Name: mammalian cell division in 3d matrices via quantitative confocal reflection microscopy
Uploaded: 2017-11-29T14:00:00.000+00:00
Duration: 10 min 22 s
Description: Watch this Scientific Journal Video about Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy at JoVE.com

这项工作得到了 NIH 赠款 R01CA174388 和 U54CA143868 的支持。作者要感谢约翰·霍普金斯大学的安玛塔拉普拉奖, 支持魏通陈这一材料是根据国家科学基金会研究生研究金资助 No. 1232825 的工作。

所提供的议定书遵循了 HIRB 机构审查委员会的准则。
1. H2B-mCherry 作为细胞有丝分裂标记物的稳定表达
<ol><li>
人胚肾 293T (HEK 293T) 细胞中慢粒子的生成
	<ol>
<li>在 10 cm 细胞培养皿上 HEK 293T 细胞, 在细胞培养基中的密度为 5 x 106细胞/皿中 (Dulbecco 氏改良的鹰培养基 (DMEM), 含高糖 (4.5 克/升), 丙酮酸钠, 10% 胎牛血清 (FBS) 和1%青霉素-链霉素 (笔/链球菌))。孵育24小时, 在37° c 和5% 二氧化碳 (CO2)。所需的细胞在转染日的70-100 约 70-80%。 注: HEK 293T 细胞用于产生病毒粒子, 这将被用于传感器 MDA-MB-231 细胞, 以生成细胞线稳定表达 H2B-mCherry。确保细胞均匀分布在整个盘子里。细胞的加入对板块有很好的下降, 轻轻地来回移动板块可以帮助均匀分布。细胞也可以在其他大小的盘子或烧瓶中播种, 这将导致收集不同数量的病毒颗粒。</li>
		<li>染 HEK 293T 细胞使用转染试剂与三质粒 (慢载体, CMV ΔR 8.91, pMDG-VSVG)。用甘油激酶启动子 (PGK) 慢载体克隆 H2B-mCherry 质粒编码。CMV ΔR 8.91 包含三需要的 HIV 基因,堵嘴,政客, 和启示录。pMDG-VSVG 含有 VSV-G 包络基因。 注: 有多种转染剂可供选择。<ol>
<li>在使用前, 允许转染试剂瓶达到室温 (RT)。简单地倒置或漩涡瓶。</li>
			<li>混合16µg 的 DNA, 其中包括6µg H2B-mCherry 质粒, 8 µg 的 CMV ΔR 8.91, 和2µg 的 pMDG-VSVG, 在1毫升的血清介质减少。在 RT 孵育5分钟。 注意: 三向量的数量和比率是变化和优化的不同的慢传输载体。</li>
			<li>添加48µL (使用1:3 的 dna 比例: 转染试剂) 转染试剂进入上述 dna 溶液, 然后在 RT 孵育至少15分钟。 注意: DNA 与转染试剂的比值是不同的, 并针对慢转移载体进行了优化。确保转染试剂不接触1.5 毫升离心管的侧面。</li>
			<li>将步骤 1.1.2.3 drop-wise 中的 DNA 脂质复合液添加到细胞中。轻轻地旋板, 以确保均匀分布的复杂的板块。</li>
		</ol>
</li>
		<li>在转染后约6小时, 吸入培养基, 添加新鲜细胞培养基 (DMEM 高糖 (4.5 克/升), 丙酮酸钠, 10% FBS, 1% 笔/链球菌)。</li>
		<li>收获上清 24, 48, 和 72 h post-transfection。每次收割后更换培养基 (DMEM, 4.5 克/升葡萄糖, 丙酮酸钠, 10% FBS, 1% 笔/链球菌)。</li>
		<li>过滤慢微粒通过0.45 µm 过滤器去除细胞残骸。或者, 旋下上清, 分离出细胞碎片。上清液可以马上用于转导, 或者可以储存在-80 ° c。</li>
	</ol>
</li>
	<li>
稳定表达 H2B-mCherry 的细胞系的生成 注意: 本协议详细描述了 MDA-MB-231 单元。在实验中使用之前, 人乳腺癌细胞 MDA-MB-231 (物理科学肿瘤中心, NIH) 是培养 DMEM 含有高葡萄糖 (4.5 克/升), 丙酮酸钠, 10% FBS, 和1% 笔/链球菌感染。<ol>
<li>在 35 mm 培养皿中, 在密度为 1 x 105细胞的 MDA-MB-231 细胞上板。 注: 不同细胞系的电镀密度应优化, 因此可能与 MDA-MB-231 细胞的最佳密度不同。</li>
		<li>24 h 在电镀细胞之后, 加入1毫升的病毒和1毫升的新鲜培养基 (DMEM, 高葡萄糖 (4.5 克/升), 丙酮酸钠, 10% FBS, 和1% 笔/链球菌)。</li>
		<li>将细胞孵育6小时至一夜。 注意: 病毒的体积和孵化时间都需要针对不同的细胞线进行优化。例如, 如果在添加病毒后几个小时内细胞看起来不健康, 请使用1毫升的病毒和2毫升的新鲜培养基 (DMEM、高糖 (4.5 克/升)、丙酮酸钠、10% FBS 和1% 笔/链球菌) 进行步骤1.2.2。在台阶1.2.3 中潜伏期的长短也可以减少。</li>
		<li>吸入培养基并加入10毫升新鲜培养基 (DMEM、高糖 (4.5 克/升)、丙酮酸钠、10% FBS 和1% 笔/链球菌)。孵育在24到72ĥ在37° c 和 5% CO2之间。</li>
		<li>使用荧光显微镜检查细胞中 H2B-mCherry 的表达。 注意: 如果细胞的转导效率较低, 步骤2.2 中的病毒体积和步骤2.3 中的潜伏期可以增加。</li>
	</ol>
</li>
</ol>2. 稳定表达 H2B-mCherry 的细胞的同步
<ol><li>在50到 60% 70-100 的单元格中,即板 2 x 104 MDA-MB-231 单元在24井板的每个井中。</li>
	<li>孵育文化在37° c 和 5% CO2为 24 h。</li>
	<li>用0.5 毫升培养基 (DMEM、高葡萄糖 (4.5 克/升)、丙酮酸钠、10% FBS 和1% 笔/链球菌) 取代生长培养基, 其中含有 2 mM 胸苷, 并在孵化器中留出24小时。 注意: 暴露于胸苷的细胞在细胞生长 (G1)/dna 合成 (s) 过渡阶段和整个 S 期由于对 dna 合成的抑制作用而被逮捕。对于不同的细胞株, 培养时间的长短应进行变化和优化。</li>
	<li>通过用磷酸缓冲盐 (PBS) 冲洗三次, 释放胸苷暴露的细胞。然后, 在正常细胞培养基中孵育细胞 (DMEM, 高葡萄糖 (4.5 克/升), 丙酮酸钠, 10% FBS 和1% 笔/链球菌) 为5小时。 注意: 从胸苷暴露细胞的释放允许细胞进展到细胞生长 (G2)/有丝分裂 (M) 阶段以前被逮捕的细胞在 G1/S 阶段, 和 G1 阶段之前被捕的细胞 S 阶段。在不同的细胞系中, 释放时间的长短应该变化和优化。</li>
	<li>用 250 ng/毫升的 nocodazole 12 小时阻断细胞。 注意: 所有暴露于 nocodazole 的细胞都在 G2/M 阶段被逮捕。Nocodazole 是细胞毒长时间接触 nocodazole 会导致细胞凋亡。如果观察到细胞死亡, 调整不同细胞株暴露的时间或浓度。成功同步的单元格将呈现球形形态。</li>
	<li>摇动的细胞四十五年代至1分钟使用一个轨道振动筛在150至 200 rpm。 注意: 有丝分裂细胞对基质的附着很少, 在过程中会被动摇。</li>
	<li>取出培养基以提取细胞, 将培养基移到离心管中, 然后加入0.5 毫升的新鲜培养基 (DMEM、高糖 (4.5 克/升)、丙酮酸钠、10% FBS 和1% 笔/链球菌) 到板块的每一个井。</li>
	<li>重复步骤2.6 和2.7 三次。</li>
	<li>离心收集的培养基中含有有丝分裂细胞在 800 x g 为3分钟。 注意: 此步骤用于从单元格中删除 nocodazole。</li>
</ol>3. 将同步细胞纳入胶原 i 矩阵
注: i 型胶原蛋白是人体内最丰富的蛋白质和结缔组织的 ECM, 因此被广泛用于研究真核细胞的功能是如何被3D 环境调制的17,23,24.胶原蛋白可溶于乙酸。将胶原蛋白溶液中和并升温至 20-37 ° c 后, 胶原单体聚合成网状胶原纤维。
<ol><li>准备 10x DMEM 解决方案, 溶解一包 DMEM 粉, 3.7 克碳酸氢钠 (NaHCO3) 和1克 4-(2-羟乙基)-1-piperazineethanesulfonic 酸 (HEPES) 在50毫升蒸馏水。过滤溶液, 然后在50毫升蒸馏水中溶解2克氢氧化钠颗粒, 然后准备1立方米氢氧化钠 (氢氧化钠)。过滤和分溶液成1.5 毫升离心管。 注意: 此步骤中不应使用正常的 DMEM 解决方案。增加大量的胶原蛋白溶液将稀释培养基。因此, 浓缩的 DMEM 溶液, 以确保最终浓度的 DMEM 在胶原基质将是相同的正常 DMEM。</li>
	<li>继续处理从步骤2.9 收集的单元格。在 0.25-0.5 毫升的新鲜细胞培养基 (DMEM, 高葡萄糖 (4.5 克/升), 丙酮酸钠, 10% FBS, 和1% 笔/链球菌) 中吸入培养基和 re-suspend 细胞。 注: 为了达到特定的细胞密度在胶原基质, 初始密度的细胞在悬浮不能太低。因此, 用于 re-suspend 单元格的介质的体积将取决于可用单元格的总数。</li>
	<li>将悬浮单元格解决方案的10µL 从步骤3.2 放置在例上, 并计算该解决方案中单元格的密度。</li>
	<li>
确定所有组件所需的卷以制作3D 胶原蛋白基质。500µL 2 µg/µL 胶原凝胶在这里作为一个例子。
	<ol>
<li>计算获得4万细胞/毫升 (或 2万500µL 胶原凝胶的细胞) 所需的细胞溶液的体积。这个数字在µL 是 x 50 µL 的 10x DMEM 将需要。将需要50µL 的 FBS。</li>
		<li>计算所需的胶原蛋白量。胶原蛋白 i 的浓度约4µg/µL。µL 所需的胶原蛋白含量为 Y。</li>
		<li>计算氢氧化钠的体积需要中和醋酸在胶原溶液中: Y µL 胶原蛋白 * 0.023 * 1000 = Z µL 氢氧化钠需要。</li>
		<li>确定填料溶液的体积: (500 µL 总凝胶-X µL 细胞-50 µL 10X DMEM-µL FBS-Y µL 胶原蛋白-Z µL 氢氧化钠) = R µL 注: 填料溶液为蒸馏水。如果填充溶液的计算体积为负数, 则细胞悬浮液中原细胞密度过低。细胞需要再次纺下来和悬浮在一个较小的体积培养基 (DMEM, 高葡萄糖 (4.5 克/升), 丙酮酸钠, 10% FBS, 和1% 笔/链球菌)。</li>
	</ol>
</li>
	<li>将以下顺序中的组件添加到干净的1.5 毫升离心管: 培养基中的细胞, 10x DMEM, FBS, 去离子水, 胶原蛋白, 氢氧化钠。将所有的组件放在冰上, 以减缓胶原蛋白溶液的凝胶。使用适当的移技术来防止气泡的形成。</li>
	<li>加入氢氧化钠后, 用1毫升吸管仔细混合溶液。 注: 胶原蛋白的凝胶将在加入氢氧化钠后立即开始。混合应该仔细和快速地做。</li>
	<li>一旦解决方案是好的混合, 增加500µL 的解决方案, 每井 24-井板。将24井板放在37° c 恒温箱中。在37° c 水浴中放置新鲜细胞培养基。 注: 塑料底板用于 low-magnification 活细胞显微镜, 其中透镜工作距离在1毫米以上. 玻璃底板用于高倍活细胞显微镜由于高的工作距离短放大镜头。</li>
	<li>添加500µL 5ml 培养基 (DMEM, 高葡萄糖 (4.5 克/升), 丙酮酸钠, 10% FBS 和1% 笔/链球菌) 的顶部凝胶30分钟后完成步骤3.7。</li>
</ol>4. 3D 胶原基质中细胞分裂的活细胞成像 (低放大率)
注: 电池的图像以2分钟的间隔收集, 使用装在相衬显微镜上的电荷耦合器件 (CCD) 摄像机, 该相机装有10X 物镜, 并由成像软件控制。
<ol><li>打开安装在物镜上方的活细胞单元。等到温度稳定在37° c 时, CO2的浓度为 5%, 湿度为75%。</li>
	<li>将24孔的凝胶板放入显微镜的活细胞单元。找到板的底部, 然后移动物镜, 直到显微镜聚焦约500µm 从底部的板块。 注意: 在3D 矩阵中, 胶原基质中太靠近平板底部的细胞没有完全嵌入。500µm 的成像单元将确保单元格不受边缘效果的影响16、17、25。</li>
	<li>移动舞台以查找单元格, 并选择多个位置进行成像。</li>
	<li>在2分钟间隔内设置延时实验。 注: 在2分钟间隔内可采取的最大位置数受机动阶段的移动速度和捕捉图像的曝光时间的长短的限制。</li>
</ol>5. 细胞分裂过程中的胶原网络变形 (高倍显微镜)
<ol><li>打开安装在物镜上方的活细胞单元。等到温度稳定在37° c 时, CO2的浓度为 5%, 湿度为75%。</li>
	<li>为了使不3D 胶原蛋白基质中的胶原纤维可视化, 配置一个共聚焦显微镜来捕捉仅反射光 (488-nm) 从 488 nm 激光用于照亮样品。激光使用60X 浸水目标, NA = 1.2, WD = 200 µm, 并由成像软件控制。</li>
	<li>将样品 (胶原蛋白基质中的细胞) 放入活细胞单元。找到板的底部, 然后移动物镜, 直到显微镜聚焦约100µm 从底部的板块。100µm 的成像细胞从板的底部将确保细胞不会受到边缘效应的影响。 注: 这里使用的是浸水物镜, 而不是油浸透镜, 因为油浸透镜的工作距离只有大约100µm。使用油浸透镜构成一个问题, 因为玻璃的厚度在板材的底部通常是大约100µm。</li>
	<li>移动舞台以查找单元格, 并选择多个位置进行成像。 注: 共焦显微镜光学剖面的样本, 只捕捉信号从焦平面。在延时实验中, 活细胞可能会进入和脱离焦点。为了在长时间内捕获单元格图像, Z 堆栈用于从连续的切片中以5µm 的间隔收集图像。共5片跨越25µm 的图像。</li>
	<li>以5分钟的间隔设置延时实验。 注: 5 分钟在这里使用的间隔, 而不是2分钟使用在4节, 因为多个扫描模式, 包括荧光扫描 H2B-mCherry, 胶原的反射扫描, 和 Z 堆栈扫描, 适用于每个位置。与低放大率成像相比, 使用多种扫描模式可降低扫描位置的速度。</li>
	<li>利用粒子成像测速仪 (PIV) 和亚像素分辨率20, 量化细胞附近的胶原基质的形变。对2D 图像进行这种分析, 可以清晰地显示 H2B-mCherry 和胶原纤维的信号。应用各向异性低通滤波增强胶原蛋白网络的信号20。</li>
	<li>为了测量位于 (x,y) 的子区域的局部位移, 从帧 k 到帧k+ 1, 在帧k上提取15×15像素的区域窗口 (x,y)。然后确定最佳匹配位置 (x,y) * 通过在帧k+ 1 中获得的图像中具有最大规范化相关系数的位置。变形向量计算为 (x,y) *-(x,y)。</li>
</ol>

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作者声明他们没有竞争的金融利益。

由于实验的局限性和技术上的挑战18,19, 先前对3D 单元划分的研究没有效率。在3D 胶原基质中有效研究哺乳动物细胞分裂的关键步骤是: (1) 将荧光标记的有丝分裂标记纳入细胞;(2) 细胞分裂的同步;(3) 利用活细胞成像技术、时间分辨共焦反射显微镜和定量成像分析对3D 矩阵中的分裂事件进行监测。
在2D 基底上的有丝分裂细胞可以根据它们的形态学区别于界面细胞, 即有丝分裂细胞是圆形的, 几乎不附着在基底上, 而相间的细胞则扩散出来并牢牢附着在基底上。然而, 在3D 矩阵中, 细胞形态不是有丝分裂细胞的可靠标记, 因为一些细胞在母体中几乎没有扩散, 并且在27,28中仍然是圆的。因此, 在3D 矩阵中引入有丝分裂标记来研究细胞分裂是非常必要的。我们稳定地表达 H2B-mCherry 在 MDA-MB-231 细胞, 这是一个可靠的标志, 不同的有丝分裂阶段, 包括前期, 中期, 中期, 后期, 和末期/胞。我们以前用这种方法来区分3D 矩阵中的有丝分裂细胞和界面细胞。在这个标记的帮助下, 我们还能够测量另一条细胞线, HT1080 细胞的有丝分裂期的长度, 在2D 基板上和3D 矩阵的19中划分。
有多种方式同步细胞, 包括血清饥饿29, 有丝分裂 shake-off30,31, 双胸苷阻断32, 和 nocodazole32。我们结合胸苷治疗和 nocodazole 治疗, 以有效地同步 MDA-MB-231 细胞到 G2/M 阶段。由于胸苷对 DNA 合成的抑制作用, 暴露于胸苷的细胞在 G1/S 过渡和整个 S 期都被逮捕。从胸苷暴露的细胞释放, 让细胞进展到 G2/M 阶段的细胞在 G1/S 阶段被捕, 并 G1 的细胞在 S 阶段被捕。所有暴露于 nocodazole 的细胞都在 G2/M 阶段被逮捕。rounded-up 细胞进入有丝分裂阶段, 然后 shaken-off 从板块和直接封装到胶原基质。我们表明, 大约70% 的细胞在2小时内分裂后, 它们被纳入胶原基质 (图 2)。另外, 在同步之前, 细胞可以嵌入胶原基质中, 但是, 胶原基体的交联网络呈现物理屏障, 降低生物分子扩散和对流的速率33, 34. 事实上, 我们试图使用胸苷和 nocodazole 来同步胶原基质中的细胞, 但未能获得有效的同步。这种结果可能是由于药物通过胶原基质的扩散和对流效果不佳所致。
反射是许多生物大分子的固有光学特性, 包括胶原蛋白。共焦反射显微镜技术的可视化和 quantitates 合成聚合物和3D 胶原基质制备的多孔生物材料的形貌23,24,35,36 ,37。在我们的实验室中, 我们已经建立了监测胶原纤维极性变化的技术, 因为胶原蛋白的浓度在不同的38。在此, 我们描述了基于反射共焦图像的时移视频来监测胶原纤维变形的方法, 以表示细胞-基质相互作用。本文的代表性结果表明, 有丝分裂 MDA-MB-231 细胞的基质变形明显小于相间的细胞, 这表明哺乳动物细胞与周围的矩阵时, 他们进入有丝分裂阶段19。
以前, 我们使用共焦反射显微镜来监测和量化细胞-基质相互作用之前, 期间和之后, 有丝分裂的 HT1080 细胞。我们还监测β-整合素 HT1080 细胞在界面和有丝分裂阶段的基质变形。β-整合素能显著降低细胞在相间的基质变形。然而, 在β-整合素 (KD) 细胞和 HT1080 野型细胞的有丝分裂期, 在基质变形方面没有差异19。
另一种可视化胶原纤维的方法是采用荧光共轭型胶原。我们以前使用这种方法的图像轨道在细胞生成的胶原基质19。然而, 这种方法需要用荧光染料来标记胶原蛋白, 例如荧光素异硫氰酸酯 (FITC), 既费时又效率低。另一方面, 共焦反射显微镜可以直接应用于未修饰的胶原蛋白, 以节省时间和资源, 并排除与荧光漂白相关的问题。此外, 这种方法不需要单独的荧光通道, 因此是兼容所有荧光染料。
本文提出的方法可以应用于任何类型的哺乳动物细胞, 在3D 胶原基质中分裂。通过慢转导将 H2B-mCherry 标记纳入其他哺乳动物细胞, 将遵循与本文中所描述的完全相同的程序, 尽管不同类型的细胞可能通过慢39. 细胞在转导时的密度和病毒的滴度都可以优化以提高效率。如果不能实现高的转导效率, 细胞可以通过荧光辅助细胞分类来选择。胸苷阻断和 nocodazole 应用于成功同步其他几种类型的哺乳动物细胞, 如 HeLa40。在有丝分裂阶段30,31中, 可将机械 shake-off 应用于所有圆形且几乎不附着在基底上的细胞类型。此外, 利用共焦反射显微镜对胶原纤维进行成像, 并对基质变形进行定量化, 可以直接应用于所有其他类型的哺乳动物细胞的划分。
本文所提出的方法是在3D 环境中研究哺乳动物细胞分裂和细胞-基质相互作用的一种有效而普遍的方法。这一方法促进了我们对正常组织和疾病发展的分子基础的探讨, 并加强了今后新的诊断和治疗方法的设计。

细胞有丝分裂是细胞生命的重要事件, 其调控在组织和器官发育中起着至关重要的作用。异常有丝分裂涉及自然遗传变异、人类衰老过程和癌症的进展1,2,3,4,5。与正常细胞相比, 肿瘤细胞增殖率增加是癌症的标志之一, 尽管在不同类型的肿瘤中, 甚至在患者中, 细胞行为是相当不均匀的。尽管有希望的临床研究结果, 一些新开发的有丝分裂药物没有证明是有效的在临床医学试验6,7,8,9,10 ,11。必须考虑实验和临床前模型的相关性。许多类型的正常哺乳动物和癌细胞在三维 (3D) 基质中分裂, 如成纤维细胞和纤维细胞在富含胶原蛋白的3D 结缔组织中, 以及3D 基质细胞外基质 (ECM) 中的转移癌细胞。然而, 绝大多数的哺乳动物细胞分裂实验和化验都是在 two-dimensional (2D) 基质上培养的细胞上进行的。一个工程化的3D 矩阵可以更好地概括的组织, 机械性能和生化信号的 3D ECM 的正常和病理组织12,13,14, 15,16,17。
尽管生理相关性和治疗意义18,19, 但在3D 环境中如何调节哺乳动物细胞分裂的研究仍大体未被探索。可能的原因包括在3D 矩阵中研究细胞分裂的技术难点和实验挑战。细胞有丝分裂在整个细胞周期中构成一个小的时间分数20。以前的研究表明, 许多哺乳动物细胞的增殖率, 如人类乳腺腺癌 MCF-7, 人骨肉瘤 U2OS, 和人的肝脏 HepG2, 是低得多的3D 矩阵相比, 他们的对应在2D 基板21, 22。此外, 嵌入在3D 矩阵中的细胞在活细胞成像过程中进出焦点。所有这些因素都有助于在3D 文化中利用成像技术捕获细胞分裂事件的效率极低。
ECM 和细胞之间的相互作用在调节细胞分裂方面起着至关重要的作用。在这里, 我们描述了一个有效的方法来研究哺乳动物细胞分裂3D 胶原基质。该方法包括将有丝分裂标记纳入细胞, 细胞分裂的同步, 以及使用活细胞成像技术、时间分辨共聚焦反射显微镜对3D 矩阵中的分裂事件进行监测, 以及定量成像分析。荧光标记的组蛋白 H2B 首先引入细胞中, 作为分化有丝分裂和相间细胞的标志物。然后, 细胞是同步使用胸苷阻断和 nocodazole 治疗的组合, 其次是机械 shake-off 技术。然后将同步细胞直接封装到3D 胶原基质中。多细胞的细胞分裂事件的监测有效使用 low-magnification 延时实时细胞成像。胶原纤维的形变是细胞-基质相互作用的指示物, 通过高倍的共聚焦反射显微镜进行监测。
我们以前使用这种技术来监测和量化细胞-基质相互作用之前, 期间和之后, 两个转移癌细胞株, 人浸润性导管癌 MDA-MB-231 和人纤维 HT1080 细胞, 在3D 胶原矩阵19。这里提出的方法提供了一个有效的和一般的方法来研究哺乳动物细胞分裂在3D 环境和细胞基质相互作用。在整个论文中, MDA-MB-231 单元格线被用作示例。这项协议提供了新的洞察到分子基础的发展的正常组织和疾病, 也可以允许设计新的诊断和治疗方法。

<table><tbody><tr><td>Human embryonic kidney 293T</td><td>ATCC</td><td></td><td></td></tr><tr><td>MDA-MB-231</td><td>Physical Sciences Oncology Center, NIH</td><td></td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>DMEM powder</td><td>ThermoFisher Scientific</td><td>12100-046</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Hyclone</td><td>SH30910.03</td><td></td></tr><tr><td>Penicillin-Streptomycin 100X</td><td>Sigma-Aldrich</td><td>P0781</td><td></td></tr><tr><td>Fugene HD</td><td>Promega</td><td>E2311</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Life technologies</td><td>11668-07</td><td></td></tr><tr><td>Plasmid encoding H2B-mCherry in a lentiviral vector</td><td>Addgene</td><td>plasmid 21217</td><td></td></tr><tr><td>Thymidine</td><td>Sigma-Aldrich</td><td>T1895</td><td></td></tr><tr><td>Nocodazole</td><td>Sigma-Aldrich</td><td>M1404</td><td></td></tr><tr><td>Opti-MEM</td><td>Life Technologies</td><td>31985-070</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>GibcoBRL</td><td>11810-025</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>113375-100</td><td></td></tr><tr><td>Collagen</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>NaOH</td><td>J.T. Bake</td><td>3722-01</td><td></td></tr><tr><td>Millex-HV syringe filter unit, 0.45-&amp;mu;m, PVDF, 33 mm</td><td>Millipore</td><td>SLHVM33RS</td><td></td></tr><tr><td>Nikon TE2000E epifluorescence microscope</td><td>Nikon</td><td>TE2000E</td><td></td></tr><tr><td>Cascade 1K CCD camera</td><td>Roper Scientific</td><td></td><td></td></tr><tr><td>NIS-Elements AR imaging software</td><td>Nikon</td><td></td><td></td></tr><tr><td>Nikon A1 confocal microscope</td><td>Nikon</td><td>A1</td><td></td></tr></tbody></table>

mammalian cell division in 3d matrices via quantitative confocal reflection microscopy

关于哺乳动物细胞分裂如何在3D 环境中被调控的研究, 尽管其生理相关性和治疗意义仍未得到很大的探索。缺乏探索的可能原因是实验性的限制和技术上的挑战, 使3D 文化中细胞分裂的研究效率低下。在这里, 我们描述了一个 imaging-based 的方法, 以有效地研究哺乳动物细胞分裂和细胞基质相互作用的3D 胶原基质。用荧光 H2B 标记的细胞同步使用胸苷阻断和 nocodazole 治疗的结合, 其次是机械 shake-off 技术。然后将同步细胞嵌入3D 胶原基质中。细胞分裂被监测使用活细胞显微镜。细胞分裂期间和之后的胶原纤维的变形是细胞-基质相互作用的指示物, 可以用定量共焦反射显微镜进行监测和量化。该方法提供了一个有效的和一般的方法来研究哺乳动物细胞分裂和细胞基质相互作用在一个生理相关的3D 环境。这种方法不仅为正常组织和疾病的分子基础的发展提供了新的洞察力, 而且还允许设计新的诊断和治疗方法。

本文的目的是提出一个 imaging-based 的方法来研究哺乳动物细胞分裂过程的3D 矩阵, 并量化的互动之间的细胞和3D 细胞外基质细胞分裂。为了促进细胞有丝分裂的成像, 我们利用慢转导将 H2B-mCherry 纳入 MDA-MB-231 细胞。H2B 结合荧光蛋白被用作有丝分裂标记, 以区分有丝分裂细胞与间期细胞, 并定义在细胞有丝分裂期间的不同阶段19,20,26。利用这种方法, 我们能够监测 MDA-MB-231 细胞在3D 胶原基质中稳定表达 H2B-mCherry 的整个分裂过程 (图 1A)。有丝分裂阶段始于核膜的溶解 (前期; 框架 1,图 1A);染色体的整顿 (中期: 框架 2);染色体在细胞体中间的排列 (中期; 框架 3);染色体的分离 (后期; 框架 4);重组染色体和细胞核膜, 并分离两个子细胞的体 (末期/胞, 框架 5)。
3D 矩阵中的细胞增殖率通常要比2D 基板上的单元格低得多, 这使得在3D 效率较低的19中对单元划分的监视成为了一个问题。为了提高研究3D 细胞分裂的效率, 我们采用了一种结合胸苷、nocodazole 和 shake-off 技术的方法来同步和选择有丝分裂期的 MDA-MB-231 细胞。然后将同步细胞嵌入胶原蛋白基质中。real-time 使用活细胞成像在10X 放大倍数下监测多细胞的分裂过程。大约70% 的细胞在胶原蛋白基质形成后的前2小时内分裂 (图 2), 从而允许对 3D (图 2) 的有丝分裂进行有效的监测。
为了监测细胞与其周围的胶原基质之间的相互作用, 我们将共焦反射显微镜与图像胶原纤维相结合, 并对细胞进行荧光显微成像。60X 透镜可以捕获高质量的胶原纤维图像。使用高倍透镜 (在每个视场中捕获较少的单元格) 的成像比使用 low-magnification 在10X 或20X 时的吞吐量低得多。细胞的成功同步大大提高了这种实验的效率和吞吐量, 因为大多数的同步细胞在胶原基质形成后的前2小时内分裂。在细胞分裂期间和之后的矩阵变形是可视化的 (代表性的快照显示在图 1B) 和量化使用自定义的 PIV 软件。我们量化和比较矩阵的变形期间和有丝分裂阶段的细胞同步到有丝分裂阶段。我们观察到, 在有丝分裂阶段 (图 3A) 中, 基体变形很少发生变化, 并且小于有丝分裂阶段 (图 3B) 中观测到的形变。这一结果表明, 哺乳动物细胞在有丝分裂阶段与周围基质的附着和相互作用最小。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56364/56364fig1.jpg"> 图 1: 有代表性的显微从一个高倍的 MDA-MB-231 细胞的活细胞成像视频中获得, 它嵌入在稳定表达 H2B-mCherry 的胶原基质中。(A) MDA-MB-231 细胞有丝分裂过程的不同阶段是由 H2B-mCherry 标记为红色的。(B)在有丝分裂过程中的胶原纤维 (白色) 通过时间和时间共焦反射显微镜进行可视化。缩放栏 = 20 µm.<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56364/56364fig1large.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/56364/56364fig2.jpg"> 图 2: 3D 胶原蛋白基质中同步细胞的分裂.细胞与 G2/M 相同步, 植入胶原基质。大约70% 的同步细胞在前2小时内分裂, 而没有同步的控制单元则随机分裂。误差条 = SEM (平均值的标准误差)。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56364/56364fig2large.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/56364/56364fig3.jpg"> 图 3: 在相间和有丝分裂过程中 MDA-MB-231 细胞的基质变形的定量.(A)矩阵嵌入 MDA-MB-231 单元的矩阵变形大小的变化。绿色箭头表示前期, 红色箭头表示末期。(B) MDA-MB-231 细胞在相间和有丝分裂阶段的基质变形定量, 表明在细胞分裂过程中基质变形极小。误差条 = SEM (平均值的标准误差)。* p < 0.05。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56364/56364fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy at JoVE.com

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

该协议有效地研究了3D 胶原基质中的哺乳动物细胞分裂, 结合细胞分裂的同步, 用活细胞成像技术监测3D 矩阵中的分裂事件, 时间分辨共聚焦反射显微镜和定量成像分析。

3D 基质中哺乳动物细胞分裂的定量共焦反射显微镜观察

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

mammalian-cell-division-3d-matrices-via-quantitative-confocal

Universidad San Sebastian

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Bioengineering

Unit 1

Cell-Matrix Interactions

SCIENTISTS IN ACTION

9.1K 次观看.  Johns Hopkins University. 该协议有效地研究了3D 胶原基质中的哺乳动物细胞分裂, 结合细胞分裂的同步, 用活细胞成像技术监测3D 矩阵中的分裂事件, 时间分辨共聚焦反射显微镜和定量成像分析。

视频: Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

科研

生物工程

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

MAME的模型四维活细胞成像肿瘤微环境的相互作用，影响的恶性进展

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

医学

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed- and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:&#945;-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as &#8216;CID&#8217; gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Cell divisions can be visualized in real time using fluorescently tagged proteins and time-lapse microscopy. Using the protocol presented here, users can analyze cell division timing dynamics, mitotic spindle assembly, and chromosome congression and segregation. Defects in these events following RNA interference (RNAi)-mediated gene knockdown can be assessed and quantified.

使用果蝇S2细胞进行细胞分裂的活成像

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a ...

遗传学

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

使用快速和定量的细胞内胆碱膜模型发现转移调节器

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

癌症研究

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

同心凝胶系统来研究基质微环境的三维细胞迁移的生物物理作用

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

使用微模式和定量成像绘制哺乳动物细胞的新兴空间组织图

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

发育生物学

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

神经科学

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 ...

免疫与感染

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

超分辨率成像细菌科机械

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...