Photophysical investigation of xanthenes for use in background suppression

Abstract

Fluorescence imaging is an important tool in biological imaging applications. Even with the many advances in this field endogenous background remains a challenge. Herein we present a way of circumventing background fluorescence by using dark-state photophysics of dyes. Amplitude modulation, as presented in Synchronously amplified fluorescence/photoacoustic image recovery (SAFIRe/SAPhIRe), is a technique employed to generate background free signals. This work focuses on understanding the properties of optically the modulatable dyes in the xanthene family, rose bengal, erythrosin B and Eosin Y, with the goal of designing better contrast agents for biomedical imaging. Results show previously undescribed methods for optical modulation of xanthenes that give long lived fluorescence as a result of direct repopulation of the singlet excited state from the triplet state. This technique described as optically activated delayed fluorescence (OADF) allows for fluorescence signals to persist much after (~μs) the decay of prompt fluorescence (~ns) allowing for background free detection of signal. The mechanism of modulation was investigated using both experimental and theoretical methods and modulation depth and in turn OADF generation was shown to increase with higher triplet quantum yields and at higher excitation rates. Dual laser modulation schemes have been used for photophysical characterization of dyes giving triplet lifetimes of 240μs, 420μs and 2.3ms for rose bengal, erythrosin B and eosin Y respectively. Finally, optical modulation in photoacoustic imaging was investigated and because both fluorescence and photoacoustic signal generations depend on the population of ground state molecules, similar approaches to dynamically shift population from the long-lived dark-states back to ground state will allow for background free photoacoustic imaging, (SAPhIRe). SAPhIRe was used to distinguish rose bengal loaded silica nanoparticles from blood background as well as buried ~10mm in rat muscle.