Laser-induced fluorescence quenching of red fluorescent dyes with green excitation: avoiding artifacts in PIE-FRET and FCCS analysis

TL;DR

This paper reveals that green laser pulses can unexpectedly quench red fluorescent dyes like Alexa Fluor 647, affecting fluorescence measurements in multicolor spectroscopy techniques and highlighting the need to account for such artifacts.

Contribution

It demonstrates the quenching effect of green laser pulses on red dyes and discusses its physical origin, providing insights to improve fluorescence spectroscopy accuracy.

Findings

Green laser pulses can quench red dyes without photobleaching.

Quenching involves long-lived dark states and photorefractive effects.

Measurement artifacts can be avoided by understanding this phenomenon.

Abstract

Multicolor excitation is at the core of many fluorescence spectroscopy techniques such as PIE-FRET, ALEX-FRET, or FCCS. However, the influence of the multiple laser excitations on the dye photophysics is often overlooked. Here, we show that green laser pulses can surprisingly quench the fluorescence of common red dyes Alexa Fluor 647 and Atto 647N, even when the conditions leading to photobleaching are avoided. The physical origin of this phenomenon is discussed via a long-lived dark state and/or photorefractive effects. These observations are important to avoid measurement artifacts as both the fluorophore concentration and the fluorescence brightness are affected.