A wide range of single molecule fluorescence techniques involve the use of multi-color laser excitation, such as PIE-FRET, ALEX-FRET and FCCS. However, the influence of these various laser excitations is very often overlooked. What is the influence of the green laser on the red dyes photophysics? Adding more optical power to the sample could lead to less fluorescence photons?
In our latest article “Laser-induced fluorescence quenching of red fluorescent dyes with green excitation: avoiding artifacts in PIE-FRET and FCCS analysis”, we show that surprisingly the presence of green laser pulses can indeed quench the fluorescence of common red dyes Alexa Fluor 647 and Atto 647N. Quite surprisingly, this comes despite the fact that the green laser pulses are temporaly delayed by a time much greater than the fluorophore lifetime and that photobleaching conditions are avoided. More laser excitation but less fluorescence.
* we show how to avoid potential artifacts as both the fluorophore concentration and fluorescence brightness can be affected.
* we discuss the physical origin of this phenomenon via a long-lived dark state of the red dyes and photorefractive effects in the microscope objective.
* it provides a novel approach to check the alignment of the laser beams in the confocal microscope.
Also freely available on arXiv 1806.11364
Raju just got awarded with the "Prix de Thèse" of Aix Marseille University which acknowledges the best thesis defended at the university during the past year. The prize comes with a 1000€ award (always welcome ;o) Before that, Raju also received the prize of the Ecole Doctorale Physique et Sciences de la Matière. Congratulations Raju!
From the Ecole de Physique des Houches Fluorescence markers for advanced microscopy