简化活体成像方法，用于在倒置共聚焦显微镜上长期监测上皮组织动力学
June 30th, 2023
2.3K views
该协议提供了一种新工具，可使用倒置共聚焦显微镜简化活体成像。
我们开发了一种工具，使用倒置共聚焦显微镜对活体小鼠进行活体成像。这使我们能够跟踪整个稳态过程中的皮肤细胞动力学，并比较疾病发作和进展期间的细胞行为。多光子显微镜是实现活细胞追踪的主要方法。
然而，这些显微镜的缺点包括日常成像多功能性有限、成本高和技术复杂性。我们的新工具支持使用多功能倒置共聚焦显微镜进行活体成像。通过修改设计文件，可以定制载物台插件尺寸，以适应任何品牌的倒置显微镜。
物镜孔可以调整大小和重新定位，以适应任何选择的动物模型和组织。
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概述
本研究介绍了一种新工具，用于在活体小鼠中使用倒置共聚焦显微镜进行体内成像，使研究人员能够在稳态和疾病进展期间跟踪皮肤细胞动态。该新工具克服了多光子显微镜的局限性，使体内成像更加易用和多样化。
关键研究组成部分
研究领域
- 体内成像
- 皮肤细胞动态
- 显微镜技术
背景
- 多光子显微镜的当前局限性包括高成本和技术复杂性。
- 倒置共聚焦显微镜为活体组织提供了多样化的成像选择。
- 定制设计允许适应不同的动物模型。
使用的方法
- 具有定制3D打印插件的倒置共聚焦显微镜
- 活体小鼠作为生物系统
- 可定制的成像设置，适用于各种组织
主要结果
- 成功获取了标有荧光的耳组织的Z堆叠，漂移最小。
- 长时间持续成像皮肤细胞（mCherry和GFP阳性）。
- 验证了3D打印活体成像插件的稳定成像效果。
结论
- 本研究展示了一种改进体内成像能力的创新方法。
- 该工具增强了在活体模型中研究细胞动态的能力，推动了细胞生物学研究的进展。
常见问题
新工具相对于传统方法的主要优势是什么？
新工具简化了体内成像的设置，并使用了倒置共聚焦显微镜，比多光子显微镜更具多样性。
成像插件如何适应不同的显微镜？
台架插件的设计可以修改以适应各种品牌的倒置显微镜和不同类型的组织。
这种方法可以观察到什么类型的细胞行为？
该方法允许观察稳态期和疾病进展过程中的皮肤细胞动态。
本研究使用了哪些生物模型？
研究使用活体小鼠作为体内成像实验的生物模型。
成像会话可以持续多长时间而不会损失数据质量？
系统支持延长成像会话，Z堆叠采集验证了几小时内最小漂移。
研究中使用了哪些荧光标记物？
使用mCherry和GFP荧光标记物对小鼠耳组织进行标记以便成像。
这项研究对未来研究有什么影响？
本研究增强了体内成像的方法学工具包，可能加速细胞生物学研究的进展。
Executive Industry Relevance
Long-term intravital imaging of epithelial tissue dynamics is critical for understanding cellular behaviors underlying disease initiation and progression. This streamlined approach using inverted confocal microscopy lowers technical and cost barriers, enabling broader adoption of in vivo imaging in discovery-stage biopharma research. Enhanced accessibility to stable, quantitative live-cell imaging supports predictive confidence in early target validation and mechanistic studies.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct observation of live cell behaviors in intact tissue environments.
- Supports mechanistic de-risking by tracking cellular responses during disease onset.
- Facilitates functional target validation through longitudinal monitoring of cell dynamics.
- Improves predictive confidence for early-stage portfolio triage.
Screening & Assay Development
- Provides validated, reproducible imaging of fluorescently labeled cells in vivo.
- Delivers quantitative Z-stack outputs for robust assay development.
- Supports standardization and scalability across multiple tissue types and animal models.
- Enables reliable evaluation of compound effects on tissue-level cell dynamics.
Translational & Preclinical Research
- Aligns in vivo imaging outputs with disease-relevant models for translational continuity.
- Enables risk-adjusted advancement decisions based on real-time tissue responses.
- Supports biomarker discovery by correlating dynamic cell behaviors with disease progression.
- Provides mechanistic insights that inform preclinical model selection.
Pipeline & Workflow Integration
This imaging platform integrates from early discovery through preclinical research, supporting hypothesis testing, target validation, and translational studies.
- Discovery Biology: Enables hypothesis-driven interrogation of cell dynamics in native tissue context.
- Screening: Provides reproducible, quantitative imaging outputs for assay readiness.
- Analytics: Supports longitudinal measurement of cellular behaviors and tissue stability.
- Translational Research: Bridges discovery and preclinical validation with disease-relevant imaging data.
- Enterprise Reuse: Customizable design allows adaptation across platforms and organ systems.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in live tissue studies.
- Operational Value: Streamlines imaging workflows with standardized, reusable components.
- Strategic Value: Enables better go/no-go decisions and capital efficiency by reducing technical barriers.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of discovery programs.
Implementation Considerations
- Requires expertise in live animal handling and confocal microscopy operation.
- Needs access to inverted confocal microscopes and compatible 3D-printed stage inserts.
- Demands cross-team standardization for imaging protocols and data analysis.
- Adaptable to various animal models and tissue types with design modifications.
- Practical limitations include maintaining animal viability and imaging stability over extended sessions.
Biopharma FAQs
Why does null hypothesis testing matter for intravital imaging-based target validation?
Null hypothesis testing in live imaging experiments enables objective assessment of whether observed cell dynamics differ significantly between homeostasis and disease states, supporting robust target validation decisions.
How does independent variable isolation fit into the inverted confocal imaging workflow?
Isolating variables such as genetic background or treatment conditions during imaging allows clear attribution of observed cellular changes to specific interventions, strengthening mechanistic insights in the discovery pipeline.
What do quantitative Z-stack measurements enable in live tissue imaging?
Quantitative Z-stack acquisition provides high-resolution, time-resolved data on cell position and morphology, enabling precise comparison of tissue dynamics across experimental groups.
Why are replication requirements critical for cross-functional imaging studies?
Replication ensures that observed cell behaviors are consistent and reproducible, facilitating reliable data sharing and interpretation across discovery, screening, and translational teams.
What statistical analysis capabilities are needed before implementing longitudinal intravital imaging?
Robust statistical tools are required to analyze time-lapse imaging data, assess drift, and compare dynamic cellular responses, ensuring data integrity for downstream R&D decisions.
