Pulsed interleaved excitation: principles and applications.

Area of Science:

• Biophysics
• Spectroscopy
• Biochemistry

Background:

• Pulsed interleaved excitation (PIE) is a technique for time-resolved fluorescence measurements.
• It allows for interleaved excitation of different fluorophores on the nanosecond timescale.
• PIE simplifies quantification in fluorescence techniques like Fluorescence Correlation Spectroscopy (FCS) and Förster Resonance Energy Transfer (FRET).

Purpose of the Study:

• To provide a comprehensive overview of the PIE technique's principles and versatility.
• To outline the theory for analysis using PIE, comparing continuous wave (CW)- and PIE-FCCS.
• To review key applications of PIE in scientific literature.

Main Methods:

• PIE methodology involves interleaved or alternating excitation of different fluorophores.
• It enables quasi-simultaneous, yet independent measurements.
• PIE filters spectral emission bleedthrough (crosstalk) and direct excitation without reducing signal-to-noise ratio (SNR).

Main Results:

• PIE allows for specific filtering of spectral crosstalk and direct excitation, preserving SNR.
• Absolute FRET efficiency can be determined in FCCS experiments, even with imperfect labeling.
• Combining PIE with advanced techniques like Multiple Fluorescence Detection (MFD) and scanning FFS enables accurate, species-specific spFRET analyses and false-positive-free interaction measurements.

Conclusions:

• PIE is a versatile technique that enhances the accuracy and reliability of fluorescence-based measurements.
• It significantly improves quantification in FRET and FCCS experiments.
• PIE facilitates advanced analyses, including species-specific spFRET and molecular interaction studies in various settings.