我们感谢临床 Hemostaseology 和输血医学研究所提供献血者的血液;卡门 Hässig 和科拉恩维尔·霍查为优秀的技术帮助。我们感谢 Rettig (萨尔大学) 的修改 pMAX 矢量, 罗兰 Wedlich-Söldner (Muenster 大学) 为原 LifeAct-红宝石结构, 和基督教勇克 (萨尔大学), 以产生 LifeAct-mEGFP 结构。该项目由 Sonderforschungsbereich 1027 (项目 A2 到 B.Q.) 和 894 (项目 A1 M.H.) 提供资金。光片显微镜由 DFG (广州: 256/4 19-1 FUGG) 资助。

为这项研究进行的研究与人的材料 (白细胞减少系统房间从人血液捐赠者) 由地方道德委员会授权 (声明从 16.4. 2015 (84/15;Rettig 博士教授)), 并遵循相应的指导方针。
1. 中性胶原蛋白溶液的制备 (500 µL)
<ol><li>将400µL 冷冻胶原蛋白溶液 (10.4 毫克/毫升) 转移到细胞培养罩下的无菌1.5 毫升管。慢慢加入50µL 冷冻 10x PBS (pH 7.0-7.3) 到400µL 的冷冻胶原蛋白溶液。通过轻轻地平铺管子来混合溶液。 注意: 1 部分的所有步骤都应该在细胞培养罩下完成。</li>
	<li>添加8µL 0.1 米氢氧化钠成500µL 胶原溶液从1.1。将 pH 值调整为 7.2-7.6。使用 ph 值测试条 (ph 范围: 6-10) 来确定混合物的酸碱度。 注: 不同批次胶原蛋白的体积可能有所不同。氢氧化钠溶液必须缓慢混合以避免气泡。该混合物应保持在冰上, 以避免胶原凝胶。</li>
	<li>添加2µL 的无菌 ddH2O 使最终体积为500µL. 拌匀, 并将此胶原蛋白溶液 (8.32 毫克/毫升) 储存在冰上或在4°c, 直到进一步使用。 注: 在这种情况下, 中性胶原蛋白可用于24小时整除数不建议避免气泡。</li>
</ol>2. 用毛细血管制备光片荧光显微镜样品
<ol><li>荧光用所需的主要荧光染料15或荧光蛋白11 , 如前所述, 将感兴趣的活细胞标记出来。</li>
	<li>将 1 x 106的细胞移植到细胞培养罩下的无菌1.5 毫升管中。离心管在 200 x g 8 分钟, 在200µL 培养基中丢弃上清和并用重悬颗粒。 注: 建议细胞密度为 5 x 106细胞/毫升的可视化人类免疫细胞迁移, 特别是对细胞毒性 T 淋巴细胞 (CTL) 和自然杀伤 (NK) 细胞。</li>
	<li>从1.3 中添加85.9 µL 的中性胶原溶液。入细胞悬浮从2.2。并适当混合以达到2.5 毫克/毫升的胶原蛋白浓度。把细胞/胶原蛋白混合在冰盖上。 注: 假设所需的胶原蛋白浓度为 N 毫克/毫升, 中和的胶原蛋白溶液体积 (200 µL 细胞悬浮) = 200 x N/(8.32-n)</li>
	<li>接下来, 将匹配的柱塞放入毛细管 (内径 ~ 1 毫米), 直到柱塞是1毫米的毛细管。将柱塞浸入培养基中 (图 1A)。 注意: 这一步可以帮助防止气泡时, 毛细血管被浸入细胞/胶原蛋白混合。毛细管和柱塞不必是无菌的。</li>
	<li>将毛细血管浸入细胞/胶原蛋白混合物中, 从2.3。慢慢拉回活塞 10-20 毫米 (图 1B)。用70% 乙醇喷雾湿润的纸巾擦拭毛细管的外壁, 除去剩下的胶原蛋白溶液。</li>
	<li>安装在5毫升管内壁上的粘土的毛细管, 并推动细胞/胶原蛋白混合到毛细管的边缘 (图 1C)。</li>
	<li>保持毛细管在37°c 与 5% CO2 1 h 为胶原聚合。</li>
	<li>将培养基 (大约 1-2 毫升) 添加到5毫升管中。仔细按聚合的胶原棒出到中等到约3/4 的胶原蛋白挂在培养基 (图 1D)。</li>
	<li>保持毛细血管, 像这样, 在37°c 与 5% CO2为另30分钟平衡胶原棒与培养基。 注: 之后, 胶原棒可以拉回毛细血管和进一步培养之前进一步使用。</li>
</ol>3. 使用光片显微镜进行图像采集
<ol><li>根据制造商的指示装配样品室。</li>
	<li>打开孵化和显微镜将样品室加热到37摄氏度 (仅用于活细胞成像)。</li>
	<li>将毛细管放在样品室中, 找到样品, 找到感兴趣的区域进行图像采集。</li>
	<li>激活相应的激光器。设置以下设置: 激光功率、曝光时间、z 堆栈的步长、z 堆栈的开始和结束位置以及活细胞成像的时间间隔。 注: 例如,图 2中的样本每四十年代成像6小时, 37 摄氏度, 步长为1µm (总厚度: 538 µm)。激光功率为 1%, 曝光时间为30毫秒. x y 方向的像素大小为0.23 µm。</li>
	<li>开始图像采集。</li>
</ol>4. 自动跟踪分析
<ol><li>打开文件转换器, 单击 "添加文件" 以选择要转换为软件文件格式 (*. ims) 的图像文件。单击 "浏览" 并选择一个文件夹以保存转换后的文件。单击 "全部启动"。</li>
	<li>打开分析软件。单击 "超越"。转到 "文件", 然后单击 "打开", 然后选择要分析的图像文件。 注意: 如果文件大小很大, 此步骤可能需要时间。对于单个实验, 建议不要使用大于 1 tb (tb) 的文件, 因为此类大型文件的计算能力要求很高。</li>
	<li>单击 "添加新点"。最后检查框过程整个图像。单击 "下一步"。</li>
	<li>输入 x 和 y (像素) 的坐标以定义感兴趣的区域。输入要分析的帧数 (时间和 z 位置)。单击 "下一步"。</li>
	<li>在源通道的下拉列表中, 选择包含要跟踪的对象的目标通道。输入估计的 xy 直径(µm)。单击 "下一步"。 注意: 估计 xy 直径是要跟踪的对象的 x y 维度的平均直径。</li>
	<li>单击 "质量"。设置一个阈值, 其中大多数单元格 (对象) 应包括在内。单击 "下一步"。</li>
	<li>选择所需的算法 (推荐自回归运动)。输入最大距离 (建议使用20 µm ), 最大间隙大小 (建议为3或 2)。最后单击 "处理整个图像"。 注意:最大距离和最大间隙大小是两个中断轨道的阈值。更具体地说, 在两个连续帧中, 当同一对象之间的距离超过最大距离时, 后面框架中的此对象将被视为新对象。有时在获取过程中, 同一对象可能会消失几个帧并再次出现。在这种情况下, 只有当此对象在最大间隙大小中重新出现时, 它才会被视为同一个对象。</li>
	<li>单击 "筛选器类型", 然后选择排除不想要的曲目的选项。 注意: 此步骤是可选的。</li>
	<li>单击 "下一步", 然后单击 "完成"。 注意: 根据计算性能的不同, 此步骤可能需要数小时的时间。</li>
	<li>单击 "编辑曲目" 并选择 "正确漂移"。选择适当的算法 (建议平移漂移)。选择所需的结果数据集大小(建议使用与当前大小相等的新大小)。单击"确定"。 注意: 只有当胶原蛋白在图像采集过程中漂移时, 才需要这个步骤。</li>
	<li>单击 "统计信息", 然后选择 "配置可见统计值列表"。检查要导出的兴趣选项 (如坐标、速度等)。单击"确定"。</li>
	<li>单击 "将所有统计信息导出到文件" 并输入文件名。</li>
</ol>5. 胶原蛋白基质中细胞的固定和免疫荧光染色
<ol><li>将1000µL 4% 多聚甲醛 (在 PBS 中的粉煤灰) 转换成5毫升管在化学罩下。 注: 粉煤灰应与室温保持平衡。</li>
	<li>用聚合胶原蛋白从2.9 中浸出毛细管。成粉煤灰溶液 (约5毫米), 并安装在5毫升管内壁上的毛细管与建模粘土 (如图 1C所示)。</li>
	<li>轻轻按压活塞, 直到一半的胶原棒挂在粉煤灰溶液中 (图 1D)。在室温下保持管20分钟。</li>
	<li>拉回柱塞, 以获得毛细血管内的胶原棒。取出毛细管并丢弃粉煤灰。</li>
	<li>将毛细管装入新鲜的管子中, 加入1毫升的 PBS。请确保毛细血管浸入 PBS 中。</li>
	<li>轻轻按压活塞, 直到一半的胶原棒挂在溶液中。在内壁上装上毛细管以建模粘土。</li>
	<li>在室温下保持管5分钟。</li>
	<li>重复5.4。-5.7 又2次。</li>
	<li>拉回柱塞, 以获得毛细血管内的胶原棒。丢弃 PBS。将 1-2 毫升的阻塞/通透缓冲器 (PBS + 1-% BSA + 0.1% 非离子表面活性剂) 转移到管内, 重复5.6。 注: BSA (1%) 可替换为5% 血清的动物的第二 Ab 被提出。</li>
	<li>在室温下保持管 30-60 分钟。</li>
	<li>拉回柱塞, 以获得毛细血管内的胶原棒。丢弃通透缓冲区。转移 200-500 µL 的主要抗体在阻塞/通透缓冲和驱逐杆到解决方案。</li>
	<li>在室温下保持管1小时。</li>
	<li>用 PBST (PBS + 0.1% 非离子表面活性剂) 清洗胶原棒3倍, 如5.4 所述。-5.8。</li>
	<li>在室温下, 在阻断/通透缓冲液中培养1小时的二级抗体棒。不要光。</li>
	<li>用 PBS 洗3次胶原棒, 如5.4 所述。-5.8。</li>
	<li>拉回柱塞, 以获得毛细血管内的胶原棒。将样品保存在 PBS 中, 直到成像。</li>
	<li>扫描样品, 如3所述。</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+not+men:+differences+between+mouse+and+human+immunology.">Of mice and not men: differences between mouse and human immunology.</a> J Immunol. 172 (5), 2731-2738 (2004).</li></ol>

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大多数的体外检测是在2D 表面进行的, 例如细胞培养板、培养皿或盖玻片, 而在体内细胞, 特别是免疫细胞中, 大多经历了3D 微环境。新出现的证据显示, 免疫细胞的迁移模式在2D 和3D 方案17之间有所不同。此外, 肿瘤细胞的表达谱也不同于2D 和 3 d 培养组织18,19,20。因此, 为了更好地模拟体外?...

大多数关于迁移细胞的知识来自2D 实验1,2,3, 通常是在玻璃或塑料表面进行的一种文化/成像皿。然而, 在大多数情况下, 生理场景需要3D 微环境, 其中胞外基质 (ECM) 起决定性作用。ECM 不仅提供了维持适当细胞形态所必需的3D 结构, 而且还为许多细胞4、5的最佳功能提供了生存信号或方向性提示。因此, 需要一个3D 的环境来更好地识别细胞的功能和行为在一个更好地反映生理环境的环境中。
在人体内, 大多数细胞特别是免疫细胞, 在3D 的情况下发挥作用。例如, 活化 t 细胞巡视组织寻找靶细胞, 天真的 t 细胞通过淋巴结迁移, 寻找其同源抗原呈现细胞, 在此期间, 迁移模式和机械适应相应的胞外环境3,6,7。3D 胶原凝胶已被广泛应用为一种成熟、特征良好的3D 细胞培养系统,8、9、10。我们以前的研究表明, 主要的人类淋巴细胞是高度流动的, 以平均速度迁移, 在0.25% 的胶原基基质11中, µm/分钟大约4.8。细胞骨架的重排在移植12中起关键作用。积累的证据表明, 淋巴细胞不应用只有单一的迁移模式, 但可以切换到特定的迁移行为, 取决于位置, 微环境, 细胞因子, 趋化梯度, 和细胞外信号, 调节迁移行为以不同的方式3。
为了可靠地分析免疫细胞的功能和行为, 例如, 迁移, 突起形成或水泡运输, 能够快速可靠地获取相对较大的3D 卷中的图像是很有好处的。对于3D 成像, 最近开发的光片显微技术 (也称为单平面照明显微镜) 提供了一个满意的解决方案13,14。在成像采集过程中, 生成一个薄的静态光片来照亮样品。这样, 在焦点平面上, 可以同时照亮大面积而不影响离平面单元。此功能使高采集速度与大幅降低漂白和 photocytotoxicity。本文介绍了如何使用光片显微镜对人体免疫细胞进行可视化, 以及如何分析3D 场景中的迁移。

<table><tbody><tr><td>Fibricol, bovine collagen solution</td><td>Advanced Biomatrix</td><td>&amp;nbsp;#5133-20ML</td><td>Collagen matrix</td></tr><tr><td>0.5 M NaOH Solution</td><td>Merck</td><td>1091381000</td><td>for neutralizing Fibricol solution</td></tr><tr><td>Ultra-Low melting agarose</td><td>Affymetrix</td><td>32821-10GM</td><td>Sample preparation in low c[Col]</td></tr><tr><td>Dynabeads Untouched Human CD8 T Cells Kit</td><td>Thermo Fisher</td><td>11348D</td><td>Isolation of primary human CD8&lt;sup&gt;+&lt;/sup&gt; T cells from PBMC</td></tr><tr><td>Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation</td><td>Thermo Fisher</td><td>11132D</td><td>Activation of CTL populations</td></tr><tr><td>Human recombinant interleukin-2</td><td>Thermo Fisher</td><td>PHC0023</td><td>Stimulation of cultured CTL</td></tr><tr><td>P3 Primary solution kit</td><td>Lonza</td><td>V4XP-30XX</td><td>Transfection</td></tr><tr><td>&amp;alpha;-PFN1 antibody, rabbit, IgG</td><td>Abcam</td><td>ab124904</td><td>IF</td></tr><tr><td>Alexa Fluor 633 Phalloidin</td><td>Thermo Fisher</td><td>A22284</td><td>IF</td></tr><tr><td>CellMask Orange Plasma membrane Stain</td><td>Thermo Fisher</td><td>C10045</td><td>Fluorescent cell label</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P1379-250mL</td><td>IF</td></tr><tr><td>Triton X-100</td><td>Eurobio</td><td>018774</td><td>IF</td></tr><tr><td>DPBS Dulbecco&#039;s phopsphate buffered saline</td><td>Thermo Fisher</td><td>14190250</td><td>IF</td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A9418-100G</td><td>IF</td></tr><tr><td>Goat &amp;alpha; Rabbit 568, IgG, rabbit</td><td>Thermo Fisher</td><td>A-11011</td><td>IF</td></tr><tr><td>Lightsheet Z.1 (Light-sheet microscopy)</td><td>Zeiss</td><td>N.A.</td><td></td></tr><tr><td>Cell culture hood</td><td>Thermo Fisher</td><td>HeraSafe KS</td><td></td></tr><tr><td>Cell culture incubator HERACell 150i&amp;nbsp;</td><td>Thermo Fisher</td><td>N.A.</td><td></td></tr><tr><td>Centrifuge 5418 and 5452</td><td>Eppendorf</td><td>N.A.</td><td></td></tr><tr><td>Pippettes</td><td>Eppendorf</td><td>3123000039, 3123000020, 3123000063</td><td></td></tr><tr><td>Pippette tips</td><td>VWR</td><td>89079-444, 89079-436, 89079-452&amp;nbsp;</td><td></td></tr><tr><td>15 mL tubes</td><td>Sarstedt</td><td>&amp;nbsp;62.554.002</td><td></td></tr><tr><td>Capillaries 50 &amp;micro;L</td><td>VWR (Brand)</td><td>613-3373</td><td>Zeiss LSFM sample preparation</td></tr><tr><td>Plunger for capillaries</td><td>VWR (Brand)</td><td>BRND701934</td><td>&quot;Stamps with Teflon tip&quot; LSFM sample preparation</td></tr><tr><td>MColorPhast pH stips</td><td>Merck</td><td>1095430001</td><td>to test pH of neutralized Fibricol</td></tr><tr><td>BD Plastipak 1mL syringes</td><td>BD</td><td>Z230723&amp;nbsp;ALDRICH</td><td>Alternative sample preparation</td></tr><tr><td>Modeling clay (Hasbro Play-Doh A5417EU7)</td><td>Play-Doh</td><td>N.A.</td><td></td></tr><tr><td>Imaris file converter</td><td>Bitplane</td><td>available at http://www.bitplane.com&amp;nbsp;</td><td>Convert imaging files to Imaris file format</td></tr><tr><td>Imaris 8.1.2 (MeasurementPro, Track, Vantage)</td><td>Bitplane</td><td>available at http://www.bitplane.com&amp;nbsp;</td><td>Analysis of 3D and 4D imaging data</td></tr></tbody></table>

light-sheet microscopy for three-dimensional visualization of human immune cells

在体内, 免疫细胞的活化、增殖和功能都发生在三维 (3D) 环境中, 例如在淋巴结或组织中。迄今为止, 大多数体外系统依赖于二维 (2D) 表面, 如细胞培养板或盖玻片。为了最佳地模拟体外生理条件, 我们利用一个简单的3D 胶原基质。胶原蛋白是细胞外基质 (ECM) 的主要成分之一, 被广泛用于构成3D 矩阵。对于3D 成像, 最近开发的光片显微技术 (也称为单平面光照显微镜) 具有采集速度快、穿透深度大、漂白低、photocytotoxicity 等特点。此外, 光片显微镜对长期测量特别有利。在这里, 我们描述了一个优化的协议如何建立和处理人类免疫细胞,例如, 在3D 胶原基质中的主要人细胞毒性 T 淋巴细胞 (CTL) 和自然杀伤细胞 (NK), 用于活细胞成像的光片显微镜和固定样本。给出了图像采集和细胞迁移分析的步骤。特别着重强调了取样准备和数据分析的关键步骤和因素。该协议可用于其他类型的悬浮细胞在3D 胶原基质和不限于免疫细胞。

T 细胞移出过程中的突起形成是一个高度动态的过程, 是肌动蛋白依赖。为了可视化第一人 ctl 的突起形成, 我们瞬时转染了 mEGFP 融合蛋白, 以标记在 CTL 的肌动蛋白细胞骨架, 如前所述11。在转染后的某一天, 细胞被嵌入胶原基质中。图像栈是每四十年代获得的步骤大小1µm 在37°c 使用光片显微镜。如图 2A和辅助电影 1, 在?...

Watch this Scientific Journal Video about Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells at JoVE.com

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

三维人体免疫细胞可视化的光片显微术

在这里, 我们提出了一个协议, 以可视化的免疫细胞嵌入在三维 (3D) 胶原基质中使用光片显微镜。该协议还阐述了如何在3D 中跟踪单元迁移。该协议可用于3D 矩阵中其他类型的悬浮单元。

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or ...

light-sheet-microscopy-for-three-dimensional-visualization-human

Research

JoVE Journal

Immunology and Infection

8.0K 次观看.  Saarland University. 在这里, 我们提出了一个协议, 以可视化的免疫细胞嵌入在三维 (3D) 胶原基质中使用光片显微镜。该协议还阐述了如何在3D 中跟踪单元迁移。该协议可用于3D 矩阵中其他类型的悬浮单元。

视频: Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope

A module for light sheet or single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and optimized for 3-dimensional cell cultures, e.g., multi-cellular tumor spheroids (MCTS). The SPIM excitation module shapes and deflects the light such that the sample is illuminated by a light sheet perpendicular to the detection path of the microscope. The system is characterized by use of a rectangular capillary for holding (and in an advanced version also by a micro-capillary approach for rotating) the samples, by synchronous adjustment of the illuminating light sheet and the objective lens used for fluorescence detection as well as by adaptation of a microfluidic system for application of fluorescent dyes, pharmaceutical agents or drugs in small quantities. A protocol for working with this system is given, and some technical details are reported. Representative results include (1) measurements of the uptake of a cytostatic drug (doxorubicin) and its partial conversion to a degradation product, (2) redox measurements by use of a genetically encoded glutathione sensor upon addition of an oxidizing agent, and (3) initiation and labeling of cell necrosis upon inhibition of the mitochondrial respiratory chain. Differences and advantages of the present SPIM module in comparison with existing systems are discussed.

A module for single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and optimized for 3-dimensional cell cultures. The sample is located within a rectangular capillary, and via a microfluidic system fluorescent dyes, pharmaceutical agents or drugs can be applied in small quantities.

单一平面照明模块和一个宽场显微镜微毛细管法

A module for light sheet or single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and ...

科研

工程学

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

生物化学

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

生发中心的高度已解决活体条纹照明显微镜

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

免疫与感染

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

光片荧光显微术在小鼠心脏研究中的应用

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

生物工程

Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification of lymphocyte subtypes is important for the study of immunology as well as diagnosis and treatment of various diseases. Currently, standard methods for classifying lymphocyte types rely on labeling specific membrane proteins via antigen-antibody reactions. However, these labeling techniques carry the potential risks of altering cellular functions. The protocol described here overcomes these challenges by exploiting intrinsic optical contrasts measured by 3D quantitative phase imaging and a machine learning algorithm. Measurement of 3D refractive index (RI) tomograms of lymphocytes provides quantitative information about 3D morphology and phenotypes of individual cells. The biophysical parameters extracted from the measured 3D RI tomograms are then quantitatively analyzed with a machine learning algorithm, enabling label-free identification of lymphocyte types at a single-cell level. We measure the 3D RI tomograms of B, CD4+ T, and CD8+ T lymphocytes and identified their cell types with over 80% accuracy. In this protocol, we describe the detailed steps for lymphocyte isolation, 3D quantitative phase imaging, and machine learning for identifying lymphocyte types.

We describe a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and a machine learning algorithm. Measurements of 3D refractive index tomograms of lymphocytes present 3D morphological and biochemical information for individual cells, which is then analyzed with a machine-learning algorithm for identification of cell types.

用三维定量相位成像和机器学习技术对淋巴细胞亚型进行无标签鉴定

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

医学

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

3D Visualization of Immune Cell Populations in HIV-Infected Tissues via Clearing, Immunostaining, Confocal, and Light Sheet Fluorescence Microscopy

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 million individuals infected worldwide and no widely accessible cure. Despite intensive efforts, a detailed understanding of virus and host cell interactions in tissues during infection and in response to therapy remains incomplete. To address these limitations, water-based tissue clearing techniques CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) and CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/in situ-hybridization-compatible Tissue hYdrogel) are applied to visualize complex virus host-cell interactions in HIV-infected tissues from animal models and humans using confocal and light sheet fluorescence microscopy. Optical sectioning of intact tissues and image analysis allows rapid reconstruction of spatial information contained within whole tissues and quantification of immune cell populations during infection. These methods are applicable to most tissue sources and diverse biological questions, including infectious disease and cancer.

Tissue clearing, combined with immunofluorescence microscopy, allows spatial visualization and quantification of immune-cell populations and virus proteins within intact tissues. Optical sectioning of cleared tissues with confocal and light sheet fluorescence microscopy can generate 3D models of complex tissue environments and reveal spatial heterogeneity exhibited during HIV infection.

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 ...

生物学

可定制的荧光显微镜，用于可视化细胞骨架网络

Design and Building of a Customizable, Single-Objective, Light-Sheet Fluorescence Microscope for the Visualization of Cytoskeleton Networks

Reconstituted cytoskeleton composites have emerged as a valuable model system for studying non-equilibrium soft matter. The faithful capture of the dynamics of these 3D, dense networks calls for optical sectioning, which is often associated with fluorescence confocal microscopes. However, recent developments in light-sheet fluorescence microscopy (LSFM) have established it as a cost-effective and, at times, superior alternative. To make LSFM accessible to cytoskeleton researchers less familiar with optics, we present a step-by-step beginner's guide to building a versatile light-sheet fluorescence microscope from off-the-shelf components. To enable sample mounting with traditional slide samples, this LSFM follows the single-objective light-sheet (SOLS) design, which utilizes a single objective for both the excitation and emission collection. We describe the function of each component of the SOLS in sufficient detail to allow readers to modify the instrumentation and design it to fit their specific needs. Finally, we demonstrate the use of this custom SOLS instrument by visualizing asters in kinesin-driven microtubule networks.

作者聚焦:通过光片显微镜推进远非平衡材料知识

This protocol describes in detail how to build a single-objective, light-sheet fluorescence microscope and its usage for visualizing cytoskeleton networks.

设计和构建用于细胞骨架网络可视化的可定制、单物镜、光片荧光显微镜

Reconstituted cytoskeleton composites have emerged as a valuable model system for studying non-equilibrium soft matter. The faithful capture of the ...

三维人体免疫细胞可视化的光片显微术

摘要

探索更多视频

此视频中的章节

单一平面照明模块和一个宽场显微镜微毛细管法

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

生发中心的高度已解决活体条纹照明显微镜

光片荧光显微术在小鼠心脏研究中的应用

用三维定量相位成像和机器学习技术对淋巴细胞亚型进行无标签鉴定

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

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