Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis
February 1st, 2022
Stimulated Raman scattering (SRS) microscopy is a powerful, nondestructive, and label-free imaging technique. One emerging application is stimulated Raman histology, where two-color SRS imaging at the protein and lipid Raman transitions are used to generate pseudo-hematoxylin and eosin images. Here, we demonstrate a protocol for real-time, two-color SRS imaging for tissue diagnosis.
Overview
Stimulated Raman scattering (SRS) microscopy is a label-free imaging technique that allows for real-time tissue diagnosis. This protocol demonstrates the implementation of two-color SRS imaging for stimulated Raman histology applications.
Key Study Components
Area of Science
- Neuroscience
- Biophysics
- Imaging Techniques
Background
- SRS microscopy is a nondestructive imaging method.
- It enables the generation of pseudo-hematoxylin and eosin images.
- The technique does not require tissue processing or staining.
- It can be applied to various samples including small molecules and cells.
Purpose of Study
- To optimize spectral focusing SRS microscopy.
- To demonstrate its application in real-time stimulated Raman histology.
- To provide a protocol for researchers to follow.
Methods Used
- Installation of achromatic lenses for pump beam magnification.
- Alignment of lenses at specific distances for optimal performance.
- Real-time imaging of tissue samples.
- Application of two-color SRS imaging techniques.
Main Results
- Demonstrated the speed of processing samples with SRS microscopy.
- Showed the effectiveness of two-color imaging for histology.
- Validated the technique's applicability beyond histology.
- Provided a clear protocol for implementation.
Conclusions
- SRS microscopy is a valuable tool for real-time tissue diagnosis.
- The protocol can enhance the efficiency of histological studies.
- Future applications may extend to various biological samples.
Frequently Asked Questions
Executive Industry Relevance
Real-time, two-color stimulated Raman scattering (SRS) imaging enables rapid, label-free tissue characterization, directly supporting high-confidence decision points in discovery and translational research. The protocol's ability to generate H&E-equivalent images without staining accelerates tissue analysis and de-risks biological interpretation in preclinical models. This capability enhances predictive confidence and operational efficiency across early discovery and pathology-driven workflows.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct visualization of molecular features in tissue without exogenous labels.
- Supports functional target validation by distinguishing lipid and protein distributions in situ.
- Facilitates mechanistic de-risking by providing rapid, quantitative tissue readouts.
- Improves predictive confidence for target engagement and biological effect.
Screening & Assay Development
- Prepares validated, label-free imaging systems for downstream compound screening.
- Delivers reproducible, quantitative outputs for assay standardization.
- Enables high-throughput, real-time imaging for scalable screening platforms.
- Supports reliable evaluation of compound effects on tissue composition.
Translational & Preclinical Research
- Aligns imaging outputs with disease-relevant tissue biomarkers for translational continuity.
- Provides rapid, intraoperative-style tissue assessment for preclinical validation.
- Reduces risk of late-stage biological failure by improving tissue characterization.
- Supports continuity from discovery through preclinical decision-making.
Pipeline & Workflow Integration
This SRS imaging protocol integrates from early discovery through preclinical research, enabling seamless transition from hypothesis testing to translational validation.
- Discovery Biology: Accelerates hypothesis testing and pathway clarification via direct, label-free tissue imaging.
- Screening: Provides reproducible, quantitative imaging outputs for assay readiness and compound evaluation.
- Analytics: Generates high-content, spectroscopic readouts for robust statistical comparison of experimental conditions.
- Translational Research: Bridges discovery and preclinical validation with disease-relevant, H&E-mimetic imaging.
- Enterprise Reuse: Offers a broadly applicable imaging platform for diverse tissue and molecular targets.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in tissue-based studies.
- Operational Value: Delivers rapid, standardized, and scalable imaging workflows without staining or processing.
- Strategic Value: Enables faster go/no-go decisions and reduces late-stage biological risk.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of discovery and translational programs.
Implementation Considerations
- Requires expertise in advanced optical alignment and laser instrumentation.
- Demands access to femtosecond or picosecond laser sources and lock-in detection systems.
- Necessitates rigorous cross-team standardization for reproducible imaging outputs.
- Adaptable to various tissue types and molecular targets with protocol optimization.
- Complexity of pulse chirping and modulation may limit immediate scalability without technical support.
Biopharma FAQs
Chapters in this video
0:04
Introduction
0:49
Beam Collimation
1:32
Spatial Overlap
2:34
Electrooptical Modulation (EOM)
3:35
Temporal Overlap
5:40
Signal to Noise (SNR) Characterization, Frequency Axis Calibration, and Calibration of Spectral Resolution
7:23
EOM1 Modulation and Epi-Mode SRS Imaging
8:53
Results: Optimizing Spectral Resolution, Two-Color SRS, and False Two-Color H&E Staining of SRS Images
10:14
Conclusion
