Time-resolved Measurements Using the Agilent Cary Eclipse Fluorescence Spectrophotometer

Significance of the Topic

The measurement of time-resolved fluorescence is essential for tracking labeled biomolecules with high specificity and sensitivity. It overcomes limitations such as high background from autofluorescence and offers safety advantages over radioactive methods. Lanthanide-based probes, especially europium, enable life science applications ranging from immunoassays to DNA hybridization with improved signal-to-noise ratios.

Objectives and Overview

This article reviews the principles of time-resolved measurements using the Agilent Cary Eclipse Fluorescence Spectrophotometer, describes its technical features, illustrates optimization of delay and gate times, and demonstrates practical examples with europium complexes.

Methodology and Instrumentation

Time-resolved assays on the Cary Eclipse rely on an 80 Hz Xenon flashlamp that produces short light pulses and dark periods for detecting prompt and delayed emission. Measurements cover timescales from 0.1 ms to indefinite durations. Key features include:

• Steady-state and phosphorescence modes without accessory changes
• Automatic timing control to eliminate photodegradation
• Bright, small arc flashlamp optimizing excitation intensity

Key Results and Discussion

Optimization of delay time effectively removed short-lived background fluorescence, isolating the long-lived europium emission. Adjusting gate time increased emission signal intensity without affecting background. In a solid PMMA matrix, the instrument recorded 1000 decay points over 2 ms, yielding high signal-to-noise and enabling accurate rate and lifetime calculations (k = 2.842 ms–1, τ = 0.352 ms).

Benefits and Practical Applications

• Concentration-independent lifetime measurements for environmental and conformational insights
• Sensitive detection of complex biomolecular interactions such as LRET and binding assays
• Quantification of analytes including oxygen and antibiotics
• Compatible with routine QA/QC and research laboratories due to robust sample handling

Future Trends and Potential Applications

Advances may include integration with microfluidic platforms, development of new lanthanide chelates with tailored lifetimes, and coupling time-resolved data with machine learning to improve assay throughput and multiplexing capabilities.

Conclusion

The Agilent Cary Eclipse spectrophotometer delivers accurate, reproducible time-resolved fluorescence and phosphorescence measurements without sample degradation. Its precise timing, broad dynamic range, and compatibility with lanthanide probes make it a versatile tool for advanced analytical and life science applications.

References

1. Lakowicz JR Principles of Fluorescence Spectroscopy 3rd Ed Springer 2006
2. Diamandis EP Clinical Biochemistry 1988 21 139-150