupancies and site-dependent chemical information of impurities or intentionally doped functional elements in a specimen, using energy-dispersive X-ray spectroscopy and electron energy-loss spectroscopy attached to a scanning transmission electron microscope, which is of significant interest to current materials science, particularly related to nanotechnologies. This scheme is applicable to any combination of elements even when the conventional Rietveld analysis by X-ray or neutron diffraction occasionally fails to provide the desired results because of limited sample sizes and close scattering factors of neighboring elements in the periodic table. In this methodological article, we demonstrate the basic experimental procedure and analysis method of the present beam-rocking microanalysis.

We provide a general outline of quantitative microanalysis methods for estimating the site occupancies of impurities and their chemical states by taking advantage of electron-channeling phenomena under incident electron beam-rocking conditions, which reliably extract information from minority species, light elements, oxygen vacancies, and other point/line/planar defects.

通过电子引导增强微分析对晶体材料中功能性多裤/点缺陷的定量原子场分析

A novel elemental and chemical analysis scheme based on electron-channeling phenomena in crystalline materials is introduced, where the incident ...

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass spectrometry (MS) is a popular tool but is not always sufficient to completely unveil all structural features. For example, although carbohydrates are biologically relevant, their characterization is complicated by numerous levels of isomerism. Ion mobility spectrometry (IMS) is an interesting complement because it is sensitive to ion conformations and, thus, to isomerism. 
Furthermore, recent advances have significantly improved the technique: the last generation of Cyclic IMS instruments offers additional capabilities compared to linear IMS instruments, such as an increased resolving power or the possibility to perform tandem ion mobility (IMS/IMS) experiments. During IMS/IMS, an ion is selected based on its ion mobility, fragmented, and reanalyzed to obtain ion mobility information about its fragments. Recent work showed that the mobility profiles of the fragments contained in such IMS/IMS data can act as a fingerprint of a particular glycan and can be used in a molecular networking strategy to organize glycomics datasets in a structurally relevant way. 
The goal of this protocol is thus to describe how to generate IMS/IMS data, from sample preparation to the final Collision Cross Section (CCS) calibration of the ion mobility dimension that yields reproducible spectra. Taking the example of one representative glycan, this protocol will show how to build an IMS/IMS control sequence on a Cyclic IMS instrument, how to account for this control sequence to translate IMS arrival time into drift time (i.e., the effective separation time applied to the ions), and how to extract the relevant mobility information from the raw data. This protocol is designed to clearly explain the critical points of an IMS/IMS experiment and thus help new Cyclic IMS users perform straightforward and reproducible acquisitions.

Ion mobility spectrometry (IMS) is an interesting complement to mass spectrometry for the characterization of biomolecules, notably because it is sensitive to isomerism. This protocol describes a tandem IMS (IMS/IMS) experiment, which allows the isolation of a molecule and the generation of the mobility profiles of its fragments.