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

Fourier transform infrared (FTIR) chemical imaging offers simultaneous spatial resolution and chemical identification of biological macromolecules in situ. Achieving real-time imaging of live cells in their native aqueous environment preserves physiological conditions, avoids dehydration artifacts, and provides more accurate insights into cellular processes. Recent advances in benchtop instrumentation remove reliance on synchrotron sources, making live‐cell FTIR imaging more accessible to academic and industrial laboratories.

Study Objectives and Overview

This application note demonstrates the capability of the Agilent Cary 670 FTIR spectrometer paired with the Agilent Cary 620 FTIR imaging microscope to perform label‐free, high‐resolution imaging of live Micrasterias hardyi algal cells in water. Key aims include:

• Validating real‐time chemical imaging in an aqueous background without synchrotron IR.
• Mapping biochemical distributions (proteins, lipids, carbohydrates) at the cellular level.
• Assessing spatial resolution, image quality, and measurement throughput.

Instrumentation Used

The imaging platform comprised:

• Agilent Cary 670 FTIR spectrometer with standard thermal IR source.
• Agilent Cary 620 FTIR imaging microscope with matched 15×, 0.62 NA objectives (21 mm working distance).
• 64×64 focal plane array (FPA) detector yielding 4 096 spectra per mosaic; optional 128×128 FPA for larger area.
• Liquid transmission cell with two 1 mm CaF₂ windows and a 7 µm Teflon spacer to maintain live‐cell hydration.

Methodology

Micrasterias cells were transferred from growth medium into a CaF₂ liquid cell by pipette. Measurements were conducted in transmission mode at 8 cm⁻¹ spectral resolution, co‐adding 256 scans per tile. A 3×3 tile mosaic covered a 210×210 µm field in ~1 hour (280×280 µm in ~30 minutes with the larger FPA). Pixel size on the sample plane was 1.1 µm, operating at the diffraction limit for mid‐IR wavelengths.

Main Results and Discussion

The mid‐IR spectrum of live Micrasterias revealed distinct bands assignable to proteins (∼1 640 cm⁻¹), lipids (∼1 713 cm⁻¹), carbohydrates (1 180–1 000 cm⁻¹), and C–H groups (∼2 928 cm⁻¹), with water background mathematically removed. False‐color chemical images highlighted:

• Lipid inclusions correlating with visible dark domains.
• Protein and C–H distributions defining cellular substructures.
• Carbohydrate band variations distinguishing live cells from emptied “ghost” cells.

These results confirm high chemical specificity and ≈1 µm pixel resolution, sufficient to resolve ∼8 µm cell appendages. The benchtop system enabled dynamic studies of live‐cell responses without dehydration.

Practical Benefits and Applications

Label‐free FTIR imaging of live cells in water offers:

• Preservation of native cellular chemistry and morphology.
• Non‐destructive, real‐time monitoring of dynamic events (cell division, stress responses, drug effects).
• Cost and accessibility advantages over synchrotron‐based methods.
• Potential integration into QA/QC workflows, pharmaceutical screening, and fundamental cell biology research.

Future Trends and Opportunities

Advances in mid‐IR detector technology, faster scanning strategies, and multimodal imaging integration are expected to push spatial and temporal resolution further. Emerging applications include high‐throughput drug screening, live‐cell metabolomics, and coupling FTIR imaging with complementary optical modalities for comprehensive phenotypic profiling.

Conclusion

The combination of the Agilent Cary 670 FTIR spectrometer and Cary 620 imaging microscope enables practical, label‐free chemical imaging of live cells in aqueous media on a benchtop platform. This approach preserves native chemistry, delivers micron‐scale spatial resolution, and facilitates real‐time monitoring of cellular processes without the constraints of synchrotron access.

References

No formal literature references were cited in this application note.