Biomolecules, Cells and Tissue Studied by IR-Spectroscopy

Importance of the Topic

Infrared (IR) spectroscopy, especially Fourier-transform IR (FT-IR), is a cornerstone technique in analytical chemistry and pharmaceuticals for characterizing molecular vibrations and identifying substances. In recent years, its non-invasive application to life sciences has revolutionized the analysis of proteins, cells, and tissues. By monitoring conformational changes, aggregation events, and spatial distributions of biomolecules without labels or stains, IR spectroscopy supports drug development, formulation optimization, and disease diagnostics.

Objectives and Overview of the Study

This application note (AN B407) demonstrates how FT-IR spectroscopy and imaging can be applied to:

• Monitor protein stability and conformational changes during formulation development.
• Track early-stage aggregation and denaturation in liquid and solid biopharmaceutical formulations.
• Identify microorganisms at strain level using IR fingerprinting.
• Map the distribution of drugs, proteins, lipids, and polysaccharides in tissue sections.

Methodology and Instrumentation

FT-IR spectroscopy exploits selective absorption of mid-infrared light by molecular vibrations, following Lambert–Beer law. State-of-the-art FT-IR spectrometers rapidly acquire high-quality spectra across the full mid-IR range. Modern FT-IR microscopes equipped with multielement detectors enable hyperspectral imaging with spatial resolutions down to 0.5 µm in tissue sections. Key instrumentation includes:

• FT-IR spectrometer with rapid-scanning interferometer.
• FT-IR microscope (Bruker Hyperion 3000) with multielement focal plane array detector.
• Optionally integrated fluorescence channels for simultaneous localization of labeled active agents.

Main Results and Discussion

Protein Conformation and Formulation Stability:

• FT-IR spectra reveal amide I/II band changes corresponding to α-helix-to-β-sheet transitions during denaturation.
• Early detection of conformational shifts precedes aggregate formation detected by classical size-exclusion chromatography.
• Both liquid formulations (complex buffers with sugars, polyalcohols, amino acids) and lyophilized powders can be analyzed directly.

Microbial Identification:

• Unique IR spectral fingerprints allow species- and strain-level discrimination of microorganisms without labels.
• This capability facilitates contamination tracking in pharmaceutical and food products.

Tissue Imaging:

• Hyperspectral IR imaging of 6–10 µm tissue cryosections yields full IR spectra at each 4 × 4 µm pixel.
• False-color mapping of characteristic absorption bands visualizes spatial distribution of proteins, lipids, polysaccharides, and active agents.
• Example: In a mouse kidney section, the active compound co-localizes with protein-rich regions, while lipids occupy distinct areas.

Benefits and Practical Applications

FT-IR spectroscopy and imaging offer:

• Non-destructive, label-free analysis of biomolecules in complex matrices.
• Rapid, sensitive detection of structural changes and aggregation in biopharmaceuticals.
• Spatially resolved biochemical mapping in tissues for histology, cancer research, and biomaterial characterization.
• Reliable microbial strain identification for quality assurance.

Future Trends and Potential Applications

Emerging developments in IR technology include higher-resolution focal plane arrays, faster data acquisition, and advanced chemometric algorithms for automated classification. Potential future applications encompass real-time monitoring of bioprocess streams, high-throughput screening of formulation libraries, and integration with multimodal imaging platforms for comprehensive tissue characterization.

Conclusion

FT-IR spectroscopy and imaging have become indispensable tools in analytical chemistry, particularly for life science applications. Their ability to detect molecular vibrations, monitor protein stability, identify microorganisms, and map biomolecular distributions in tissues provides critical insights for drug development, quality control, and biomedical research.

Reference

Application Note AN B407, Biomolecules, Cells and Tissue Studied by IR-Spectroscopy, Bruker Optics BOPT-01, 2021