哺乳动物细胞 原位 冷冻断层扫描的样品制备
December 15th, 2023
3.8K views
该方法为原 位 细胞冷冻断层扫描和相关光电子显微镜 （CLEM） 的电子显微镜 （EM） 网格的制备提供了一种可访问且灵活的协议。
本研究的范围是开发一种既可访问又可靠可重复的样品制备方法。通过实现这一点，直接细胞冷冻电子断层扫描的力量可用于回答各种生物学问题。电网准备的挑战是广泛的。
细胞不容易附着在网格上，操作会导致碳层损坏，从而导致可成像网格区域的丢失。此外，在不注意适当的样品制备条件的情况下，可以减少薄细胞突起的发生率。目前的协议通常需要专门的设备来解决接种的主要瓶颈，例如分别用于细胞附着和操作的微图案化和 3D 打印支架。
该协议为解决方案提供了更大的可访问性，同时仍然保持了这些专用工具用于下游应用的灵活性。
概述
该方法为原位细胞冷冻超分辨电子显微镜（cryotomography）和相关光学与电子显微镜（CLEM）的电子显微镜（EM）网格的制备提供了一种易于获取和灵活的协议。该协议解决了网格制备中的挑战，确保细胞附着并最大程度地减少对碳层的损害。
关键研究组成部分
科学领域
- 神经科学
- 细胞生物学
- 电子显微镜
背景
- 直接细胞冷冻电子显微镜断层扫描是生物研究的强大工具。
- 网格制备对成功的成像和分析至关重要。
- 当前的方法通常需要专门的设备，限制了可及性。
- 该协议旨在简化过程，同时保持灵活性。
研究目的
- 开发一种可重复和易于获取的EM网格制备方法。
- 提高冷冻电子显微镜断层扫描在生物学研究中的可用性。
- 解决细胞附着和网格完整性的常见挑战。
使用的方法
- 通过发光放电处理网格以增强细胞粘附。
- 应用牛纤连蛋白以促进细胞附着。
- HIV分子克隆的细胞接种和转染程序。
- 网格的急速冷冻以进行冷冻电子显微镜断层扫描成像。
主要结果
- 细胞成功附着在EM网格上，损伤最小。
- 使用冷冻相关显微镜观察HIV产生细胞。
- 在制备过程中有效保持网格完整性。
- 多次试验中展示了该协议的可重复性。
结论
- 开发的协议提高了EM网格制备的可及性。
- 它允许使用冷冻电子显微镜断层扫描有效地研究细胞过程。
- 未来的应用可以利用这种方法进行各种生物学探究。
常见问题
什么是冷冻电子显微镜断层扫描？
冷冻电子显微镜断层扫描是一种允许在接近原始条件下进行生物样品三维成像的技术。
为什么网格制备很重要？
正确的网格制备对于确保细胞附着和最大程度地减少损害至关重要，这会影响成像质量。
牛纤连蛋白如何帮助细胞附着？
牛纤连蛋白提供了一种粘性表面，增强细胞对EM网格的粘附。
该协议解决了哪些挑战？
它解决了与细胞附着、网格完整性和对专门设备的需求相关的问题。
这种方法可以用于其他类型的细胞吗？
是的，根据实验需求，该协议可以适应各种类型的细胞。
该协议的主要应用是什么？
该协议主要用于研究细胞过程和相互作用，使用冷冻电子显微镜断层扫描。
Executive Industry Relevance
Robust sample preparation for in situ cryotomography of mammalian cells is critical for enabling high-resolution structural studies in native cellular contexts. This protocol addresses key bottlenecks in grid handling, cell attachment, and reproducibility, directly impacting the reliability of downstream imaging and integrative analyses. Improved accessibility and standardization in sample preparation enhance predictive confidence and portfolio decision-making in early discovery and translational research.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct visualization of cellular ultrastructure for mechanistic de-risking.
- Supports functional target validation by preserving native cell environments.
- Facilitates hypothesis-driven interrogation of molecular assemblies in situ.
Screening & Assay Development
- Prepares validated biological samples for correlative imaging workflows.
- Standardizes grid preparation to ensure reproducibility and quantitative imaging outputs.
- Improves readiness for high-content screening and integrative microscopy platforms.
Translational & Preclinical Research
- Aligns imaging outputs with disease-relevant cellular models for translational continuity.
- Enables structural assessment of viral and cellular interactions in preclinical systems.
- Supports risk-adjusted advancement by providing high-fidelity morphological data.
Pipeline & Workflow Integration
This sample preparation protocol integrates at the interface of discovery biology and advanced imaging, supporting workflows from early mechanistic studies to preclinical model validation.
- Discovery Biology: Provides reproducible sample preparation for hypothesis testing and pathway clarification.
- Screening: Delivers standardized, high-quality grids for quantitative imaging and assay development.
- Analytics: Enables acquisition of robust structural data for comparative analysis across experimental conditions.
- Translational Research: Bridges discovery and preclinical research by maintaining cellular context in imaging outputs.
- Enterprise Reuse: Establishes a flexible, accessible protocol adaptable to diverse cell types and imaging modalities.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces ambiguity in structural target validation.
- Operational Value: Enhances reproducibility, standardization, and scalability of sample preparation workflows.
- Strategic Value: Improves go/no-go decision quality and capital efficiency by reducing imaging failures.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of structurally validated targets.
Implementation Considerations
- Requires expertise in cell culture, grid handling, and microscopy techniques.
- Needs access to glow discharge devices, biosafety cabinets, and plunge-freezing instrumentation.
- Demands cross-team standardization for reproducible grid preparation and imaging quality.
- Adaptable to various adherent mammalian cell types with protocol modifications as needed.
- Grid fragility and manipulation remain practical limitations requiring careful handling.
Biopharma FAQs
Why does null hypothesis testing matter for grid preparation reproducibility?
Null hypothesis testing ensures that observed differences in imaging quality or cell attachment are statistically significant, supporting reliable target validation and reducing false positives in early discovery.
How does independent variable isolation improve cell seeding on EM grids?
Isolating variables such as fibronectin concentration or incubation time allows teams to optimize cell attachment and minimize grid damage, enhancing reproducibility across experiments and supporting robust assay development.
What do quantitative dependent variable measurements enable in cryotomography workflows?
Quantitative measurements of cell density and grid integrity enable standardized adjustments to blotting and freezing parameters, ensuring consistent imaging outputs and facilitating cross-study comparisons.
Why are replication requirements critical for cross-functional imaging teams?
Replication ensures that sample preparation protocols yield consistent results across different operators and labs, supporting collaborative assay development and enterprise-wide adoption of imaging standards.
What statistical analysis capabilities are required before implementing new grid preparation protocols?
Teams must be able to analyze variance in cell attachment, grid integrity, and imaging outcomes to validate protocol robustness and inform go/no-go decisions for broader R&D integration.
