Fluorescence Lifetimes and Quantum Yields of Rhodamine Derivatives: New Insights from Theory and Experiment Marika Savarese, †,‡ Anna Aliberti, ‡ Ilaria De Santo, ‡ Edmondo Battista, ‡ Filippo Causa, ‡ Paolo A. Netti, ‡ and Nadia Rega* ,†,‡ † Dipartimento di Scienze Chimiche, Universita ̀ di Napoli ‘Federico II’, Complesso Universitario di M.S.Angelo, via Cintia, I-80126 Napoli, Italy ‡ Center for Advanced Biomaterials for Health Care@CRIB, Istituto Italiano di Tecnologia, Largo Barsanti e Matteucci 53, I-80125 Napoli, Italy * S Supporting Information ABSTRACT: Although lifetimes and quantum yields of widely used fluorophores are often largely characterized, a systematic approach providing a rationale of their photophysical behavior on a quantitative basis is still a challenging goal. Here we combine methods rooted in the time-dependent density functional theory and fluorescence lifetime imaging microscopy to accurately determine and analyze fluorescence signatures (lifetime, quantum yield, and band peaks) of several commonly used rhodamine and pyronin dyes. We show that the radiative lifetime of rhodamines can be correlated to the charge transfer from the phenyl toward the xanthene moiety occurring upon the S 0 ← S 1 de-excitation, and to the xanthene/ phenyl relative orientation assumed in the S 1 minimum structure, which in turn is variable upon the amino and the phenyl substituents. These findings encourage the synergy of experiment and theory as unique tool to design finely tuned fluorescent probes, such those conceived for modern optical sensors. 1. INTRODUCTION Rhodamine dyes are xanthene derivatives presenting photo- physical properties well suited for a wide range of applications. Due to the advance of techniques based on fluorescence signaling and encoding, 1-5 a renewed interest in synthesis strategies and spectroscopic characterization of these systems has been recently shown in the literature. 6-10 As a matter of fact, a high degree of comprehension is required for a full control and design of modern optical sensors, such as fluorescence encoding multiplex systems. 3-5 On the other hand, the photophysical behavior of rhod- amines is not fully understood or established, in spite of the large amount of studies published along the decades. 11-19 For example, the rationale underlying trends of both radiative and nonradiative decays with respect to structural arrangements has not been identified, and several models have been debated to interpret the quantum yield behavior. 12,14,16-23 Time-dependent density functional theory (TD-DFT) 24-27 provides an important tool to interpret experimental trends in photochemistry. On the other hand, assessment of TD-DFT as predictive of structure/properties relationships on a quantita- tive basis is still the subject of an intense research effort. 28-31 In this work we combine calculations based on TD-DFT with fluorescence lifetime imaging microscopy (FLIM) 32 data to determine and systematically analyze fluorescence lifetime and quantum yield of several commonly used rhodamine dyes. Comparison of calculated and experimental lifetimes allowed us to obtain values of the quantum yield that are in very good agreement with data previously reported in literature. We demonstrate that radiative decay rates are modulated by the interactions involving the two main moieties of the rhodamine, namely, the phenyl and the xanthene rings. These interactions sensibly change upon electronic excitation to the fluorescent state and are finely tuned by the solvent and by both the xanthene and phenyl substituents. The correlation between radiative lifetime and relative orientation of the two molecular moieties is evident already when analyzed on the basis of calculated minimum energy structures, once the excited state energy surface is accurately defined at the quantum mechanical level including dispersion energy terms and average solvent effects. The present results point at the combination of TD- DFT and FLIM as a very promising support to perform a rational and well controlled design of fluorescent probes. In the following section we briefly describe our experimental procedure and computational protocol. We illustrate and discuss the results in section 3 and give our conclusions in section 4. Received: March 5, 2012 Revised: May 31, 2012 Published: June 6, 2012 Article pubs.acs.org/JPCA © 2012 American Chemical Society 7491 dx.doi.org/10.1021/jp3021485 | J. Phys. Chem. A 2012, 116, 7491-7497