Time-resolved Measurements Using the Agilent Cary Eclipse Fluorescence Spectrophotometer

Importance of the Topic

Time‐resolved fluorescence spectroscopy provides critical insights into molecular interactions and environments by separating short‐lived background signals from longer‐lived probe emissions. This capability greatly enhances sensitivity and selectivity in biochemical and life science assays, enabling precise quantification and detection of biomolecules without interference from autofluorescence.

Objectives and Article Overview

This technical overview presents the principles and advantages of the Agilent Cary Eclipse Fluorescence Spectrophotometer for time‐resolved measurements. It explains how pulsed xenon illumination and precise timing parameters eliminate background signals, reviews applications in lanthanide‐based assays, and demonstrates instrument performance through worked examples involving europium (Eu3+) probes.

Methodology and Instrumentation

The Cary Eclipse employs an 80 Hz xenon flashlamp to provide intense, short light pulses with a small arc size, ensuring high brightness for fluorescence excitation. Two detection modes are available:

• Prompt fluorescence mode captures steady‐state emission immediately after each lamp pulse.
• Phosphorescence (delayed fluorescence) mode introduces a user‐defined delay before detection, allowing short‐lived background signals to decay.

Key timing parameters include:

• Delay time: interval between lamp pulse and the start of data collection.
• Gate time: duration over which emitted light is collected.

The main instrument used is the Agilent Cary Eclipse Fluorescence Spectrophotometer with WinFLR software for data acquisition and lifetime analysis.

Main Results and Discussion

Worked examples demonstrate the separation of background emission from Eu3+ ligand signals by adjusting delay and gate times. Increasing delay time from 0 to 100 μs eliminates short‐lived background peaks around 450 nm while preserving the Eu3+ emission at 615 nm. Adjusting gate time from 40 to 200 μs enhances the intensity of the long‐lived Eu3+ signal without affecting prompt fluorescence. Time‐resolved decay measurements of Eu3+ in a PMMA block collected with 50 μs and 2 μs gate times show excellent signal‐to‐noise and allow decay‐curve averaging. Lifetime fitting yields a decay constant (k1) of 2.842 ms⁻¹ and a lifetime (TAU1) of 0.352 ms, demonstrating rapid data collection of millisecond‐scale decays.

Benefits and Practical Applications

Time‐resolved assays using the Cary Eclipse offer:

• Elimination of sample photodegradation and autofluorescence interference.
• Highly reproducible timing for quantitative lifetime and intensity measurements.
• Simultaneous acquisition of prompt and delayed emissions from a single sample without hardware changes.
• Enhanced sensitivity for immunoassays, receptor‐ligand binding studies, enzyme assays, DNA hybridization, and cell‐based assays.

The independence of lifetime measurements from concentration and intensity provides information on local viscosity, polarity, molecular conformations, and binding events.

Future Trends and Opportunities

Advances in pulsed‐light sources and detector electronics will push time‐resolved spectroscopy towards sub‐microsecond lifetimes and higher throughput. Integration with automation and microfluidic platforms will enable rapid screening of ligand‐biomolecule interactions. Emerging applications include Förster resonance energy transfer (FRET) assays, high‐content imaging with temporal resolution, and real‐time monitoring of dynamic processes in live cells.

Conclusion

The Agilent Cary Eclipse Fluorescence Spectrophotometer offers a versatile and reliable platform for time‐resolved fluorescence and phosphorescence measurements. Its precise control over delay and gate times, combined with robust software for lifetime analysis, ensures maximal sensitivity and specificity in complex biological and material science applications.

References

• Lakowicz JR. Chapter 3: Fluorescence Anisotropy. In Principles of Fluorescence Spectroscopy, 3rd ed. Springer Science+Business Media, New York (2006).
• Diamandis EP. Clin Biochem. 1988;21:139–150.