Anisotropy Measurement Using the Agilent Cary Eclipse Fluorescence Spectrophotometer

Significance of the Topic

Fluorescence anisotropy is a non destructive analytical technique that yields precise information on molecular size, shape, and mobility. Its intrinsic robustness against sample degradation and environmental fluctuations makes it essential for studies in life sciences, materials research, polymer science and beyond.

Objectives and Study Overview

This technical overview describes how the Agilent Cary Eclipse Fluorescence Spectrophotometer, combined with Cary WinFLR software, enables accurate anisotropy measurements without photodegradation. Authors Dr Fabian Zieschang, Katherine McNamara and Kevin Grant demonstrate key applications and compare results across different sample environments.

Methodology and Instrumentation

All measurements employ polarized excitation and emission beams using interchangeable polarizers mounted before and after the sample. The Cary Eclipse lamp flashes only when acquiring data points, preventing continuous illumination and sample damage. Cary WinFLR software guides the user step by step, records spectra, performs anisotropy calculations and toggles measurement modes.

Main Results and Discussion

Two example studies illustrate method performance:

• A rhodamine B probe in PMMA, ethylene glycol and water shows anisotropy values that correlate with medium viscosity: highest in rigid PMMA, intermediate in viscous glycol and lowest in fluid water.
• Temperature dependent analysis of rhodamine B in PMMA and glycol reveals that anisotropy remains constant in the rigid thermoplastic across –10 to 100 °C, whereas it decreases in glycol as molecular mobility increases with temperature. Intensity changes alone would mislead interpretation without anisotropy data.

Benefits and Practical Applications

Fluorescence anisotropy on the Cary Eclipse system offers:

• Non destructive measurement of photosensitive samples
• High precision and repeatability free from lamp induced photobleaching
• Insights into molecular interactions, dynamics and environment without complex calibration

Future Trends and Possibilities

Advances may include high throughput anisotropy screening, integration with microfluidics and single particle analysis. Coupling anisotropy with time resolved and super resolution techniques could open new avenues in biophysics, nanomaterials and drug discovery.

Conclusion

The Agilent Cary Eclipse Fluorescence Spectrophotometer enables reliable anisotropy measurements across diverse sample types. Its flash lamp design and intuitive software ensure accurate, photobleaching free data, driving progress in polymer science, molecular biology, immunology and materials research.

Reference

1. Gavin P, Prescott M, Fyfe DJ, Comerford JJ. Minimizing photobleaching of Blue Fluorescent Protein using the Agilent Cary Eclipse fluorescence spectrophotometer. Agilent Technologies Application Note 5990-7791EN (2011).
2. Lakowicz JR. Chapter 10 Fluorescence Anisotropy. In Principles of Fluorescence Spectroscopy, 3rd Edn. Springer Science Business Media, LLC, New York (2006).