Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion
September 25th, 2019
7.3K views
Nanoscale imaging of clinical tissue samples can improve understanding of disease pathogenesis. Expansion pathology (ExPath) is a version of expansion microscopy (ExM), modified for compatibility with standard clinical tissue samples, to explore the nanoscale configuration of biomolecules using conventional diffraction limited microscopes.
Overview
Expansion pathology (ExPath) is a novel imaging technique that enhances the visualization of biomolecules in clinical tissue samples with nanoscale precision. By expanding tissue samples, ExPath allows for the separation and observation of previously overlapped fluorescently labeled biomolecules using conventional optical microscopes.
Key Study Components
Area of Science
- Neuroscience
- Pathology
- Imaging Techniques
Background
- Conventional light microscopy is limited by diffraction, hindering the observation of nanoscale structures.
- Expansion microscopy techniques have been adapted for clinical applications.
- ExPath enables the study of disease-related biomolecules in various tissue types.
- This method can provide insights into complex diseases such as cancer and autoimmune disorders.
Purpose of Study
- To develop a low-cost imaging method for clinical tissue sections.
- To facilitate the observation of nanoscale configurations of biomolecules.
- To enhance understanding of disease pathogenesis.
Methods Used
- Infusion of clinical tissue sections with a water swellable hydrogel.
- Physical expansion of treated samples by approximately one hundred fold.
- Use of conventional optical microscopy to observe expanded samples.
- Demonstration of critical steps, including gel chamber construction and sample handling.
Main Results
- Successful separation and observation of biomolecules that were previously overlapped.
- Demonstrated applicability to a broad array of tissue types.
- Enabled new insights into the pathogenesis of various diseases.
- Provided a visual demonstration of the method's critical steps.
Conclusions
- ExPath is a valuable tool for researchers studying disease-related biomolecules.
- This method does not require investment in new imaging hardware.
- ExPath fosters new diagnostic insights into complex diseases.
Frequently Asked Questions
What is expansion pathology?
Expansion pathology (ExPath) is a technique that allows for nanoscale imaging of biomolecules in clinical tissue samples.
How does ExPath improve imaging?
ExPath circumvents the diffraction limit of light microscopy by expanding tissue samples, allowing for better separation of biomolecules.
What types of diseases can ExPath help study?
ExPath can be applied to various diseases, including cancer, brain diseases, and autoimmune disorders.
Is new imaging hardware required for ExPath?
No, ExPath can be performed using conventional optical microscopes, eliminating the need for new hardware.
What are the critical steps in the ExPath process?
Key steps include the construction of the geling chamber and proper handling of the gelled samples.
Can ExPath be applied to different tissue types?
Yes, ExPath is versatile and can be used on a broad array of tissue types.
Executive Industry Relevance
Expansion pathology (ExPath) addresses the diffraction limit barrier in conventional optical microscopy, enabling nanoscale visualization of biomolecules in clinical tissue sections without new hardware investment. This capability supports early discovery workflows by revealing subtle pathological changes and molecular configurations relevant to disease mechanisms. By providing nanoscale precision using standard microscopes, ExPath enhances target validation confidence and mechanistic de-risking in preclinical research.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of nanoscale biomolecule configurations in disease-related targets such as Alpha Actinin 4 and Vimentin.
- Operational Value: Uses conventional wide-field or confocal microscopes, reducing dependency on specialized super-resolution equipment.
- Strategic Value: Supports hypothesis testing and pathway clarification through direct visualization of molecular interactions in tissue context.
Screening & Assay Development
- Scientific Value: Produces expanded, isotropic tissue-hydrogel hybrids that separate overlapped fluorescent signals for quantitative analysis.
- Operational Value: Compatible with FFPE and frozen tissue sections, enabling standardized preparation across diverse sample types.
- Strategic Value: Facilitates assay readiness by generating reproducible, flat, transparent gels suitable for high-content imaging.
Translational & Preclinical Research
- Scientific Value: Maintains antigenicity and structural integrity post-expansion, allowing multiplexed labeling of proteins, nucleic acids, and carbohydrates.
- Operational Value: Demonstrated applicability to kidney, breast, and neural tissues, supporting cross-disease target validation.
- Strategic Value: Provides mechanistic insights into pathogenesis of cancer, autoimmune, and neurodegenerative diseases through nanoscale imaging.
Pipeline & Workflow Integration
ExPath integrates into the discovery continuum from early target validation through preclinical evaluation by enabling nanoscale imaging of clinically relevant tissue specimens.
- Discovery Biology: Supports hypothesis testing via direct observation of biomolecule localization and co-localization in expanded tissue sections.
- Screening: Enables assay standardization through reproducible expansion factors (3-4.5x) and minimal signal distortion when protocols are followed.
- Analytics: Achieves effective resolution of ~63 nm with 0.95 NA objective, enabling quantitative measurement of nanoscale biomolecular distributions.
- Translational Research: Connects discovery findings to preclinical continuity by preserving tissue architecture and enabling validation in disease-relevant systems.
- Enterprise Reuse: Establishes a scalable, low-cost imaging capability applicable across multiple projects and disease areas without capital equipment expenditure.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence through direct nanoscale visualization of target engagement and pathway modulation.
- Operational Value: Reproducibility and standardization via defined steps for gelation, digestion, and expansion across tissue types.
- Strategic Value: Reduced biological risk in lead identification by confirming target presence and distribution at nanoscale resolution.
- Portfolio Impact: Informs go/no-go decisions through enhanced mechanistic understanding of disease models.
Implementation Considerations
- Requires expertise in histology, immunohistochemistry, and hydrogel-based sample preparation.
- Needs standard laboratory equipment including incubators, shakers, microscopes, and basic reagents (hydrogel monomers, Proteinase K, buffers).
- Demands cross-team standardization for consistent tissue processing, particularly in gel chamber construction and orientation tracking.
- Requires optimization across model systems due to variability in tissue density, fixation status, and antigen retrieval efficiency.
- Practical limitations include potential cracking or signal loss if anchoring or homogenization is inadequate, as noted in kidney and breast tissue controls.
Biopharma FAQs
Why does nanoscale separation of biomolecules matter for target validation?
Nanoscale separation via ExPath resolves overlapped fluorescent signals, enabling clear visualization of molecular targets like Alpha Actinin 4 and Vimentin in tissue sections. This precision supports confident target validation by revealing true biomolecule localization and co-localization patterns obscured in conventional imaging.
How does isotropic expansion fit into the discovery pipeline for target de-risking?
Isotropic expansion uniformly scales tissue sections ~3-4.5x in water, preserving spatial relationships while separating nanoscale biomolecules. This allows discovery teams to assess target distribution and pathway involvement in clinically relevant samples without artifacts from anisotropic distortion.
What quantitative measurements does expanded tissue imaging enable for assay development?
Expanded tissue sections permit quantitative fluorescence intensity measurements and spatial mapping of biomolecules at ~63 nm effective resolution. These outputs support assay standardization by providing measurable, reproducible readouts for target expression and localization across samples.
Why are replication requirements critical for cross-functional collaboration in ExPath workflows?
Replication ensures consistent expansion, minimal distortion, and reliable signal retention across tissue types and operators. Standardized replication supports cross-functional teams in pathology, discovery, and preclinical science by establishing trustworthy, comparable nanoscale imaging data.
What statistical analysis capabilities are needed before implementing ExPath in discovery workflows?
Implementation requires capability to analyze spatial distribution, co-localization coefficients, and intensity distributions from expanded tissue images. These analyses enable objective comparison of biomolecule patterns across conditions, supporting statistical rigor in target validation and mechanistic studies.
Chapters in this video
0:05
Title
1:19
Preparation of Archived and Freshly Prepared Clinical Tissue Slides for ExPath
5:16
Sample Digestion and Imaging
6:40
Results: Multicolor Super-resolution Tissue Imaging using Expansion Pathology
8:18
Conclusion
4:05
In Situ Polymerization of Specimens
