Imaging of Live Cells in Water Using an Agilent 620 FTIR Microscope and an Agilent Cary 670 FTIR System Equipped with Standard Thermal Source

Importance of the Topic

FTIR chemical imaging offers simultaneous spatial and molecular insights into live cells under native, aqueous conditions. By preserving hydration and biochemical integrity, this approach enables real-time investigation of dynamic cellular processes such as metabolism, stress response, and cell division without labels or stains.

Objectives and Study Overview

This work demonstrates that a benchtop Agilent Cary 670 FTIR spectrometer coupled to an Agilent Cary 620 FTIR imaging microscope, both equipped with a standard thermal source, can perform live-cell imaging in water. The study focuses on Micrasteris hardyi algal cells to compare performance against synchrotron-based systems.

Methodology and Instrumentation

Sample Preparation:

• Micrasteris hardyi cells transferred into a liquid transmission cell featuring two 1 mm CaF₂ windows and a 7 µm Teflon spacer.

Instrument Configuration:

• Agilent Cary 670 FTIR spectrometer with thermal IR source.
• Agilent Cary 620 FTIR imaging microscope with matched 15×, 0.62 NA objectives and 21 mm working distance.
• 64×64 FPA detector (4 096 pixels), 1.1 µm pixel size in high-magnification transmission mode. Optionally 128×128 FPA for faster area coverage.
• Spectral resolution: 8 cm⁻¹; scans: 256 co-added per tile.
• 3×3 mosaic covering 210×210 µm in approximately one hour.

Main Results and Discussion

Infrared spectra of live algal cells reveal distinct bands for proteins, lipids, carbohydrates, and CH groups, with water contributions mathematically removed. Chemical images generated by integrating:

• CH stretch (∼2 928 cm⁻¹) highlights overall biomass.
• Lipid ester carbonyl (∼1 713 cm⁻¹) maps lipid inclusions, correlating to visible dark spots.
• Amide I band (∼1 640 cm⁻¹) visualizes protein distribution.
• Carbohydrate band (∼1 109 cm⁻¹) discriminates live cells from ‘ghost’ cells.

Composite RGB overlays combine lipid, CH, and protein signals. The 1.1 µm pixels approach the mid-IR diffraction limit (4–10 µm), enabling clear visualization of subcellular features and appendages.

Benefits and Practical Applications

By eliminating the need for synchrotron sources, this benchtop FTIR platform reduces cost and access barriers. Applications include:

• Monitoring cellular responses to environmental stressors (salinity, temperature, chemicals, drugs).
• Investigating cell division and metabolic dynamics in real time.
• Label-free chemical phenotyping in life science, pharmaceutical, and QA/QC contexts.

Future Trends and Opportunities

Advances may include faster, larger FPA detectors for higher throughput live-cell mapping; integration with microfluidic platforms for dynamic perturbation studies; coupling with machine learning for automated spectral classification; and expanding to mammalian and microbial systems to broaden biomedical and environmental applications.

Conclusion

This study establishes that a thermal-source benchtop FTIR microscope system can image live cells in water with high chemical specificity and micro-scale resolution. The accessible design preserves native cellular chemistry, opens new possibilities for dynamic life science research, and removes the reliance on synchrotron infrastructure.

Reference

No formal literature references were provided in the source application note.