Resolution of multiple fluorescence lifetimes in heterogeneous systems by phase-modulation fluorometry

Area of Science:

• Photophysics and Fluorescence Spectroscopy
• Biophysical Chemistry
• Computational Chemistry

Background:

• Fluorescent systems often exhibit multiexponential decay, complicating lifetime analysis.
• Traditional methods for resolving heterogeneous lifetimes can be limited in accuracy and scope.

Purpose of the Study:

• To present novel computer procedures (FIT programs) for analyzing phase and modulation lifetime data.
• To demonstrate the utility of these programs in resolving lifetimes in complex fluorescent systems.

Main Methods:

• Development of FIT programs based on lifetime resolution theory for phase-modulation measurements.
• Utilizing a Monte Carlo approach with simultaneous analysis of phase and modulation data at three frequencies.
• Application to defined binary (carbazole, pyrene) and ternary (carbazole, pyrene, POPOP) systems.

Main Results:

• FIT programs provide accurate lifetime resolution, with results within 5% of independently measured values for binary and ternary systems.
• Fractional fluorescence contributions were determined within 10% of expected values.
• Demonstrated successful application to a biological system (dansyl label on myosin) involving Förster energy transfer.

Conclusions:

• Phase-modulation measurements, when analyzed with appropriate FIT programs, can accurately resolve lifetimes in binary and selected ternary fluorescent systems.
• These computational tools offer a simple, efficient, and effective method for analyzing complex fluorescence decay data.
• The methodology allows for quantification of distance changes in biological systems based on lifetime analysis.