Measuring Photophysical Transition Rates with Fluorescence Correlation Spectroscopy and Antibunching.

Area of Science:

• Photophysics
• Spectroscopy
• Physical Chemistry

Background:

• Accurate measurement of photophysical rate constants is crucial for understanding molecular behavior.
• Existing methods may have limitations in quantifying parameters like intersystem crossing and triplet state lifetimes.
• Fluorescence correlation spectroscopy (FCS) and fluorescence antibunching are powerful spectroscopic techniques.

Purpose of the Study:

• To develop and validate a new method for measuring photophysical rate constants of fluorescent molecules.
• To combine microsecond time-scale FCS with nanosecond time-scale antibunching measurements.
• To enable absolute quantification of intersystem crossing rates and triplet state lifetimes.

Main Methods:

• Combining fluorescence correlation spectroscopy (FCS) on the microsecond time scale with fluorescence antibunching measurements on the nanosecond time scale.
• Quantifying the average excitation rate of fluorescent molecules using antibunching.
• Analyzing the microsecond temporal decay of FCS curves to determine photophysical rates.
• Theoretical analysis to estimate bias due to spatial averaging within the detection volume.

Main Results:

• The combined method allows for absolute quantification of intersystem crossing rates and triplet state lifetimes.
• The developed method demonstrates a maximum bias of less than 5% in most cases.
• Photophysical rate constants were successfully measured for Rhodamine 110 and ATTO 655.

Conclusions:

• The novel method provides accurate and absolute measurements of key photophysical parameters.
• This technique enhances the understanding of molecular photodynamics for fluorescent dyes.
• The approach is applicable to widely used fluorescent molecules like Rhodamine 110 and ATTO 655.