Name: optical screening of novel bacteria-specific probes on ex vivo human lung tissue by confocal laser endomicroscopy
Uploaded: 2017-11-29T13:00:00.000+00:00
Duration: 9 min 24 s
Description: Watch this Scientific Journal Video about Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy at JoVE.com

我们要感谢工程和物理科学研究理事会 (EPSRC, 英国) 跨学科研究合作赠款 EP/K03197X/1, 卫生署和惠康信托通过健康创新挑战基金 (HICF).供资参考编号: 0510-069。

所有的人肺组织都是在知情同意下获得的, 该研究得到了区域伦理委员会的批准。
1. 生物样品的制备
<ol><li>
探头的制备
	<ol>
<li>在无菌 dH2O 中, 用一个精细的天平称量 freeze-dried 探头复合11, 组成每个探头的 1 mM 库存解决方案 (例如、素 AM、UBI-3、UBI-10、等)。根据探针的称重质量和分子量计算 dH2O 的体积。</li>
	</ol>
</li>
	<li>
细菌培养物的制备 注意: 对于此方法, 使用金黄色葡萄球菌作为样本菌株。任何适当的细菌菌株都可以选择。任何商业染料, 标签细菌与适当的激发和发射光谱可以选择积极标签的细菌。<ol>
<li>使用无菌回路从新鲜的性肉汤 (LB) 琼脂板中选择所需菌株的单一菌落。通过将循环的末端浸入到培养基中, 在50毫升离心管中将菌落接种到10毫升的 LB 中。孵育在37° c, 250 rpm 为16小时 (或隔夜)。</li>
		<li>确定过夜文化的 OD595通过在1毫升试管中添加100µL 过夜文化到900µL 磅。在分光光度计中测量 595 nm 的 OD (使用试管和1毫升磅作为空白)。将获得的 od 乘以10以获取过夜文化的 od。</li>
		<li>一夜文化。通过将光学密度调整为 595 nm (OD595) 到0.1 在10毫升的新鲜磅. 计算要添加到10毫升新鲜磅的过夜文化所需的容量, 以调整 OD595到0.1。孵育文化在37° c, 250 rpm 直到文化到达 mid-log 阶段 (OD595 0.6-0.8), 大约 4 h。</li>
		<li>测量培养光密度 (步 1.2.2) 并且收获 1 x 108殖民地形成单位 (CFU) (OD595 ~ 1-1 x 108 CFU/ml) 细菌文化入1.5 毫升微 (例如, 如果细菌培养是 OD 的5950.6, 收集1.67 毫升)。离心培养在 1万 x g 在室温下为1分钟的颗粒细菌。在磷酸缓冲盐水 (pbs) 中, 用溶液 (移) 在1毫升 PBS 中, 离心以上, 丢弃上清, 再重复清洗颗粒。在清除上清液时, 注意不要取出细菌颗粒。重1毫升 PBS 中的最后一团。 注: 为每个标签程序准备尽可能多的样品。协议可以在这里暂停1小时, 后跟步骤1.2.5.1、1.2.5.2 或1.2.5.3。</li>
		<li>细菌染色<ol>
<li>要用素 AM 标记细菌, 将染料添加到最终浓度为1µM 的细菌培养液中。孵育文化为30分钟在37° c 以摇晃在300转每分钟。用离心法在 PBS 中洗涤1-4 细菌悬浮液3次, 1.2.4 去除过量染料。重在1毫升 pbs, 稀释100µL 1:1 在 pbs 获得200µL OD595 0.5 素 AM 染色细菌。该协议可以在这里暂停长达1小时。</li>
			<li>用测试探针 (例如, UBI-3 或 UBI-10) 对细菌进行标记, 在 pbs 中稀释100µL 细菌培养 1:1, 以获得 OD595 0.5 的200µL pbs。将其中任一探头添加到最终浓度为10µM. 反转微几次, 以确保彻底混合的细菌和探针。 注: 成像应在添加探针后立即进行, 以模拟临床情况。</li>
			<li>为制备控制不细菌样本, 用 PBS 稀释100µL 1:1 的培养基, 获得200µL 的不 OD595 0.5 样本。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
人肺组织的体外制备 注: 肺癌患者经手术切除后, 可获得人肺组织标本。所有用于成像的组织都是从癌细胞生长的正常肺组织样本中获得的。样品从操作的剧院被采取了新鲜和存放在管内或离心管在-80 ° c 直到用途。<ol>
<li>在成像前, 将人体的肺组织标本从冰箱的干冰中取出。在室温下, 允许组织稍微解冻;刚好足够用手术刀切成 1 x 4 mm2节。 注意: 解冻的程度是重要的;太冷冻和组织将不会切片没有剥落, 太解冻和组织太软切片。</li>
		<li>使用镊子, 将切片的人肺组织放置在一个96很清楚的平底组织培养板的井中。将任何未使用的人肺组织立即归还储存容器, 并将其放置在干冰上, 以便输送回-80 ° c 冷冻柜。</li>
		<li>用吸管将100µL PBS 添加到每个肺组织标本中。使用吸管的尖端, 以确保所有的组织覆盖在 PBS (而不是粘在墙的井)。组织将略有膨胀, 并可能漂浮。将 PBS 放在样品上几分钟, 让血液从组织中渗入溶液中。尽可能多地删除 PBS。组织可阻断吸管尖端的末端;尝试角板/尖端放置, 以防止这一点。</li>
		<li>吸管100µL 的不, 素 AM 标记, 或测试探针标记细菌的每一个井含有肺组织。还设置了肺组织和100µL PBS 的控制。对这些控制井, 没有细菌的探针可以被增加测量任何增加的背景荧光和/或非特异的激活探针的肺组织单独。还应包括肺组织和 PBS 的井。</li>
	</ol>
</li>
</ol>2. 用 FCFM 的设备进行成像
<ol><li>
设置清洗设备 注: 在校准前准备20分钟的系统, 允许激光预热。<ol>
<li>按系统变压器背面的开/关开关, 然后打开指定的计算机。按下激光扫描装置 (路易斯安那州) 前面的开/关按钮。确认绿色指示灯的外观, 指示设备已打开。</li>
		<li>双击 "软件" 图标。输入登录详细信息并等待路易斯安那州立会初始化 (10-三十年代)。</li>
		<li>为使用新的 FCFM 成像纤维, 安装与提供的 CD 是必要的。将安装光盘插入计算机 cd 驱动器, 并按照屏幕说明进行操作。</li>
		<li>用纤维清洁剂清洁 FCFM 成像光纤连接器单元。将清洁带上的接头在向前移动时擦除灰尘/污垢。从路易斯安那州立会的前面取下保护性的黄色帽子。</li>
		<li>通过逆时针旋转银轮毂, 直到它停止。将 FCFM 成像光纤连接器插入中心, 并将连接器的平面侧向上朝上。保持纤维到位, 顺时针旋转银色轮毂, 直到它点击两次。通过进一步45°顺时针旋转银色轮毂来完成连接。 注意: 如果未识别光纤, 请检查 FCFM 成像光纤是否已安装并以正确的方向连接。</li>
		<li>按照将弹出的说明完成 FCFM 成像光纤校准。有3步骤: (1) FCFM 成像纤维测试 (步骤 2.1.7-2.1.8), (2) 背景获取 (步骤 2.1.9), (3) 光纤检测 (步骤 2.1.10)。</li>
		<li>按屏幕上的 "启动激光" 按钮。激光将中心。</li>
		<li>选择新鲜的小瓶 (黄色: 校准; 红色: 清洁; 蓝色: 冲洗) 从校准套件和按照屏幕上的说明: 将 FCFM 成像纤维的远端放置到黄色小瓶中, 观察显示器上荧光的增加, 然后将纤维尖进入红色小瓶 (不搅拌)。等待荧光 (如计算机显示器上所示) 消失。最后用蓝色瓶子冲洗纤维尖。 注意: 如果荧光不消失, 清洁 FCFM 成像光纤与8% 小时2O2和镜片清洗组织, 并重新开始。重复这个过程, 直到得到满意的结果 (图像质量是明确的, 没有污点的痕迹是明显的)。</li>
		<li>将 FCFM 成像纤维放入蓝色小瓶中。按 ' 开始激光 ', 然后 ' 计算 ', 当这成为一个选项。</li>
		<li>将 FCFM 成像纤维放入黄色小瓶中。按 ' 开始激光 ', 然后 ' 计算 ', 当这成为一个选项。</li>
		<li>在自动校准过程中, 将 FCFM 成像光纤的远端放置于 > 十年代的红色小瓶中, 然后用该软件所示的 > 4 s 的蓝色瓶进行清洁。</li>
	</ol>
</li>
	<li>
数据收集与整理
	<ol>
<li>在安装之后, 将会打开一个选择存储位置和文件前缀的窗口。为数据存储选择所需的文件夹, 并相应地命名该前缀。</li>
		<li>放置脚踏板, 以便操作员可以方便地访问它们。左踏板: 激光开/关;中心踏板: 暂停;右踏板: 记录/停止。 注意: 激光控件也可以通过屏幕上的控件进行访问。</li>
		<li>单击 "启动" 屏幕或按左脚踏板打开激光。这将开始获取和获取图像使用100% 激光功率和帧速率为12帧/秒 (默认设置)。 注意: 对于其他应用程序, 如果需要, 这些设置可以在屏幕上更改, 具体取决于示例类型。</li>
		<li>图像的每一个细菌悬浮样品。<ol>
<li>插入 FCFM 成像纤维的远端, 并缓慢地通过悬浮物移动纤维来审问样品。</li>
			<li>通过按右脚踏板或选择屏幕记录控件来记录任何长度的视频 (最多10分钟), 因为光纤在样品周围缓慢移动。</li>
			<li>使用8% 小时2O2和样品之间的镜片清洗组织清洁 FCFM 成像光纤的远端。 注: 典型的视频长度为 10-三十年代是足够的体外成像。</li>
		</ol>
</li>
		<li>图像每一个肺组织样本。<ol>
<li>将 FCFM 成像纤维的远端插入样品中, 确保纤维端与组织之间的直接接触。轻轻地将成像纤维的末端移到周围以询问样品。 注意: 将纤维的末端从组织中抬离, 将组织从焦平面上移除;然而, 这可能是用来图像标记的细菌, 没有附着在组织。</li>
			<li>通过按右脚踏板或选择屏幕记录控件来记录任何长度的视频 (最多10分钟), 因为光纤在样品周围缓慢移动。 注: 通常情况下, 三十年代的视频长度对于组织上的活体成像是足够的。</li>
			<li>用透镜清洁组织和 8% H2O2在样品之间清洁 FCFM 成像纤维的远端。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
关闭系统
	<ol>
<li>通过按左脚踏板或单击 on-screen 按钮来关闭激光开关。</li>
		<li>将 FCFM 成像光纤从该设备上断开, 然后逆时针旋转, 直到停止。从路易斯安那州立中心通过轻轻拉动纤维连接器从路易斯安那州立 FCFM 的成像纤维。</li>
		<li>使用8% 小时2O2和镜片清洁组织清洗和消毒 FCFM 成像光纤。将保护帽返回到 FCFM 成像纤维的近端和路易斯安那州立会的前部。将纤维轻轻地放在存储盒中。</li>
		<li>关闭数据捕获软件并将所有保存的文件复制到外部 USB 设备。关闭计算机, 然后按前面板 i/o 3 秒, 直到绿灯消失。</li>
		<li>根据当地规定处理人体肺组织和细菌。</li>
	</ol>
</li>
</ol>3. 数据分析
<ol><li>通过在 "软件" 面板上的 "转到" 图标上选择适当的目录, 打开软件并导入要分析的文件。或者, 可以将文件拖放到软件仪表板中。</li>
	<li>双击每个视频文件打开它们。该视频将自动播放与优化的颜色查找表格 (表) 和颜色表调整。通过单击强度刻度栏上方的 "魔杖" 按钮, 禁用自动强度缩放。当按钮周围没有黑色阴影时, 该功能将被禁用。 注意: 必须禁用自动强度缩放, 以防止在每个视频中持续进行对比度增强, 从而无法比较和分析来自同一数据集的视频。</li>
	<li>通过移动最小和最大条形来选择所需的强度缩放, 以提供最佳对比度。在选择强度缩放时使用直方图工具, 以确保捕获最广泛的动态范围。 注意: 请确保动态范围的图像不饱和 (即限制图像的白色区域, 这表示饱和度), 这样就不会遗漏低强度特征。</li>
	<li>完成所需的缩放后, 右键单击列出为 "默认 (绿色)" 的下拉菜单按钮。选择保存查找的选项。将 "查找" 保存到所需的位置。</li>
	<li>对于数据集内的其他视频, 通过右击 "默认 (绿色)" 下拉菜单并选择 "加载查找表" 来应用相同的查找表。选择适当的文件, 将一致的强度缩放应用于数据集中的所有视频。</li>
	<li>通过点击 "电影卷轴" 按钮来导出处理过的视频。选择所需的视频格式,例如"用于演示目的", 它将生成. mpg 文件。按 "导出" 并选择文件位置以保存视频文件。可以通过单击 "照相机" 按钮导出单个帧的快照。可以保存. png、. bmp 或. jpg 文件。选择文件目标并按 "保存"。 注: 然后可以将视频导入到任何软件中, 以便准备演示文稿或进一步量化。被标记的细菌被形象化为绿色 ' 闪动 ' 点在录影。肺组织结构将是明显的作为有序的荧光股, 肺泡空间出现黑色。</li>
</ol>

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爱丁堡分子成像的创始人。获得顾问公司的技术咨询。
爱丁堡分子成像的创始人主任。

下呼吸道感染占疾病的第二高负担全球12,13, 并有大量增加的感染, 归因于耐药性细菌已报告14。肺炎仍然是住院的常见原因。在 ICU, 肺炎的发展因诊断不确定而复杂化, 并与极高的死亡率15有关。在肺炎爆发期间, 细菌在远端肺的肺泡间隙内增殖, 这是一个相对不育的区域, 在健康方面微生物最小。
此方法描述相对较晚阶段的前体验证特定于细菌的光学成像探头11, 但在开始此验证步骤之前的设计、合成和探头评估是必需的, 如前所示11.
real-time 是一种新兴的临床技术, 用于诊断疾病状态原位。它提供了许多优势比传统的技术, 调查疑似肺部病理, 这可能涉及活检和收集洗液。活检是侵入性的, 并可能导致发病率和死亡率在通风病人, 和收集的灌洗液经常污染的细菌从上呼吸道。然而, 尽管有许多协调一致的努力10, 但由于有可能提供疾病的功能性信息的兼容成像探头的可用性较差, 在肺炎检测中的使用却有些有限。与光学试剂相结合, 与目前的标准做法相比, 其诊断肺炎的速度更快, 创的可能性更小。
该协议的关键步骤是在示例准备和设置的平台。获得与最终临床应用相关的人体组织也很重要, 如本研究中所示的人肺组织。有必要使用人体组织, 因为组织自身荧光的程度表现出较大的间变异, 因此可能误导细菌探针的敏感性。此外, 获取和使用人类肺组织的伦理学是必不可少的。从技术层面上说, 正确的清洁、将成像纤维附着在图像处理平台上, 以及校准对于良好的分辨率和一致的成像是至关重要的, 因为这样可以确保每一个肺组织样本中都添加等量的细菌。为了进一步扩大这一方法的效用, 筛选面板的探针, 重复的程序与一系列的病原体, 如那些可能是致病剂的肺炎是必要的。
这项技术的最大限制是, 经临床批准的清洗设备只有一个激光器 (488 nm)。因此, 目前, 用于探针设计的荧光的选择是有限的与该系统的使用, 虽然临床认可的单一颜色的设备确实存在的激发波长 660 nm 和近红外。这是非常可取的, 有第二个激光线在同一设备内实现, 使探针开发与光谱独特的荧光, 以提高细菌探针灵敏度的组织自发荧光水平。虽然双色清洗设备正在开发中, 但它们不是经临床批准的, 或者它们的成本是显著的16。
体外使用病原菌和ex 体内人肺组织筛测电位探针可以弥合传统的体外分离技术, 如流式细胞仪和 CLSM, 以及临床应用。这一步提供了信心, 当选择有前途的化合物, 以进行结合临床的影像;并将提供指示测试探针是否保持目标特异性, 或演示任何不相干标记, 如直接绑定到组织, 或显示不稳定与宿主蛋白水解酶。另外, 将每个激活探针直接添加到人体肺组织和细菌样本中, 以充分刻画 real-time 中探针结合和活化的速度。
我们相信, 我们的管道快速筛选新的细菌特定的探针, 以评估其潜在的成像在远端肺部的病人将导致更快的翻译到诊所。这主要是因为细菌特定的探针可以通过导管插入支气管镜的工作通道在肺部局部传递, 这意味着微量 (< 100 µg) 的数量可以被传递。因此, 系统的交付和分布的化合物是不关心的情况下, 在许多其他感染目标的身体内, 或与核成像。此外, 在如此小的剂量下提供成像探头可以降低毒性相关并发症的风险 (尽管需要进行毒性筛查)。在注入探针之后, 导管就可以被 FCFM 纤维和同样的肺部区域所取代, 就像我们在这种方法中所做的那样。在探头安装探针之前, 应迅速进行成像, 然后将探针冲刷到无法探测到的浓度。
同样重要的是要注意, 通过这种技术筛选疾病鉴定探针不应局限于细菌成像剂, 但也可以扩展到对其他目标, 如炎症的探针的验证。这种方法也应适用于身体内的其他疾病部位, 通过 FCFM 成像是允许的。

这项技术描述了一个快速筛选的过程, 以评估细菌特异性光学成像剂在前体内人肺组织通过使用 FCFM 的快速鉴定化合物的潜在临床实用的可视化在远端肺部的细菌原位。
在抗菌素耐药性上升的时代, 迫切需要对抗菌药物进行定量配给1。为此, 开发的诊断方法, 以确定细菌感染的高特异性, 灵敏度, 并在 real-time 是高度寻求2。目前确诊危重病人肺炎的技术, 如重症监护病房 (icu), 往往依赖于解释非特异性的临床或放射学特征和细菌培养技术, 从吸气液/组织, 这可能需要长达3天的结果。此外, 从流体灌输到远端肺和检索的细菌培养容易受到更多的近端气道3的污染, 而且由于伴随的抗菌疗法或抽样技术的不良, 往往是文化上的阴性。此外, 分子技术, 如聚合酶链反应是过于敏感, 当使用吸气液, 冒着过度的病人。一种新兴的诊断方法是分子光学成像, 使原位组织的分子病理学成为可能;然而, 光学成像化合物的开发和验证是必需的。然而, 通过激活光学探针直接可视化细菌, 可能是一种非常有力的方法, 可以研究肺炎在患者中的存在和演变, 而且重要的是, 可以用来研究宿主-病原体之间的相互作用。对治疗的反应在 real-time原位.
是一个已建立的多疾病调查程序4, 包括胃肠病学领域的5, 肿瘤科6,7, 并为审讯呼吸道和肺泡囊8,9. 它利用纤维成像束使病变组织的点结构成像, 通过临床内窥镜的工作通道, 并与组织表面直接接触, 并通过共聚焦显微镜成像。然而, 仍然存在的一个限制是需要通用的对比剂。因此, 使用疾病特定的探针, 如特定的细菌剂, 可以极大地扩大这种模式的效用, 直接可视化细菌在疑似感染部位。通过启用 real-time、高分辨率成像和诊断电位, 光学代理提供了许多优于其他技术的优势。此外, 光学探头提供了多路复用的前景, 用于审讯多个目标, 都以较低的成本实现。许多光学代理正在为此目的而开发, 但在诊所10内尚未成功实施。我们合成了一个专门针对细菌的小分子化学探针库, 并研制了一种快速有效的检测细菌性肺炎探头功能的管道,原位11。
为了确定合适的探头候选者, 必须在审讯前在对人体肺组织的 FCFM: i) 水溶解度, ii) 的探针之前完成以下先决条件临床上快速标记的特异性和选择性相关的细菌, iii) 高信噪比, 和 iv) 抵抗退化在肺环境之内。后者是由支气管肺泡灌洗液 (BALF) 的急性呼吸窘迫综合征 (ARDS), 这是一个条件的特点是蛋白水解和炎症环境的肺部在 ICU。此外, 探针必须有一个适当的荧光, 以检测由临床认可的光学在人肺肺泡组织的影像设备。
对每一个先决条件进行审问的管道如下 (在每个阶段, 只有通过的探针被结转到下一个): (1) 综合研究了探针库;(2) 每个探针被添加到一个活细菌的小组共聚焦激光扫描显微镜 (CLSM), 以确保细菌标记;(3) CLSM co-cultures 与原人中性粒细胞建立的哺乳动物细胞的细菌标记选择性;(4) CLSM 和基质辅助激光解吸/电离-飞行时间 (MALDI) 质谱法测定 ARDS 患者 BALF 中存在的细菌的稳定性和成功标记;(5) 通过 CLSM 确定候选者的最佳浓度, 确保维持哺乳动物细胞细菌的选择性;(6) 候选人被 FCFM 在悬浮和在前体人肺肺泡组织中的影像, 以确保稳定性, 而信噪比是足够的检测。步骤6在本协议中详细描述。先前已报告了步骤 1-5 的方法11。

<table><tbody><tr><td>1-14 Microfuge</td><td>SciQuip</td><td>90616</td><td>Small benchtop microcentrifuge</td></tr><tr><td>96-well plate</td><td>Corning</td><td>3370</td><td>Assay plate</td></tr><tr><td>Calcein AM</td><td>Sigma Aldrich</td><td>17783&amp;nbsp;</td><td>Commercial fluorescent dye</td></tr><tr><td>Cellvizio 488 nm Research CLE</td><td>Mauna Kea Technologies</td><td>LC-0001-488</td><td>Confocal laser endomicroscopy device</td></tr><tr><td>Cletop-S</td><td>Cletop</td><td>14110601</td><td>Fibre cleaner</td></tr><tr><td>Eppendorf (1.5 mL)</td><td>Eppendorf</td><td>30120086</td><td>1.5 mL microfuge tube</td></tr><tr><td>Eppendorf Research Plus Pipettes</td><td>Fisher Scientific</td><td>11568663</td><td>Micro pipettes</td></tr><tr><td>Falcon tube (50 mL)</td><td>Scientific Lab Supplies</td><td>352070</td><td>50 ml centrifugation tube</td></tr><tr><td>Gibco Phosphate Buffered Saline</td><td>Thermo Fisher Scientific</td><td>10010023</td><td>PBS - wash media</td></tr><tr><td>IC-Viewer</td><td>Mauna Kea Technologies</td><td>LW-0001</td><td>Data collection and processing software for the research CellVizio 488 nm system</td></tr><tr><td>Incu-Shake midi</td><td>Sciquip</td><td>SQ-4020</td><td>Floor standing shaking incubator</td></tr><tr><td>Lysogeny Broth&amp;nbsp;</td><td>Sigma Aldrich (Miller)</td><td>L3522</td><td>LB Growth media for S. aureus</td></tr><tr><td>non-standard research AlveoFlex 488 nm</td><td>Mauna Kea Technologies</td><td>MP-0002-AF3</td><td>Fibred confocal fluorescence microscopy fibre</td></tr><tr><td>Quanti Kit 488 nm</td><td>Mauna Kea Technologies</td><td>LQ-0005</td><td>Calibration kit for the CellVizio 488 system</td></tr><tr><td>S. aureus ATCC 25923</td><td>ATCC</td><td>25923</td><td>Bacterial strain used in this study</td></tr><tr><td>Semi-micro spectrophotometry cuvette</td><td>Sigma Aldrich&amp;nbsp;</td><td>C5416-100EA</td><td>For spectrophotometry&amp;nbsp;</td></tr><tr><td>Thermomixer comfort</td><td>Eppendorf</td><td>41102422</td><td>Benchtop heater with shaking</td></tr><tr><td>UV 1101 Biotech photometer</td><td>Biochrom WPA</td><td></td><td>Spectrophotometer</td></tr></tbody></table>

optical screening of novel bacteria-specific probes on ex vivo human lung tissue by confocal laser endomicroscopy

提高细菌检测的速度和准确度对于患者的分层和确保适当使用抗菌素是非常重要的。为了实现这一目标, 需要发展诊断技术, 以识别细菌存在的 real-time 在点。在宿主内直接识别细菌的光学成像是一种有吸引力的方法。对化学探针设计和验证的几次尝试都进行了调查, 但是还没有成功地将其转化为临床。在这里, 我们描述了一个方法,前体内验证细菌特定的探针, 以确定细菌在远端肺部, 成像纤维共聚焦荧光显微镜 (FCFM)。我们的模型使用了前体人肺组织和一个临床认可的共焦激光内 () 平台, 以筛选新的细菌特异的成像化合物, 密切模仿的成像条件, 预计将遇到的病人。因此, 这种技术的筛选化合物提供了潜在的临床可的信心。

在本研究中, 我们已经证明了一种方法, 快速筛选新的细菌特异探针在一个前体内人肺泡肺组织模型感染使用临床认可的设备。
FCFM 是非常适合获得结构信息的远端肺部, 因为这个地区 (由于高丰度的弹性蛋白和胶原) 是自然高度荧光时兴奋与 488 nm 激光8。相反, 肺泡空间不荧光, 因此使组织结构和空气空间之间的高对比度可视化 (图 1)。
增加与疾病相关的探针或造影剂, 如特定细菌的探针, 应能使有关疾病过程的功能信息在 real-time 中获得。我们以前曾描述的合成和初步的体外筛选的一个细菌专用探针库11;在细菌的特异性, 蛋白水解稳定性, 并在时间内的菌膜滞留确定。在这项研究中发现了一种有希望的细菌特异探针 (UBI-10), 以及在细菌细胞膜 (UBI-3) 中表现不佳的保留。这些与用于标记为S. 金黄色葡萄球菌的商业 counterstaining (素 AM) 的控制相比较。
不、素 AM、UBI-3 和 UBI-10 标记为S. 金黄色葡萄球菌, 在悬浮时由 FCFM 与 100% 488 nm 激光功率和帧速率12帧/秒 (图 2) 进行了成像。在 PBS 的未标记细菌被成像的地方, 没有任何荧光信号被探测到。这与被标记的细菌被成像的对比。当细菌悬浮与 UBI-3 或 UBI-10 的 FCFM, 很明显, 一般的背景荧光的解决方案是提高相比, PBS 只控制, 这是因为工作日 (探针荧光) 确实发出少量的在水溶液中的荧光信号, 然而, 明亮的点斑点在整个溶液中是清晰可见的, 而无需清洗步骤。这是由于在极地环境中的 NDB 的荧光信号的增加即, 细菌膜。素 AM 不是激活探针, 因此需要在细菌染色后清洗一步, 去除溶液中游离探针的高荧光背景。像 UBI-3 和 UBI-10 标记的细菌, 在溶液中检测出带有素 AM 的细菌, FCFM 为亮绿色的点状斑点。由于数据是以视频格式收集的, 这些点在核心和 in-and-out 的焦点之间移动时看起来 "闪烁", 这种方法是成像标记细菌的特征特性。
被标记的细菌随后被添加到小切片的前体人肺组织和 FCFM (图 3) 再次成像。只有在肺组织中添加了 PBS 或未标记的金黄色葡萄球菌时, 才检测到肺组织 autofluorescent 结构 (被视为胶原蛋白和弹力蛋白的亮绿色链和肺泡间隙的暗区)。在这些控制条件下, 没有检测到点斑点。同样, 只有肺组织结构 (和无点斑点) 被形象化为肺组织情况与S. 金黄色葡萄球菌加上 UBI-3;表明该探针未在细菌细胞膜即内稳定保存, 在肺组织内有原生的水解酶 (如先前所示的11), 它被冲出并/或被降解。
然而, 明亮的斑点点是可看见的在两个素 AM 标记的正面控制s. 金黄色葡萄球菌样品和与最有希望的细菌特异探针 (UBI-10) s. 金黄色葡萄球菌样品。尽管有强烈的组织自发荧光 (图 3), 但 "闪烁" 点是可见的。因此, 由 FCFM 获得的结果与体外筛选的细菌特异性探针的 CLSM, 并证明了临床相关的检测方法的影像感染 real-time。
结果表明, 肺是一个适当的器官系统的成像由 FCFM 由于其独特的自发荧光。明亮的独特的结构允许被清洗的操作员确定他们在肺泡空间。这些地区, 加上黑暗的肺泡气囊提供了完美的背景下成像荧光标记细菌与高对比度。
虽然在这项研究中提出的细菌的检测是定性的, 通过可视化明亮的斑点点, 它可以通过使用一个辅助软件来定量地确定有点点数的帧的数量, 以便进一步刻画探测库。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56284/56284fig1.jpg"> 图 1:人肺组织自发荧光的静态图像.用纤维共聚焦荧光显微镜 (FCFM), 在 488 nm 激发, 100% 激光功率, 12 帧/秒的共焦激光内 () 图像的人肺组织, 使用的是人类的细胞, 在前体. 弹性蛋白和胶原蛋白是高度荧光 (假彩色绿色),而肺泡空间不是, 并显示为黑色区域。此图已从阿克拉姆et al中修改。11<a href="//cloudflare.jove.com/files/ftp_upload/56284/56284fig1large.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/56284/56284fig2.jpg"> 图 2:共聚焦激光内标记的金黄色葡萄球菌的图像暂停.纤维共焦荧光显微镜 (FCFM) 用于图像 pre-labeled 细菌, 在 488 nm 激发, 100% 激光功率, 12 帧/秒. 标记细菌显示为高荧光点 (假彩色绿色)。此图已从阿克拉姆et al中修改。11 <a href="//cloudflare.jove.com/files/ftp_upload/56284/56284fig2large.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/56284/56284fig3.jpg"> 图 3:共焦激光内体外人肺组织, 带有标记的金黄色葡萄球菌.纤维共焦荧光显微镜 (FCFM) 用于图像的前体人肺组织和标记的金黄色葡萄球菌, 在 488 nm 的励磁, 100% 激光功率, 和12帧/秒. 标记的细菌显示为高荧光点的斑点 (伪彩色绿色) 在肺组织标本中标有素 AM 或 UBI-10。最高的对比是观察细菌在肺泡间隙中的影像。此图已从阿克拉姆et al中修改。11<a href="//cloudflare.jove.com/files/ftp_upload/56284/56284fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy at JoVE.com

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

这项技术描述了一个有效的筛选过程, 以评估细菌特异性光学成像剂在前体内人肺组织, 通过纤维共聚焦荧光显微镜快速鉴定小分子化学具有可翻译潜力的候选者。

激光内在人肺组织中的新型细菌特异探针的光学筛选

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

optical-screening-novel-bacteria-specific-probes-on-ex-vivo-human

Research

JoVE Journal

Immunology and Infection

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

7.4K 次观看.  University of Edinburgh. 这项技术描述了一个有效的筛选过程, 以评估细菌特异性光学成像剂在前体内人肺组织, 通过纤维共聚焦荧光显微镜快速鉴定小分子化学具有可翻译潜力的候选者。

视频: Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

高分辨率光纤内窥镜为原位细胞成像

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

科研

生物工程

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

Ex 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

癌症研究

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

视频率扫描共聚焦显微镜和内窥镜

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1 and gastrointestinal tracts,2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10 Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.7
The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use.11 Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent&mdash;most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.12 Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.10 Recent availability of a &lt; 1 mm imaging probe13 opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities14 that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides15 and antibodies for endoscopic imaging of disease processes with molecular specificity.

Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.

基于探测器的激光共聚焦激光显微内镜对泌尿道的技术

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

医学

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

多峰成像和光谱学纤维束显微内窥镜纲要非侵入性，<em&gt;在体内</em&gt;组织分析

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

体外肺转移及微环境的实时成像

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空 体内 成像

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and immunological tolerance, while in immunocompromised patients, conidia can colonize airways and cause severe invasive respiratory disorders. Various cells in different airway compartments are involved in the immune response that prevents fungal invasion; however, the spatio-temporal aspects of pathogen elimination are still not completely understood. Three-dimensional (3D) imaging of optically cleared whole-mount organs, particularly the lungs of experimental mice, permits detection of fluorescently labeled pathogens in the airways at different time points after infection. In the present study, we describe an experimental setup to perform a quantitative analysis of A. fumigatus conidia distribution in the airways. Using fluorescent confocal laser scanning microscopy (CLSM), we traced the location of fluorescently labeled&#160;conidia in the bronchial branches and the alveolar compartment 6 hours after oropharyngeal application to mice. The approach described here was previously used for detection of the precise pathogen location and identification of the pathogen-interacting cells at different phases of the immune response. The experimental setup can be used to estimate the kinetics of the pathogen elimination in different pathological conditions.

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

We describe the method for quantitative analysis of the distribution of Aspergillus fumigatus conidia (3 &#181;m in size) in the airways of mice. The method also can be used for the analysis of microparticles and nanoparticle agglomerate distribution in the airways in various pathological condition models.

聚焦激光扫描显微镜为基础的定量分析 阿斯珀吉卢斯熏蒸 孔尼迪亚分布在全山光学清除鼠肺

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and ...

免疫与感染

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.

激光内在人肺组织中的新型细菌特异探针的光学筛选

摘要

探索更多视频

此视频中的章节

高分辨率光纤内窥镜为原位细胞成像

Ex 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

视频率扫描共聚焦显微镜和内窥镜

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

基于探测器的激光共聚焦激光显微内镜对泌尿道的技术

多峰成像和光谱学纤维束显微内窥镜纲要非侵入性，

体外肺转移及微环境的实时成像

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空体内成像

聚焦激光扫描显微镜为基础的定量分析阿斯珀吉卢斯熏蒸孔尼迪亚分布在全山光学清除鼠肺

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

激光内在人肺组织中的新型细菌特异探针的光学筛选

摘要

探索更多视频

此视频中的章节

高分辨率光纤内窥镜为原位细胞成像

Ex 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

视频率扫描共聚焦显微镜和内窥镜

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

基于探测器的激光共聚焦激光显微内镜对泌尿道的技术

多峰成像和光谱学纤维束显微内窥镜纲要非侵入性，

体外肺转移及微环境的实时成像

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空 体内 成像

聚焦激光扫描显微镜为基础的定量分析 阿斯珀吉卢斯熏蒸 孔尼迪亚分布在全山光学清除鼠肺

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

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空体内成像

聚焦激光扫描显微镜为基础的定量分析阿斯珀吉卢斯熏蒸孔尼迪亚分布在全山光学清除鼠肺