Fig. 1. Principle of fluorescence anomalous phase advance (FAPA). (a) Three-state quantum system of fluorophores consisting of a ground state G, an excited state E, and a dark state D. (b) Time-dependent fluorescence signal traces in the absence of any dark states (green) and in the presence of a dark state (red) under a modulated laser excitation (black). The red trace appears ahead of the excitation (i.e., phase advance).
Research-Dark State Sensing and Imaging
In 2011 we reported an interesting observation: unlike conventional frequency-domain fluorescence lifetime measurement in which the fluorescence trace always lags behind the modulated excitation source, the detected fluorescence traces can actually exhibit fluorescence anomalous phase advance (FAPA) as if the fluorescence is emitted “ahead” of the source (Fig.1). We attribute FAPA to the asymmetric modulation distortion via photoinduced dark state (including triplet state or radical state) hysteresis. After establishing this FAPA effect, we made use of it to map out the spatial distribution of fluorophore’s dark state lifetime. This is the first time that the dark state is directly used for sensing local environment.
To gain deeper insight into FAPA, we generalized the framework of phase fluorometry by considering the intricate interplay between a short-lived fluorescent state and a long-lived dark state. FAPA exists only in a narrow modulation frequency range that is resonant with the intrinsic time scale of dark-state lifetime. Thus FAPA effect offers a specific and convenient reporter for sensing and imaging dark state dynamics, complementary to the widely used fluorescence lifetime spectroscopy and microscopy.
1. X. Zhu and W. Min. "Frequency-domain phase fluorometry in the presence of dark states: a numerical study", Chem. Phys. Lett., 516, 40, (2011). [PDF]
2. E. Gatzogiannis, X. Zhu, Y.-T. Kao, W. Min. "Observation of frequency-domain fluorescence anomalous phase advance due to dark state hysteresis", J. Phys. Chem. Lett., 2, 461, (2011). [PDF]