Name: measuring the confluence of ipscs using an automated imaging system
Uploaded: 2020-06-10T13:30:00.000+00:00
Duration: 11 min 39 s
Description: Watch this Scientific Journal Video about Measuring the Confluence of iPSCs Using an Automated Imaging System at JoVE.com

该研究得到了Bambino Gesù基金会和Ricerca Corrente（意大利卫生部）对C.C.的资助。 我们要感谢Enrico Bertini博士（神经科学系，神经肌肉和神经退行性疾病部门，分子医学实验室，Bambino Gesù儿童研究医院），Stefania Petrini博士（共聚焦显微镜核心设施，研究实验室，Bambino Gesù儿童研究医院），Giulia Pericoli（肿瘤血液学，基因和细胞治疗科，儿童研究医院Bambino Gesù） Roberta Ferretti（Bambino Gesù儿童研究医院肿瘤血液学、基因和细胞治疗科）进行科学讨论和技术帮助。玛丽亚·芬奇是"英国癌症儿童奖学金"的获得者。

1. 涂覆 96 孔板
注意:在同一板中测试不同的涂层，但不同的孔（见 补充文件）。
<ol><li>在DMEM中以1:100稀释基质胶。向96孔板中每孔加入100μL，并在室温下孵育1小时。之后，除去溶液并用 100 μL DMEM 洗涤孔两次。</li>
	<li>在PBS（含钙和镁）中稀释层粘连蛋白（20μg/ mL，LN-521）。向孔中加入100μL并在4°C下孵育过夜。第二天在接种细胞之前用DMEM进行两次洗涤。</li>
	<li>在稀释缓冲液中稀释玻连蛋白（10μg/mL）。向 96 孔板中每孔加入 100 μL，并在室温下孵育 1 小时。在电镀细胞之前，用PBS（不含钙和镁）清洗孔。</li>
	<li>在ddH2O中稀释HU-纤连蛋白（30μg/ mL），向孔中加入100μL并在室温下孵育45分钟。之后，在接种细胞之前用培养基清洗孔。</li>
</ol>2. 培养中iPSCs的维持
注意:iPSC 是商业购买的。iPSCs来源于健康的人成纤维细胞，并使用游离体技术重新编程。
<ol><li>从-80°C冰箱或液氮中，在37°C水浴中解冻冷冻保存的iPSC。在将其移入生物安全柜之前，用70%乙醇清洁装有细胞的小瓶。</li>
	<li>用 1000 μL 移液器在 15 mL 无菌锥形管中逐滴将细胞悬液加入 5 mL 预热的细胞培养基（例如 mTeSR1）中。</li>
	<li>在室温（RT）下以304× g 离心细胞5分钟。</li>
	<li>取出培养基并将细胞沉淀重悬于 4 mL 细胞培养基中。</li>
	<li>将细胞悬液接种到 6 孔板的两个孔中（每个 6 孔细胞培养皿 105 个 iPSC），其中小鼠胚胎成纤维细胞 （MEF） 先前已接种。在DMEM中以2.4 x 104 / cm2 的密度接种iPSCs前两天播种MEF（含有10%胎牛血清，1%L-谷氨酰胺和1%青霉素 - 链霉素）。</li>
	<li>接种后，用10μM的ROCK抑制剂Y-27632补充细胞培养基。</li>
	<li>在 MEF 上培养 iPSC 前 4-5 周，然后在无饲养层条件下（无 MEF 条件），使用 mTeSR1 中感兴趣的包衣之一（参见步骤 1 和 表 1）。</li>
	<li>当 iPSC 汇合 70-80% 时，使用 0.5 mM EDTA 处理在室温下 1:4 传代 3-5 分钟。 为 6 孔板添加 1 mL 的 0.5 mM EDTA（或其他类型的板按比例）。在无饲养器条件下转移到新孔中，并在37°C，5%CO 2，20%O2下孵育。</li>
	<li>每天用新鲜的mTeSR1更换培养基，每2天分裂一次细胞。</li>
</ol>3. 细胞汇合的表征
<ol><li>使用96孔板进行实验。</li>
	<li>在无饲养层条件下培养至少 1 个月后，每孔接种 10，000 个细胞，以确保 MEF 未传代。使用一次性计数玻片用光学显微镜对细胞进行计数。</li>
	<li>一式三份进行实验。因此，在三个孔中测试每个播种条件。</li>
	<li>从接种后的第1天开始，在明场模式下使用细胞仪进行自动图像采集。每24小时进行一次自动图像采集，持续5天。有关实验参数的详细信息，请参阅 补充文件。</li>
	<li>使用自动对比和自动曝光来更好地可视化细胞。</li>
	<li>设置汇合分析的分析设置（参见 补充文件），以应用每孔60%、80%和100%的掩模，以评估由于孔边界的光折射引起的焦点变化。使用上面提到的不同掩模分析设置来分析每个时间点的细胞汇合度。</li>
</ol>4. 统计分析
<ol><li>将定量结果报告为平均值的标准误差 （SEM） ±平均值。</li>
	<li>为了比较不同涂层条件的总体差异，使用相同的样本获取数据并执行学生配对样本t检验。小于 0.05 的 P 值被认为具有统计显著性，并且所有报告的 p 值都是双侧的。</li>
</ol>5. 细胞骨架微丝的表征
<ol><li>用4%多聚甲醛（4%PFA）在PBS中固定细胞在室温下10分钟，然后在PBS中洗涤两次（总共10分钟）。</li>
	<li>在室温下向每个孔中加入 100 μL 封闭溶液（由 5% BSA、0.1% Triton 在 PBS 中组成）1 小时。</li>
	<li>除去封闭溶液并用PBS洗涤样品两次10分钟。</li>
	<li>每个样品加入 100 μL 鬼笔环肽偶联工作溶液，并在室温下孵育 1 小时。</li>
	<li>用PBS洗涤细胞两次（室温下10分钟）。</li>
	<li>用Hoechst 33342染色细胞核，在PBS中稀释1:10000，在室温下10分钟。</li>
	<li>取出Hoechst溶液，用PBS洗涤细胞两次，每次10分钟。</li>
	<li>用H2O洗涤样品，并在化学罩下干燥。</li>
	<li>加入 100 μL 封片剂（即 PBS:甘油，1:1）以覆盖细胞并保留样品荧光。</li>
	<li>在配备白光激光 （WLL） 源和 405 nm 二极管激光器的激光扫描共聚焦显微镜上观察 Ex/Em 493/517 nm 处的细胞。使用HC PLAPO 40x油浸物镜采集连续共聚焦图像。对所有检查样品使用相同的激光功率、分束器、滤光片设置、针孔直径和扫描模式。</li>
</ol>

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作者没有什么可透露的。

使用iPSCs进行疾病建模和未来的药物筛选以及它们在精准医学中的可能应用使其成为一项具有重要意义的技术，因此我们认为有必要清楚地了解更类似于胚胎干细胞生理状况的体外培养条件。在这种情况下，我们使用野生型iPSCs测试了不同的ECM涂层，以了解允许细胞保持健康和未分化状态的条件。除此之外，一个关键点是在MEF和基质胶的异种成分中培养iPSC，这可能解释了一式三份之间的实验变异?...

诱导多能干细胞（iPSC）是从体细胞中获得的，可以分化成不同的细胞类型。它们通常被用作模拟疾病发病机制或进行药物筛选的系统，并且还提供了用于个性化医疗的潜力。由于iPSCs具有巨大的潜力，因此充分表征它们以用作可靠的模型系统非常重要。我们之前展示了在缺氧环境中生长iPSCs的重要性，因为这些细胞依赖于糖酵解，而有氧环境会导致氧化还原失衡1。iPSCs也容易受到其他培养条件的影响，特别是细胞外环境。优化培养条件是保持其健康和增殖的关键问题。健康的iPSC培养将产生健康的分化细胞，这些分化细胞通常是用于了解特定人类疾病或细胞过程的分子，细胞和功能特征的模型的终点。
在这项研究中，已经使用一个简单的方案来测试在不同孔中使用不同包衣条件的iPSCs的汇合度。iPSCs需要鼠胚胎成纤维细胞（MEF）的饲养层才能正确附着，但是iPSCs和MEF的共存使得难以进行RNA或蛋白质提取等分析，因为存在两个细胞群。为了避免饲养层，属于细胞外基质（ECM）的不同蛋白质已被用于重建天然细胞生态位并具有无饲养层iPSC培养物。特别是，Matrigel是从Engelbreth-Holm-Swarm（EHS）小鼠肉瘤中提取的可溶基底膜制剂，其富含细胞外基质蛋白（即层粘连蛋白，胶原蛋白IV，硫酸乙酰肝素蛋白聚糖，entactin/nidogen和生长因子）2，3。其他使用的包被条件是纯化的蛋白质，这些蛋白质在构建ECM中具有已知的相关性:已知层粘连蛋白-521由胚胎内部细胞团中的人多能干细胞（hPSCs）分泌，它是出生后体内最常见的层粘连蛋白之一4，5，6，7，8，9， 10，11;玻连蛋白是一种无异种细胞培养基质，已知可支持hPSC12，13，14，15，16的生长和分化;纤连蛋白是一种ECM蛋白，对脊椎动物发育以及胚胎干细胞在多能状态下的附着和维持很重要17，18，19，20，21，22，23，24，25。由于存在不同的包被条件，我们根据它们对iPSC汇合的影响来比较它们。

<table><tbody><tr><td>10 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td><td>Corning</td><td>4488</td><td>Tool</td></tr><tr><td>15 mL high-clarity polypropylene (PP) conical centrifuge tubes</td><td>Falcon</td><td>352097</td><td>Tool</td></tr><tr><td>1x PBS (With Ca&lt;sup&gt;2+&lt;/sup&gt;; Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Thermofisher</td><td>14040133</td><td>Medium</td></tr><tr><td>1x PBS (without Ca&lt;sup&gt;2+&lt;/sup&gt;; Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Euroclone</td><td>ECB4004L</td><td>Medium</td></tr><tr><td>5 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td><td>Corning</td><td>4487</td><td>Tool</td></tr><tr><td>Cell culture microplate, 96 WELL, PS, F-Bottom</td><td>Greiner Bio One</td><td>655090</td><td>Support</td></tr><tr><td>Cell culture plate, 6 well</td><td>Costar</td><td>3516</td><td>Support</td></tr><tr><td>DMEM (Dulbecco&#039;s Modified Eagle&#039;s Medium- high glucose)</td><td>Sigma</td><td>D5671</td><td>Medium</td></tr><tr><td>EDTA</td><td>Sigma</td><td>ED4SS-500g</td><td>Reagent</td></tr><tr><td>Epi Episomal iPSC Reprogramming Kit</td><td>Invitrogen</td><td>A15960</td><td>Reagent</td></tr><tr><td>FAST - READ 102</td><td>Biosigma</td><td>BVS100</td><td>Tool</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>10270106</td><td>Medium</td></tr><tr><td>Fibronectin</td><td>Merck</td><td>FC010</td><td>Coating</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td>Reagent</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td>MILLIQ</td><td></td></tr><tr><td>Hoechst</td><td>Thermofisher</td><td>33342</td><td>Reagent</td></tr><tr><td>Laminin 521</td><td>Stem Cell Technologies</td><td>77003</td><td>Coating</td></tr><tr><td>L-Glutamine (200 mM)</td><td>Gibco</td><td>LS25030081</td><td>Reagent</td></tr><tr><td>Matrigel</td><td>Corning Matrigel hESC-Qualified Matrix</td><td>354277</td><td>Coating</td></tr><tr><td>Mouse embryonic fibroblasts (MEF)</td><td>Life Technologies</td><td>A24903</td><td>Coating</td></tr><tr><td>MTESR1 Medium</td><td>Stem Cell Technologies</td><td>85851</td><td>Medium</td></tr><tr><td>MTESR1 Supplement</td><td>Stem Cell Technologies</td><td>85852</td><td>Medium</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Gibco</td><td>15140122</td><td>Reagent</td></tr><tr><td>Phalloidin</td><td>Sigma</td><td>P1951</td><td>Reagent</td></tr><tr><td>Vitronectin</td><td>Stem Cell Technologies</td><td>7180</td><td>Coating</td></tr><tr><td>Y-27632</td><td>Sigma</td><td>Y0503</td><td>Reagent</td></tr></tbody></table>

measuring the confluence of ipscs using an automated imaging system

本研究的重点是了解在不同ECM包衣底物上生长iPSC如何影响细胞汇合。已经建立了实时评估iPSC汇合的协议，而无需对单细胞悬液中的细胞进行计数，以避免任何生长扰动。使用高内涵图像分析系统以自动方式评估 4 种不同 ECM 上的 iPCS 汇合度。使用不同的分析设置来评估贴壁iPSC的细胞汇合度，并且仅观察到使用60%、80%或100%面膜的微小差异（在层粘连蛋白24和48小时）。我们还表明，与基质胶，玻连蛋白和纤连蛋白相比，层粘连蛋白导致最佳汇合。

在这项研究中，我们研究了在不同包衣条件下生长时的iPSCs汇合。使用细胞仪，我们能够在5天内获得一式三份的简单信息结果。由于iPSCs几乎不附着在塑料容器上，并且需要涂层来支持其增殖，因此我们决定监测人iPSC的汇合，因为它表明细胞培养物的健康状况，并可能反映其分化潜力。体外扩增后，我们将iPSCs接种在不同的ECM底物上，并通过观察在明场中获得的样品图像并使用鬼笔环肽染色（用?...

Watch this Scientific Journal Video about Measuring the Confluence of iPSCs Using an Automated Imaging System at JoVE.com

Measuring the Confluence of iPSCs Using an Automated Imaging System

该方案的目标是比较不同的细胞外基质（ECM）包被条件，以评估差异涂层如何影响诱导多能干细胞（iPSC）的生长速率。特别是，我们的目标是创造条件以获得iPSC培养物的最佳生长。

使用自动成像系统测量 iPSC 的汇合度。

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

measuring-the-confluence-of-ipscs-using-an-automated-imaging-system

Research

JoVE Journal

Biology

2.0K 次观看.  Ospedale Pediatrico Bambino Gesù, IRCCS. 该方案的目标是比较不同的细胞外基质（ECM）包被条件，以评估差异涂层如何影响诱导多能干细胞（iPSC）的生长速率。特别是，我们的目标是创造条件以获得iPSC培养物的最佳生长。

视频: Measuring the Confluence of iPSCs Using an Automated Imaging System

A Practical Guide on Coupling a Scanning Mobility Sizer and Inductively Coupled Plasma Mass Spectrometer (SMPS-ICPMS)

A large variety of analytical methods are available to characterize particles in aerosols and suspensions. The choice of the appropriate technique depends on the properties to be determined. In many fields information about particle size and chemical composition are of great importance. While in aerosol techniques particle size distributions of gas-borne particles are determined online, their elemental composition is commonly analyzed offline after an appropriate sampling and preparation procedure. To obtain both types of information online and simultaneously, a hyphenated setup was recently developed, including a Scanning Mobility Particle Sizer (SMPS) and an Inductively Coupled Plasma Mass Spectrometer (ICPMS). This allows first to classify the particles with respect to their mobility diameter, and then to determine their number concentration and elemental composition in parallel. A Rotating Disk Diluter (RDD) is used as the introduction system, giving more flexibility regarding the use of different aerosol sources. In this work, a practical guide is provided describing the different steps for establishing this instrumentation, and how to use this analysis tool. The versatility of this hyphenated technique is demonstrated in example measurements on three different aerosols generated out of a) a salt solution, b) a suspension, and c) emitted by a thermal process.

A Practical Guide on Coupling a Scanning Mobility Sizer and Inductively Coupled Plasma Mass Spectrometer SMPS-ICPMS

In this work a practical guide is provided, describing the different steps to establish the coupling of SMPS and ICPMS systems, and how to use them. Three descriptive examples are presented.

耦合扫描流动性测定仪和电感耦合等离子体质谱仪（SMPS-ICPMS）的实用指南

A large variety of analytical methods are available to characterize particles in aerosols and suspensions. The choice of the appropriate technique depends ...

科研

化学

Scanning Light Scattering Profiler (SLPS) Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

The scanning light scattering profiler (SLSP) methodology has been developed for the full-angle quantitative evaluation of forward and backward light scattering from intraocular lenses (IOLs) using goniophotometer principles. This protocol describes the SLSP platform and how it employs a 360&#176; rotational photodetector sensor that is scanned around an IOL sample while recording the intensity and location of scattered light as it passes through the IOL medium. The SLSP platform can be used to predict, non-clinically, the propensity for current and novel IOL designs and materials to induce light scatter. Non-clinical evaluation of light scattering properties of IOLs can significantly reduce the number of patient complaints related to unwanted glare, glistening, optical defects, poor image quality, and other phenomena associated with the unintended light scattering. Future studies should be conducted to correlate SLSP data with clinical results to help identify which measured light scatter is most problematic for patients that have undergone cataract surgery subsequent to IOL implantation.

Scanning Light Scattering Profiler SLPS Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

This protocol describes the scanning light scattering profiler (SLSP) that enables the full-angle quantitative evaluation of forward and backward scattering of light from intraocular lenses (IOLs) using goniophotometer principles.

基于扫描光散射分析器（SLPS）的方法来量化评估来自眼内镜的前向和后向光散射

The scanning light scattering profiler (SLSP) methodology has been developed for the full-angle quantitative evaluation of forward and backward light ...

工程学

Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living cells in real time. In this protocol, we cover the fundamental steps to realize an iSCAT microscope and complement it with additional imaging channels to monitor the viability of a cell under study. Following this, we use the method for real-time detection of single proteins as they are secreted from a living cell which we demonstrate with an immortalized B-cell line (Laz388). Necessary steps concerning the preparation of microscope and sample as well as the analysis of the recorded data are discussed. The video protocol demonstrates that iSCAT microscopy offers a straightforward method to study secretion at the single-molecule level.

We present a protocol for the real-time optical detection of single unlabeled proteins as they are secreted from living cells. This is based on interferometric scattering (iSCAT) microscopy, which can be applied to a variety of different biological systems and configurations.

通过 iscat 显微镜对活细胞分泌的单一蛋白质进行无标签成像

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

免疫与感染

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

发育生物学

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

使用一个荧光显微镜进行多种成像模式

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

生物工程

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

三维超F-微丝高分辨率显微镜通过干涉光敏定位显微镜（iPALM）

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

生物学

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

共聚焦显微镜通过图像相关谱揭示细胞表面受体聚集

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high throughput. In general, the acquisition of single-cell time courses from cultured cells is hampered by cell motility and diversity of cell shapes. Adhesive micro-arrays standardize single-cell conditions and facilitate image analysis. LISCA combines single-cell microarrays with scanning time-lapse microscopy and automated image processing. Here, we describe the experimental steps of taking single-cell fluorescence time courses in a LISCA format. We transfect cells adherent to a micropatterned array using mRNA encoding for enhanced green fluorescent protein (eGFP) and monitor the eGFP expression kinetics of hundreds of cells in parallel via scanning time-lapse microscopy. The image data stacks are automatically processed by newly developed software that integrates fluorescence intensity over selected cell contours to generate single-cell fluorescence time courses. We demonstrate that eGFP expression time courses after mRNA transfection are well described by a simple kinetic translation model that reveals expression and degradation rates of mRNA. Further applications of LISCA for event time correlations of multiple markers in the context of signaling apoptosis are discussed.

Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site.

单细胞阵列的活细胞成像 （LISCA） - 量化细胞动力学的多功能技术

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...

使用自动成像系统测量 iPSC 的汇合度。

摘要

探索更多视频

此视频中的章节

耦合扫描流动性测定仪和电感耦合等离子体质谱仪（SMPS-ICPMS）的实用指南

基于扫描光散射分析器（SLPS）的方法来量化评估来自眼内镜的前向和后向光散射

通过 iscat 显微镜对活细胞分泌的单一蛋白质进行无标签成像

在髓系细胞动态实时成像

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

使用一个荧光显微镜进行多种成像模式

三维超F-微丝高分辨率显微镜通过干涉光敏定位显微镜（iPALM）

共聚焦显微镜通过图像相关谱揭示细胞表面受体聚集

单细胞阵列的活细胞成像（LISCA） - 量化细胞动力学的多功能技术

使用自动成像系统测量 iPSC 的汇合度。

摘要

探索更多视频

此视频中的章节

耦合扫描流动性测定仪和电感耦合等离子体质谱仪（SMPS-ICPMS）的实用指南

基于扫描光散射分析器（SLPS）的方法来量化评估来自眼内镜的前向和后向光散射

通过 iscat 显微镜对活细胞分泌的单一蛋白质进行无标签成像

在髓系细胞动态实时成像

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

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