Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy
July 15th, 2021
We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.
Overview
This study presents a protocol for preparing ribbons of serial sections for Array Tomography analysis using automated imaging in a scanning electron microscope (SEM). The method facilitates the effective screening and retrieval of localized, rare events within large sample volumes, significantly reducing data recording times compared to traditional whole-tissue block methods.
Key Study Components
Research Area
- Electron microscopy
- Image acquisition techniques
- Sample preparation methodologies
Background
- Complexity of electron microscopy data analysis
- Importance of targeted imaging for rare structures
- Need for improved efficiency in sample processing
Methods Used
- Preparation of ultrathin serial sections
- Utilization of an ultramicrotome and diamond knives
- Automated SEM imaging protocols
Main Results
- Streamlined protocol for preparing and imaging serial sections
- Reduced imaging time with enhanced data analysis capabilities
- Effectiveness of the method in identifying target structures
Conclusions
- The protocol demonstrates significant advantages for acquiring electron microscopy data
- Provides a diagnostic tool for scientific inquiries into cellular structures
Frequently Asked Questions
Executive Industry Relevance
Array tomography in SEM enables precise localization of target structures within large sample volumes, reducing unnecessary data acquisition and improving efficiency in discovery workflows. This approach supports rapid identification of rare cellular events, enhancing predictive confidence in target validation and mechanistic de-risking. The method facilitates translational continuity by preserving samples for repeated imaging and enabling scalable, reproducible analysis across discovery stages.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses through precise localization of target areas in serial sections.
- Operational Value: Reduces data acquisition time by focusing imaging on relevant fractions of sample volume.
- Predictive Value: Supports biological de-risking by providing nanometer-resolution structural insights for organelle-level questions.
Screening & Assay Development
- Scientific Value: Facilitates preparation of validated biological systems for downstream SEM imaging workflows.
- Operational Value: Enables array sectioning on wafers for standardized, repeatable sample preparation.
- Scalability: Supports creation of section libraries ready for repeated imaging and automated acquisition.
Translational & Preclinical Research
- Translational Continuity: Conserves array tomography samples during imaging, allowing reuse for validation studies.
- Mechanistic De-risking: Provides quantitative structural data on mitochondria, nuclei, and microvilli to inform preclinical decisions.
- Predictive Confidence: Enables alignment of imaging data with disease-relevant systems through high-resolution parameter screening.
Pipeline & Workflow Integration
The array tomography workflow integrates into the discovery continuum by enabling early target localization, supporting lead identification through targeted high-resolution imaging, and informing preclinical validation via reproducible structural analysis.
- Discovery Biology: Supports hypothesis testing and pathway clarification by identifying rare events such as mitotic divisions in tissue samples.
- Screening: Delivers assay readiness through low-resolution overview imaging to localize areas of interest before high-resolution acquisition.
- Analytics: Generates quantitative dependent variable measurements via mosaic maps and aligned stacks for comparative condition analysis.
- Translational Research: Connects discovery to preclinical continuity through rendered sections that support biomarker alignment and structural validation.
- Enterprise Reuse: Positions the method as a reusable capability via section libraries stored in tightly-closed boxes and oven-stabilized for repeated imaging.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in target validation through nanometer-resolution imaging of cellular and structural details.
- Operational Value: Standardization and reproducibility via automated SEM imaging protocols and section finder tools.
- Strategic Value: Better go/no-go decisions by reducing late-stage biological risk through early structural de-risking.
- Portfolio Impact: Risk-adjusted prioritization enabled by efficient acquisition of volume information from large sample surfaces.
Implementation Considerations
- Requires expertise in ultramicrotome operation, diamond knife handling, and wafer preparation techniques.
- Depends on instrumentation including ultramicrotome, plasma cleaner, syringe-based humidity control, and SEM with optical camera.
- Necessitates cross-team standardization of section thickness (50–100 nm), cutting speed (6–1 mm/s), and water basin protocols.
- Involves adaptation considerations for varying block size, tissue homogeneity, and resin type affecting ribbon straightness.
- Includes practical limitations such as the need for manual ribbon detachment using eyelash tips and water retraction timing for delicate samples.
Biopharma FAQs
Chapters in this video
0:04
Introduction
0:46
Array Generation
1:42
Sample Sectioning
2:23
Array Sectioning and Transfer
3:53
Scanning Electron Microscopy (SEM)
4:16
Imaging Region Definition
6:37
Auto Function Configuration and Image Acquisition
7:27
Results: Representative Targeted Acquisition of Volume Information
9:02
Conclusion
