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Bioengineering

用于小鼠角膜和康康菲瓦实时成像的自定义多光子显微镜平台

Published: May 17, 2020
doi:
10.3791/60944
, , , ,
1Department of Biomedical Engineering,National Taiwan University, 2Institute of Pharmacology, College of Medicine,National Taiwan University, 3Molecular Imaging Center,National Taiwan University, 4Department of Dermatology,National Taiwan University Hospital, and College of Medicine, 5Research Center for Developmental Biology and Regenerative Medicine,National Taiwan University, 6Department of Ophthalmology,Chang Gung Memorial Hospital, 7College of Medicine,Chang Gung University

Summary

这里展示的是一个多光子显微平台，用于实时小鼠眼表面成像。荧光转基因小鼠能够可视化细胞核、细胞膜、神经纤维和眼表面的毛细血管。从胶原结构派生的非线性二谐波生成信号为频闪结构提供了无标签成像。

Abstract

传统的组织分析和细胞培养系统不足以完全模拟体内的生理和病理动力学。多光子显微镜 （MPM） 已成为体内细胞水平上生物医学研究最流行的成像模式之一，其优点包括高分辨率、深层组织渗透和极小光毒性。我们设计了一个 MPM 成像平台，具有定制的鼠标眼架和用于体内眼表面成像的立体轴舞台。双荧光蛋白报告鼠标能够可视化细胞核、细胞膜、神经纤维和眼表面的毛细血管。除了多光子荧光信号外，获得第二谐波生成 （SHG） 可同时进行胶原蛋白生成结构。该平台可用于病毒内成像，可准确定位整个眼表面，包括角膜和结膜。

Introduction

眼表面结构，包括角膜和结膜，保护其他更深的眼组织免受外部干扰。角膜是眼睛的透明前部，既充当折射透镜，可将光线引导到眼睛中，也作为保护屏障。角膜上皮是角膜的最外层，由不同的表面细胞层、翼细胞和基底细胞组成。角膜频闪由嵌入角细胞的复杂包装胶原蛋白组成。角膜内皮是一层扁平六角细胞，通过泵送功能1保持角膜频闪，在保持角膜的透明度方面具有重要作用。林巴斯形成角膜和结膜之间的边界，是角膜上皮干细胞的蓄水池2。高度血管化的结膜有助于润滑眼睛产生粘液和眼泪3。

角膜表面结构的细胞动力学由组织学分析或体外细胞培养学进行常规研究，可能不足以模拟体内细胞动力学。因此，非侵入性实时成像方法可以弥补这种差距。由于其具有高分辨率、最小光损和深成像深度等优点，MPM已成为生物研究4、5、6、7、8,等不同,6,7领域的有力模式。对于角膜成像，MPM 提供来自细胞内NAD（P）H的固有自荧光的细胞信息。第二次谐波生成（SHG）信号来自非分色类型我胶原纤维在飞秒激光扫描下提供胶原蛋白生成结构，无需额外的染色程序9。此前，我们和其他团体利用MPM对动物和人类角膜进行成像，9、10、11、12、13、14、15。,10,11,12,13,14,15

在特定细胞群中表现出荧光蛋白的转基因小鼠系已广泛应用于细胞生物学的各种研究，包括发育、组织平衡、组织再生和致癌。我们使用标有荧光蛋白的转基因小鼠菌株，通过MPM对角膜9、10、,10毛囊10和表皮10进行体内成像。双荧光小鼠株与细胞膜标记与tDTomato和细胞核标记与EGFP是由两个小鼠株培育：R26R-GR（B6;129-Gt（ROSA）26Sortm1Ytchn/J，#021847）16和16tm1Ytchn/JmT-mG（Gt（ROSA26）ACTB-ttomato-EGFP，#007676ACTB-tdTomato-EGFP17R26R-GR转基因小鼠线包含双荧光蛋白报告器结构，包括H2B-EGFP融合基因和mCherry-GPI锚点信号融合基因，插入Gt（ROSA）26Sor位点。mT-mG转基因菌株是一种细胞膜靶向的tdTomato和EGFP荧光Cre-reporter小鼠。在Cre重组之前，具有tdTomato荧光表达的细胞膜蛋白广泛存在于各种细胞中。这种转基因小鼠菌株使我们能够可视化核-EGFP和膜与tdTomato没有Cre激发。两只雌性（R26R-GR+/+）和一只雄性（mT-mG+/+）转基因小鼠一起繁殖，以产生足够的小鼠进行实验。+/+他们的后代与R26R-GR+/–;mT-mG+/-基因型，双荧光小鼠株，在这项研究中被使用。+/-+/-与前面描述的9、10号荧光记者鼠标线相比，10这种双荧光测量仪的成像时间缩短了50%。

在这项工作中，我们描述了使用我们的成像平台和双荧光转基因小鼠逐步进行眼表面体内成像的详细技术方案。

Protocol

所有动物实验都是按照台湾国立大学动物护理与使用委员会（IACUC）和长贡纪念医院批准的程序进行的。 1. 多光度显微镜设置 构建一个基于直立显微镜的系统，具有浸入水 20x 1.00 NA 目标（图 1A）。 使用 Ti：蓝宝石激光（可调波长）作为激励源。将EGFP的激光输出波长设置为 880 nm，为 tdTomato 将激光输出波长设置为 940 nm（图…

Representative Results

使用这个实时成像平台，鼠标眼表面可以在细胞水平上可视化。为了可视化眼表面的单个细胞，我们采用了双荧光转基因小鼠，其EGFP在细胞核中表达，tdTomato在细胞膜中表达。富含胶原蛋白的角膜频闪被SHG信号突出显示。 在角膜上皮，表面细胞，翼细胞和基底细胞（图2）被可视化。在双荧光转基因小鼠中，我们能够将单个细胞从基底层映射到角膜和边?…

Discussion

这个定制的MPM成像平台与控制软件用于小鼠上皮器官的病毒内成像，包括皮肤10，毛囊10和眼表面9，10（109图1A）。自项目开始以来，定制系统一直用于灵活地更改各种实验的光学元件。这种成像方法适用于商用 MPM 产品。该协议描述了通过MPM成像平台对小鼠眼表面进行病毒内成像的详细方法。使…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢台湾科技部（106-2627-M-002-034，107-2314-B-182A-089）的赠款支持， 108-2628-B-002-023，108-2628-B-002-023），台湾国立大学医院（NTUH108-T17）和台湾长荣纪念医院（CMRPG3G1621，CMRPG3G1622，CMRPG3G1623）。

Materials

AVIZO Lite software Thermo Fisher Scientific Version: 2019.3.0
Bandpass filters Semrock FF01-434/17
FF01-500/24
FF01-585/40
Dichroic mirrors Semrock FF495-Di01-25×36 FF580-Di01-25×36
Galvano Thorlabs GVS002
Jade BIO control software SouthPort Corporation Jade BIO
Oxybuprocaine hydrochloride Sigma O0270000
PMT Hamamatsu H7422A-40
Polyesthylene Tube BECTON DICKINSON 427401
Stereotaxic mouse holder Step Technology Co.,Ltd 000111
Ti: Sapphire laser Spectra-Physics Mai-Tai DeepSee
Upright microscopy Olympus BX51WI
Vidisic Gel Dr. Gerhard Mann Chem-pharm. Fabrik GmbH D13581
Zoletil Virbac VR-2831
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Tags

Multiphoton MicroscopyLive ImagingMouse CorneaMouse ConjunctivaDual Fluorescent Transgenic MouseEGFPTdTomatoSHGStereotaxic Mouse HolderEyelid RetractionEye GelZ-stack Imaging

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Wu, Y., Ye, R., Pan, M., Lin, S., Tan, H. A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva. J. Vis. Exp. (159), e60944, doi:10.3791/60944 (2020).
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