单一平面照明模块和一个宽场显微镜微毛细管法
Summary
为单一平面照明显微镜的模块（SPIM）中描述了很容易适应的倒宽视场显微镜和3维细胞培养物进行了优化。样品位于一个矩形的毛细管，并通过微流体系统的荧光染料，药物制剂或药物可以以小批量应用。
Abstract
这是很容易适应于一个倒置的宽视场显微镜和三维细胞培养物的优化，对于光片或单个平面照明显微镜（SPIM）中描述的模块， 例如，多细胞肿瘤球体（MCTS）。在SPIM激发模块的形状和偏转，使得样品被光片照射垂直于显微镜的检测路径的光。该系统的特点是利用一个矩形毛细管的用于保持（和在一个先进的版本也被用于旋转微毛细管的方法）的样品中，由照明光片的同步调整并用于荧光检测以及物镜通过微流体系统的应用的荧光染料，在少量的药物制剂或药物的适应性。给出的协议与该系统的工作，和一些技术细节的报告。代表性的结果包含的向上（1）的测量在添加氧化剂的需要抑制细胞生长的药物（阿霉素）和其部分转化为降解产物，（2）氧化还原测量通过使用遗传编码谷胱甘肽传感器，以及（3）启动和细胞坏死的经抑制的标签线粒体呼吸链。本SPIM模块与现有系统相比，差别和优点进行了讨论。
Introduction
除了 公认的方法（共焦或多光子激光扫描显微术1-4，结构照明显微镜5,6）光片或单个平面照明显微镜（SPIM）已被证明是三维成像7,8的一种有价值的方法， 9。特别令人感兴趣的是其应用到三维细胞培养物， 例如，多细胞肿瘤球体（MCTS），它们越来越多地用于药物发现研究10,11。再者，SPIM是当即使在长期暴露或重复测量低光剂量必须保持样品的可行性，因为用于样品仅此平面被曝光的每个平面的测定的优先方法。这是相对于其他显微技术，其中对于检测各焦平面的整个样品被照射，使记录的许多平面时的光量总和起来，并可能损坏样品12。 </P>
光片显微镜或SPIM是根据与观察路径的样品在垂直方向上或者通过使用圆柱形透镜的或者通过扫描激动人心的激光束（用于见参考文献8评论）的照明。这通常需要特殊的样品室13,14或矩阵， 例如，琼脂糖7,15，在特殊的高性价比的显微镜来实现。作为替代那些系统中可比简单的照明装置，SPIM已经开发并适于常规的倒置显微镜16（参见图1）。它由激光束扩大到直径为8毫米，集中由一个圆柱形镜头（焦距：50毫米，数值孔径：0.08），以6-10微米厚的片状光通过景深约100微米。样品位于600至900微米内径的矩形毛细管放置在显微镜物镜的len的前S代表荧光检测。这些主要功能是目前完成，通过使用先进的微毛细管的方法，用于保持和转动用于荧光检测（相同光路中的样本，照射光片的同步调整（在轴向）和物镜的优化位移的长度，所需要的机械进给的校正），和一微流体系统的适配为应用荧光染料，药物制剂或药物，从而减少所需的数量和费用。
Protocol
Representative Results
Discussion
本手稿描述光片或单一平面照明显微镜（SPIM）装置，其3维电池系统进行了优化， 例如，多细胞肿瘤球体（MCTS）。三个示例性的应用包括：（1）吸收细胞生长抑制药物和其部分转化为降解产物（其对化疗疗效贡献仍有待评估）中，氧化还原状态的（2）通过采用遗传编码谷胱甘肽传感器在测量另外一种氧化剂，和（3）启动和细胞坏死时抑制线粒体呼吸链的标记。
本SPIM…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这个项目是由土地巴登 – 符腾堡州，以及欧洲联盟，的Europäischer全宗资助献给死去大区ENTWICKLUNG。作者感谢赖维蒂希（ILM乌尔姆）提供的U251-MG-L106-Grx1-roGFP2细胞系和克劳迪娅·欣策的熟练的技术援助。
Materials
| Name of the Material/Equipment | Company | Catalog Number | Comments/Description(optional) |
| microtiter plate | Orange Scientific | 4430100 | for cell spheroid growing |
| agarose | Carl Roth GmbH | 3810.1 | for cell spheroid growing |
| MCF-7 cell line | CLS Cell Lines Service GmbH | 300273 | cell line |
| U251-MG-L106 cell line | cell line | ||
| DMEM | Biochrom AG (Merck Millipore) | FG0435 | culture medium |
| DMEM/Ham's F-12 | Biochrom AG (Merck Millipore) | FG4815 | culture medium |
| FCS | Biochrom AG (Merck Millipore) | S0615 | cell culture supplement |
| penicillin/streptomycin | Biochrom AG (Merck Millipore) | A 2213 | antibiotics |
| hygromycin B | PAA Laboratories | P02-015 | antibiotic |
| EBSS | Sigma-Aldrich Inc. | E3024 | cell culture supplement |
| doxorubicin | Sigma-Aldrich Inc. | D1515 | fluorescent dye (CAUTION: acute toxicity) |
| green cytotoxicity dye | Promega GmbH | G8742 | CellTox – fluorescent cytotoxicity dye |
| rotenone | Sigma-Aldrich Inc. | R8875 | cellular inhibitor (CAUTION: acute toxicity) |
| hydrogen peroxide (H2O2) | Sigma-Aldrich Inc. | 95302 | reagent for oxidation (CAUTION: acute toxicity) |
| capillary | VitroCom | 8260-050 | sample preparation |
| microscope | Carl Zeiss Jena | Axiovert 200M | |
| AxioCam MRc CCD-camera | Carl Zeiss MicroImaging GmbH | 426508-9901-000 | CCD-camera |
| AxioVision data aquisition software | Carl Zeiss MicroImaging GmbH | version 4.8.2. | |
| laser diode | Pico Quant GmbH | LDH-P-C-470 | used with driver PDL800-B |
| perestaltic pump | Ismatec Labortechnik | MS-1 Reglo | re-callibrated to reduce the minimum pump speed by 1/10 |
| silicone tubes | IDEX Health & Science GmbH | TYGON R3607 |
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