Targeted Studies Using Serial Block Face and Focused Ion Beam Scan Electron Microscopy
August 10th, 2019
Here, we present a protocol for efficiently combining serial block face and focused ion beam scanning electron microscopy to target an area of interest. This allows for efficient searching, in three dimensions, and locating rare events in a large field of view.
Overview
This study introduces a protocol that efficiently combines serial block face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) for three-dimensional ultrastructural investigation in biological samples. It is applicable to various biological model systems, allowing researchers to locate rare events in large fields of view.
Key Study Components
Research Area
- 3D ultrastructure investigation
- Microscopy techniques for biological samples
- Protocol development for targeting areas of interest
Background
- Utilization of SBF-SEM and FIB-SEM imaging modalities
- Sample preparation efficiency enhanced by microwave assistance
- First application demonstrated with root tips of Arabidopsis thaliana
Methods Used
- Serial block face and focused ion beam scanning electron microscopy
- Arabidopsis thaliana root tips as biological models
- Sample preparation involving multiple chemical treatments and dehydration steps
Main Results
- Successful imaging of the tissue revealing spatial orientations of cells
- Detailed imaging of specific cellular structures
- Correlative data between SBF-SEM and FIB-SEM methods
Conclusions
- The study presents a robust protocol for integrating two advanced imaging techniques.
- This approach enhances the understanding of complex cellular architectures in biological research.
Frequently Asked Questions
Executive Industry Relevance
This protocol enables biopharma R&D teams to perform targeted 3D ultrastructural analysis of rare cellular events within complex tissues, supporting mechanistic de-risking in early discovery. By combining SBF-SEM for contextual overview and FIB-SEM for high-resolution detail, it improves predictive confidence in target validation and pathway elucidation. The workflow enhances translational continuity from discovery through preclinical stages by providing reproducible, quantitative structural data.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses through 3D visualization of subcellular architecture in disease-relevant systems.
- Operational Value: Supports biological de-risking by clarifying spatial relationships between cells and intracellular structures prior to lead identification.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems for downstream assay development by establishing baseline ultrastructural phenotypes.
- Operational Value: Enhances assay standardization and reproducibility through correlative imaging workflows that reduce variability in sample preparation.
Translational & Preclinical Research
- Scientific Value: Provides disease-relevant structural insights that align with translational biomarker discovery and mechanistic de-risking.
- Operational Value: Enables risk-adjusted advancement decisions by delivering quantitative, high-resolution data for preclinical model validation.
Pipeline & Workflow Integration
The method integrates into the discovery continuum by supporting hypothesis testing in early biology, enabling assay readiness through standardized preparation, and delivering quantitative readouts for cross-functional analytics.
- Discovery Biology: Facilitates target validation by revealing spatial orientations of cells and intracellular connections critical for pathway clarification.
- Screening: Ensures assay readiness via microwave-assisted processing that improves reproducibility and scalability of sample preparation.
- Analytics: Generates aligned 3D TIFF datasets that allow teams to compare structural conditions and track ROI-specific changes over time.
- Translational Research: Supports preclinical continuity by providing correlative SBF-SEM and FIB-SEM data that bridge discovery-scale context with subcellular detail.
- Enterprise Reuse: Establishes a reusable imaging capability for targeted ROI analysis across multiple projects and model systems.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in target validation through reduction of mechanistic ambiguity in 3D ultrastructure.
- Operational Value: Standardization and reproducibility via microwave-assisted processing and correlative workflow.
- Strategic Value: Improved go/no-go decisions by enabling capital-efficient, data-driven target prioritization.
- Portfolio Impact: Risk-adjusted advancement decisions supported by quantitative, reproducible structural outputs.
Implementation Considerations
- Requires expertise in electron microscopy techniques including SBF-SEM and FIB-SEM operation and sample preparation.
- Dependent on access to integrated SEM platforms with ultramicrotome and focused ion beam capabilities.
- Necessitates cross-team standardization of fixation, staining, and infiltration protocols for reproducible results.
- Involves adaptation considerations when applying the workflow to diverse biological models beyond plant systems.
- Limited by the labor-intensive nature of sample preparation despite microwave-assisted time reduction.
Biopharma FAQs
Chapters in this video
0:04
Title
0:51
Sample Fixation and Processing for Electron Microscopy
3:51
Prepare Embedded Samples for Imaging
5:03
Imaging in the SBF-SEM (Serial Block Face Scanning Electron Microscopy) and Data Processing
6:22
Imaging in the FIB-SEM (Focused Ion Beam SEM)
7:49
Results: SBF-SEM and FIB-SEM Data
8:46
Conclusion
