An excitation emission fluorescence lifetime spectrometer using a frequency doubled supercontinuum laser source

Area of Science:

• Biophysical Chemistry
• Spectroscopy
• Protein Analysis

Background:

• Accurate fluorescence analysis of complex proteins requires multi-dimensional techniques.
• Measuring intrinsic fluorescence from tyrosine (Tyr) and tryptophan (Trp) necessitates tuneable UV excitation.
• Simultaneous multi-dimensional wavelength and time-resolved measurements of protein fluorescence are challenging.

Purpose of the Study:

• To assemble and validate a novel Excitation Emission Fluorescence Lifetime Spectrometer (EEFLS).
• To enable simultaneous multi-dimensional wavelength and time-resolved measurements of intrinsic protein fluorescence.
• To improve the characterization of complex intrinsic emission from proteins.

Main Methods:

• Developed a novel EEFLS using a pulsed, frequency-doubled Super-Continuum Laser (SCL) source.
• Integrated a 16-channel multi-anode Time Correlated Single Photon Counting (TCSPC) measurement system.
• Collected 4-dimensional Excitation Emission Fluorescence Lifetime Matrix (EEFLM) data (λex/λem/I(t)/τ).

Main Results:

• The EEFLS enabled collection of near-complete lifetime and intensity maps in the 260-350 nm (excitation) and 300-500 nm (emission) range.
• Achieved high spectral resolution (1-2 nm) and an Instrument Response Function (IRF) of ~650 ps for nanosecond lifetime measurements.
• Demonstrated stable UV power output (<2% variation over 9 hours) and reproducible measurements (RSD <1.5%).

Conclusions:

• The novel EEFLS system provides accurate and efficient multi-dimensional analysis of intrinsic protein fluorescence.
• The generated EEFLM data enhances the characterization of complex protein emission.
• The system's performance was validated against conventional methods using p-terphenyl and tryptophan standards.