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
May 17th, 2020
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。
该协议使用多滤显微镜对双荧光转基因小鼠模型中小鼠眼表面的整个结构进行实时成像。定制多光子显微平台与双荧光转基因小鼠相结合，实现了眼表面和结构的实时可视化。要设置多光子显微镜，请选择浸入式 20X、1.00 NA 目标，并在直立显微镜上将钛蓝宝石激光器设置为激励源。
将 EGFP 的激光输出波长设置为 880 纳米，将 tdTomato 的激光输出波长设置为 940 纳米。包括两个双色镜，用于分离SHG EGFP和EGFP tdTomato，并使用434-17、510-84和585-40纳米带通滤波器，以光谱方式分离SHG EGFP和tDTomato信号。要设置眼架，在确认麻醉的8-12周大小鼠对踏板反射反应缺乏反应后，将鼠标放在加热显微镜阶段，并插入温度监测探头。
将鼠标放入自定义的立体鼠标支架中，并将耳条插入外听觉肉质中。使用三点固定固定固定固定头架，并在眼表面涂抹 0.4% 盐酸氧丙酸和盐水溶液。用 PE 管回路盖住一对五号 Dumont 钳子的尖端。
三分钟后，执行手动眼睑缩回，以确认眼球突出，并小心地沿眼睑边缘放置一个PE支架管环，以暴露眼表面。并使用支架的旋钮用钳子稳定眼球。然后，用眼凝胶覆盖角膜表面，折射率为 1.338。
对于角膜表面的 Z 串行成像，使用汞光源对靶向场进行成像并打开显微镜软件。选择适当的照片乘数和数字增益，以可视化眼表面的细胞结构，并设置第一张和最后一张幻灯片，以便采集图像堆栈。将图像分辨率设置为 512 x 512 像素，将 Z 步长设置为 1 微米。
然后，单击"开始"收集 Z 串行图像，获取一个带 880 纳米的实时图像，用于 SHG EGFP 信号采集，并在每个区域内以 940 纳米的激发获取一个实时图像。在整个图像中，使用步进电动舞台支架旋转眼球，以便在整个角膜表面进行成像。获取所有图像后，将 Z 串行图像加载到斐济并选择中位数 3D 滤镜插件。
消除背景噪音后，选择取消锐化蒙版滤镜包斐济锐化图像，然后单击自动亮度对比度自动优化图像质量。处理后，将图像保存为串行图像序列，然后将图像序列导出到适当的 3D 重建软件程序中。在所有多光子显微镜图像中，在通过快照捕获 3D 结构图像之前，分别以伪绿色、红色和青色呈现 EGFP tdTomato 和 SHG 信号。
多光子显微镜允许在双荧光转基因小鼠的角膜上皮上对表面翼和下层细胞进行可视化。基底层的单个细胞可以映射到表面层以及单个六边形表面细胞。正如tdTomato信号的细胞质表达所揭示的，富含细胞内细胞内的膜蛋白系统，包括高尔吉装置和内质神经细胞分散在翼细胞内。
在胶原状频闪中，这些小鼠的EGFP荧光膜勾勒出硬质形状的皮质状。嵌入在大学频闪中的皮质体比内皮细胞内更松散的空间。此外，角膜频闪内的薄分支神经也可以通过膜瞄准tdTomato信号和角膜内皮细胞单层显示一个相对均匀的六边形形状连接在蜂窝图案。
林皮上皮由一到两层上皮细胞组成。双荧光报告器转基因菌株还能够成像结膜内的毛细血管，促进毛细血管的3D结构的重建，勾勒出血管内皮。在不伤害的中等压力下稳定眼球对于获得高质量的图像至关重要，因为压力不足或过大会损害图像质量。
此NVivo图像成像平台可用于眼表面以下结构的可视化，并可应用于各种眼科疾病的原位研究。
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概述
本文介绍了一种多光子显微镜平台，用于使用双荧光转基因小鼠模型进行活体小鼠眼表面成像。该系统允许实时观察眼表面内的各种细胞结构。
关键研究组成部分
科学领域
- 神经科学
- 眼科学
- 显微镜学
背景
- 多光子显微镜是一种强大的成像技术。
- 荧光转基因小鼠提供了细胞成分的增强可视化。
- 理解眼表面结构对各种研究应用至关重要。
- 无标记成像技术可以补充荧光方法。
研究目的
- 开发一种活体小鼠眼表面成像的方法。
- 利用定制的多光子显微镜进行增强可视化。
- 展示双荧光转基因小鼠在成像中的能力。
使用的方法
- 设置具有特定物镜和激光设置的多光子显微镜。
- 使用二向色镜和带通滤波器进行信号分离。
- 在加热显微镜台上对麻醉小鼠进行活体成像。
- 实时观察眼表面结构。
主要结果
- 成功成像细胞核、膜和神经纤维。
- 使用二次谐波发生进行基质结构的无标记成像。
- 展示多光子平台的有效性。
- 实时可视化能力得到验证。
结论
- 多光子显微镜平台对眼部成像有效。
- 双荧光转基因小鼠增强了眼部结构的可视化。
- 该方法可以推进眼部生物学和相关领域的研究。
常见问题
Executive Industry Relevance
This multiphoton microscopy platform enables live, label-free imaging of mouse ocular surface structures, providing critical insights into corneal and conjunctival biology for ophthalmic drug discovery. By visualizing cellular dynamics, stromal architecture, and vascular networks in vivo, the method supports mechanistic de-risking of therapeutic targets in preclinical models of ocular disease. The platform’s ability to image across the entire ocular surface with accurate positioning enhances predictive confidence in target validation and assay development pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by visualizing cell nuclei, membranes, nerve fibers, and capillaries in live ocular tissue.
- Operational Value: Supports functional target validation through real-time observation of stromal architecture via second harmonic generation and cellular dynamics in dual fluorescent reporters.
- Predictive Value: Enhances portfolio triage by providing quantitative, reproducible readouts of structural and cellular changes in disease-relevant ocular models.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems for downstream workflows by enabling high-resolution imaging of epithelial, stromal, endothelial, and vascular compartments.
- Operational Value: Addresses assay standardization and reproducibility through precise Z-stack acquisition, spectral separation of SHG and fluorescent signals, and stabilized eyeball positioning.
- Strategic Value: Highlights screening readiness and platform reuse by allowing imaging across the entire corneal and conjunctival surface with minimal phototoxicity.
Translational & Preclinical Research
- Scientific Value: Discusses disease relevance by enabling reconstruction of 3D capillary architecture in the conjunctiva and visualization of limbal epithelial layers.
- Operational Value: Describes continuity from discovery through preclinical validation by supporting intravital imaging with accurate positioning across ocular surface regions.
- Predictive Value: Addresses risk-adjusted advancement decisions by providing label-free stromal imaging and cellular mapping that inform mechanistic understanding of ocular pathology.
Pipeline & Workflow Integration
The method integrates into the discovery continuum from early target validation through preclinical imaging, supporting hypothesis testing and mechanistic de-risking in ophthalmic research.
- Discovery Biology: Explains how the method supports hypothesis testing, pathway clarification, and biological de-risking by enabling live imaging of cellular and structural dynamics in the ocular surface.
- Screening: Describes assay readiness, reproducibility, and quantitative outputs through Z-serial imaging, spectral signal separation, and stabilized imaging conditions.
- Analytics: Highlights measurements, readouts, or statistical outputs that help teams compare conditions, including SHG for collagen architecture and tdTomato/EGFP for membrane and nuclear localization.
- Translational Research: Connects the method to preclinical continuity or biomarker alignment by enabling 3D reconstruction of capillaries and epithelial layer mapping in disease-relevant models.
- Enterprise Reuse: Frames the method as a reusable capability rather than a single-use technique, adaptable to various ophthalmic disease models and imaging endpoints.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence, target validation, reduction of mechanistic ambiguity in ocular surface biology.
- Operational Value: Standardization, reproducibility, and scalability through customized eye holder, stereotaxic staging, and controlled imaging parameters.
- Strategic Value: Better go/no-go decisions, capital efficiency, and reduced late-stage biological risk by enabling early visualization of target engagement and tissue response.
- Portfolio Impact: Risk-adjusted prioritization and advancement decisions through quantitative, label-free, and fluorescent readouts across corneal and conjunctival compartments.
Implementation Considerations
- Required scientific expertise in multiphoton microscopy, ocular anatomy, and transgenic model handling.
- Instrumentation and analytical infrastructure needs including titanium sapphire laser, dichroic mirrors, band pass filters, motorized stage, and 3D reconstruction software.
- Cross-team standardization requirements for imaging protocols, signal detection settings, and image processing pipelines (e.g., Fiji plugins for noise reduction and contrast enhancement).
- Adaptation considerations across model systems, including adjustment of eyelid stabilization, ocular surface coupling gel, and excitation wavelengths for different fluorescent reporters.
- Practical limitations supported by source material: stabilizing the eyeball with moderate pressure without injury is critical, as insufficient or excessive pressure compromises image quality.
