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>

提交人不申报任何金融或商业利益冲突。

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生理条件, 在3D 环境下进行实验, 例如3D 胶原基质, 具有很大的优越性。新开发的方法, 特别是光片荧光显微镜, 在受控环境条件下, 能够在3D 矩阵系统中进行可靠和可重复的实验。
与传统的激光扫描共焦显微术, 照亮整个探头, 与光片显微镜, 只有一薄的激光光被产生照亮样品。检测摄像头与照明平面垂直。由于这种技术, 只有在焦平面上的部分暴露在激光。因此, 可以将光漂白和毒性最小化。此外, 与点扫描显微术相比, 采用光片显微镜 (100-1000 倍快) 的采集率大大提高, 信噪比也得到了改善。结合本论文所用的光片显微安装的孵化室, 本显微镜是一种强大的选择, 用于图像采集的大型3D 卷, 以最小的漂白和照片损伤的时间延长 (数天) 下孵化条件。
对于3D 胶原基质的样品制备, 最关键的一点是避免气泡。考虑到它的高粘度, 当气泡被引入到胶原溶液中, 很难从溶液中去除。气泡的存在会诱发胶原基质聚合的异质性;此外, 它可以阻挡光的路径, 这将降低图像的质量。因此, 当转移或混合胶原溶液时, 必须轻轻吸管, 不要排出吸管尖端的最后一滴。而且, 胶原蛋白在室温下开始慢慢聚合。因此, 在混合溶液之前, 要防止聚合, 应始终将含胶原的管子放在冰上。
我们在使用光片显微镜时遇到的一个重大挑战是数据处理。光片显微镜产生的数据可以很容易地达到 500 gb 甚至数 tb 的大小, 特别是用于长期实验。对这些数据进行分析需要几天时间, 有时由工作站计算能力很高。由于所分析图像的大小不比原始数据小, 所以存储容量很快就成为光片显微镜应用的限制因素。因此, 对于跟踪实验或如果需要复杂的图像分析, 强烈建议将每个实验的文件大小限制为可由可用的处理单元在合理的时间框架内处理的值。
本文介绍的光片荧光显微镜有一些特点, 如果不仔细处理, 可能会影响结果。利用毛细血管是特别有利的无障碍照明的样品。由于胶原棒挂在培养基中, 可能发生胶原棒漂移在图像采集, 特别是长期测量。必须纠正这种漂移, 以避免对结果的误解。当然, 市面上还有其他的光板显微镜设置, 不需要毛细血管来装样品。此外, 与光片显微镜, 除了事实相比, 传统的线扫描共焦显微镜毒性已经大大减少, 毒性仍然是一个关注长期成像21。
除了光片显微镜, 还有其他方法为3D 荧光成像, 其中主要应用是共焦显微镜, 广域荧光显微镜, 多光子显微镜。在穿透深度方面, 共焦和广域荧光显微镜位于类似的范围内, 通常限于 100 nm22。由于多光子显微镜使用的波长越长, 其穿透深度可提高到 0.5-1 毫米23的范围。相比之下, 光片显微镜光学切片所产生的多维图像的尺寸可达几毫米24。在横向分辨率方面, 一个优雅的实验表明, 光片显微镜提供了一个更好的三维空间分辨率相比, 共聚焦荧光显微镜在大样本25。近年来, 光片显微镜已与双光子技术相结合, 进一步提高了与普通光子光片显微镜相比的穿透深度, 比点扫描双光子显微镜26快十倍。
考虑到为细胞提供微体系结构的3D 矩阵, 除了本文所用的牛胶原蛋白外, 还有许多其他的选择, 如从小鼠肉瘤细胞、琼脂糖、合成水凝胶等衍生出的超细胞基质 (ECM)。生物相容高分子材料。小鼠肉瘤 ECM 是一种蛋白质混合物, 包括胶原 IV、层粘连蛋白以及多种生长因子。与合成材料、生物 ECMs (胶原蛋白和小鼠肉瘤 ECM) 相比, 一方面呈现出更具生理学意义的语境;但另一方面, 质量和组成生物 ECMs 可能不同批次和公司。相比之下, 合成材料的重现性更有保障。值得注意的是, 生物化学和机械性能的微调, 以及所需的修改, 是允许合成材料, 但不为生物 ECMs10,27。
总之, 本文介绍了用光片荧光显微镜构建细胞生物学实验3D 胶原基质的一种协议, 以及如何在3D 胶原基质中嵌入一个人 ctl 来可视化和分析 ctl 迁移。重点介绍了样品制备、图像采集和分析的要点。结果表明: 光片显微镜能较好地显示高动态突起和细小肌动蛋白结构的形成, 并具有良好的时空分辨率。此外, 这里描述的分析协议使我们能够以客观和自动化的方式可靠地跟踪细胞。这种方法对于人类免疫学研究特别有利, 因为小鼠和人类在许多免疫学功能和反应性方面有极大的差异28。

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>&#160;#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+ 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>&#945;-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&#39;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 &#945; 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&#160;</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&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Sarstedt</td>
			<td>&#160;62.554.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillaries 50 &#181;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>&#34;Stamps with Teflon tip&#34; 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&#160;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&#160;</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&#160;</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, 在迁移过程中, 人类 CTL 形成柠檬形的主要突起, 流苏状的细纺锤状结构。在这里所示的实验中, 不仅有一个单一的细胞被成像, 而且是一个大的体积 (450 x 450 x 538 µm3) (图 2B)。因此, 在这里显示的光学质量和分辨率是以低放大目标 (20X/1.0 DIC 为 IR) 获得的。因此, 通过更高的 NA 目标, 可以进一步改善该决议。
在图 2和电影 1中所示的实验中, CTLs 被自动跟踪, 如协议第4部分所述。在图 2B中描述了 CTL 的轨迹。分析了两种迁移参数: 速度和持久性。持久性定义为位移除以总轨道长度。分析表明, 在3D 中, CTL 的移动性非常多样: 速度范围从 0.01-0. 19 µm 与几乎20倍的差异, 持久性范围从 0-0.7 (图 2C)。据报道, 在小鼠体内间质迁移中 T 细胞的平均速度为4µm/分钟16。最近, 我们已经确定了 CTL 的平均速度为 4-5 µm/分钟11使用本文所描述的方法。这些结果表明, 光片显微镜是一个强有力的工具, 以可视化细胞的行为, 例如, 细胞迁移和细胞的相互作用。在体内, 许多因素和复杂成分的非均质 ECM (包括可影响迁移的可溶性因子) 会改变 T 细胞的行为。
胶原基质的应用不限于活细胞。固定和染色也可以在母体中进行。图 3显示了3D 胶原凝胶中固定样本的例子, 其中内源性 perforin1 (PFN1) 和肌动蛋白染色。样品从单面 (单面照明) 或两侧 (双侧照明) 照明。我们观察到, 在不同 z 位置的细胞均匀染色, 表明我们的协议中描述的程序达到令人满意的抗体渗透到胶原凝胶。更重要的是, 在固定的 CTL 表现出与活细胞成像相同的形态学 (比较图 3和图 2B), 表明这一方案也很好地维护了形态学。另外, 从单面或两侧的光照对焦平面的图像质量没有显著的影响。考虑到相对于双侧照明的单侧照明方式照射到激光器的面积较小, 建议采用单面照明。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57651/57651fig1.jpg"> 图 1: 在样品准备过程中处理毛细血管的图示.(A)将柱塞放入毛细管中, 以及如何用培养基将柱塞润湿。(B)把细胞/胶原蛋白混合到毛细血管中。(C)使用建模粘土来安装毛细血管。柱塞和胶原棒分别在灰色和红色框中描述。(D)处理胶原棒, 以平衡培养基或染色。柱塞和胶原棒分别在灰色和红色框中描述。<a href="/files/ftp_upload/57651/57651fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57651/57651fig2v2.jpg"> 图 2: 跟踪3D 胶原蛋白中的主要人体 CTL, 并可视化肌动蛋白动力学.(A)第一人 CTL 肌动蛋白的最大强度投射。肌动蛋白被标记在 CTL 如前所述11。第一天后转染细胞植入0.5% 胶原基质。显示一个代表性单元格。刻度条为5µm. 在突出边缘的细微肌动蛋白结构由箭头突出显示。(B) CTL 的轨道为6小时。轨迹被软件 automatedly 跟踪 (颜色代码: 蓝色 = 开始, 红色 = 轨道的结尾, 450 x 450 x 538 µm3容量)。刻度条为50µm. (C)量化速度和持久性。一个点代表一个单元格。结果以4个捐献者的平均扫描电镜显示。<a href="/files/ftp_upload/57651/57651fig2v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57651/57651fig3v2.jpg"> 图 3: 嵌入3D 胶原基质的 CD8+ T 细胞内源性 profilin1 (PFN1) 和肌动蛋白染色.主要的人 CD8+ T 细胞嵌入0.25% 胶原蛋白。在粉煤灰固定后, 样品被染色使用 anti-PFN1 (橙色) 和罗丹明 (紫色) 和可视化的光片显微镜在1µm 的总体积 440 x 440 x 1000 µm3。样品从单面 (单面照明) 或两侧 (双侧照明) 照明。显示3D 重建的最大强度投影 (MIP)。刻度条是50µm.<a href="/files/ftp_upload/57651/57651fig3v2large.jpg" target="_blank">请点击这里查看这个数字的大版本</a>。
<img alt="Movie 1" src="/files/ftp_upload/57651/57651mov1.jpg"> 电影 1: T 细胞迁移过程中突起的产生.影片中显示的单元格取自图 2A所示的卷。细胞嵌在胶原基质中 (0.5%)。图像是每四十年代获得6小时在37°c 使用光片显微镜。<a href="/files/ftp_upload/57651/Movie_1.avi" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>

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

Nanyang Technological University

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

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

科研

免疫与感染

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

脂肪组织免疫细胞的分离

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

免疫突触由双色时间选通受激发射损耗（STED）纳米显微可视化

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

细菌细胞的三维成像，实现精确的细胞表现和精确的蛋白质定位

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...