通过共聚焦显微镜重建单细胞先天荧光特征
Summary
在这里，提出了一种方案，用于光学提取和编目来自分布在三维空间中的每个个体活细胞的先天细胞荧光特征（即细胞自发荧光）。该方法适用于以单细胞分辨率研究各种生物系统的先天荧光特征，包括来自细菌，真菌，酵母，植物和动物的细胞。
Abstract
这里描述的是共聚焦反射显微镜辅助的单细胞先天荧光分析（CRIF），这是一种微创方法，用于重建分布在三维（3D）空间中的群体中每个个体活细胞的先天细胞荧光特征。细胞的先天荧光特征是由细胞内各种生物分子发出的荧光信号的集合。先前的研究表明，先天荧光特征反映了各种细胞特性和生理状态的差异，是细胞表征和鉴定的丰富信息来源。传统上，先天荧光特征是在群体水平上进行分析的，需要克隆培养，但不是在单细胞水平上。CRIF特别适用于需要3D分辨率和/或选择性提取单个细胞荧光信号的研究。由于荧光特征是细胞的先天特性，CRIF也适用于完整和单个细胞的类型和/或生理状态的无标记预测。这种方法可能是简化细胞分析的强大工具，其中异质性群体中每个单个细胞的表型可以在显微镜下通过其自发荧光特征直接评估，而无需细胞标记。
Introduction
cell1内的各种生物分子发出自发荧光信号，细胞的先天荧光特征由这些信号的组装组成。这种特征性荧光反映了各种细胞特性以及生理状态的差异。先天荧光的分析是微创的，可以补充传统的，更具侵入性的微生物探针，这些探针会留下从轻度代谢修饰到完全细胞破坏的一系列痕迹。虽然DNA或细胞含量提取2，3，荧光原位杂交4以及将荧光报告基因引入基因组等传统技术可有效确定细胞类型或生理状态，但它们通常需要对细胞进行操作或侵入性标记。
对各种活的和完整的微生物菌落的先天荧光的研究表明，包括散装微生物培养悬浮液5，6，活性污泥7，哺乳动物组织8，9和哺乳动物细胞1，10，先天荧光分析有助于对细胞类型和生理状态进行无标记分析。传统上，先天荧光特征是在群体水平上而不是在单细胞水平上进行分析的，因此需要克隆培养。相比之下，此处描述的食灶性反射显微镜辅助单细胞无光泽分析（CRIF）技术11重建并编目了每个活微生物细胞的先天细胞荧光特征。此外，CRIF可以系统地整理分布在三维（3D）空间中的群体中单个微生物细胞的先天荧光特征。
Protocol
1. 样品的制备 将带有孔的1毫米厚硅胶垫圈放在载玻片上。 将1毫米厚的0.8%（w / v）琼脂糖板坯放在硅胶垫圈的孔中。 将任意微生物细胞培养物的细胞密度稀释至600nm（OD660）= 1.0的光密度。 将5μL等分试样的细胞悬浮液放在琼脂糖板上。 用玻璃盖板轻轻盖住。 2. 显微镜的设置 注意：CRIF技术结合了共聚焦反?…
Representative Results
图1A显示了细菌细胞的典型单细胞荧光特征，以传统光谱图（顶部）和热图（中间）的形式呈现。图1B显示了叠加在土壤细菌群的原始CRM图像上的精确2D细胞分割的结果（假单胞菌KT2440）12。由此产生的群体先天荧光特征在图1D中以热图的形式呈现。请注意，在成功进行细胞分割后，种群内的变异性相对较小…
Discussion
该方法中有两个关键点需要密切关注以获得可重复的结果：1）通过激发波长和实验保持显微镜物镜下的激光功率输出一致，以及2）执行准确的细胞分割。
在比较不同实验之间的先天荧光特征时，第一点尤其重要。避免简单地将相同的”百分比输出”设置应用于激发波长（即，对所有405、488、514和530 nm激光线使用5%功率输出），因为激光线之间的最大功率输出差异可达一个数量?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了日本文部科学省（18K04843）向Y. Yawata，JST ERATO（JPMJER1502）到N. Nomura的科学研究资助的部分支持。
Materials
| Agarose | Wako Chemicals | 312-01193 | |
| Beam splitters | Carl Zeiss, Nikon | MBSInVis405, MBS458, MBS488, MBS458/514, MBS488/543, or MBS 488/543/633 beam splitters (Carl Zeiss) | |
| Confocal microscope | Carl Zeiss, Nikon | Model LSM 880 (Carl Zeiss), Model A1R (Nikon) | |
| Cover slips | Matsunami Glass | C024601 | |
| Glass slides | Matsunami Glass | S011120 | |
| Half-reflection mirror | Carl Zeiss, Nikon | NT80/20 | |
| Laser power meter | Thorlabs | PM400 (power meter console) and S175C (sensor) | |
| LB Broth | Nacalai tesque | 20066-95 | For bacteria culture |
| Image analysis software | The MathWorks | MATLAB version 2019a or later, Image Processing Toolbox is needed | |
| Microscope objective | Carl Zeiss, Nikon | 440762-9904 | e.g. 63x plan Apochomat NA = 1.4 (Carl Zeiss) |
| Microscope software | Carl Zeiss, Nikon | ZEN (Carl Zeiss),NIS-elements (Nikon) | |
| PBS(-) | Wako Chemicals | 166-23555 | |
| Programming language | Python and libraries, modules (numpy, scikit-learn, scikit-image, os, glob, matplotlib, tkinter) are rquired to run the supplied PCA script. | ||
| Silicone gasket | ThermoFisher Scientific | P24744 | |
| Workstation | A high-performance workstation with discrete GPUs is recommended. | ||
| Yeast extract-peptone-dextrose (YPD) agar medium | Sigma-Aldrich | Y1500-250G | For yeast culture |
| YPD medium | Sigma-Aldrich | Y1375-250G |
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Cite This Article
Hirayama, T., Takabe, K., Kiyokawa, T., Nomura, N., Yawata, Y. Reconstruction of Single-Cell Innate Fluorescence Signatures by Confocal Microscopy. J. Vis. Exp. (159), e61120, doi:10.3791/61120 (2020).