Wavelength selective modulation in femtosecond pump–probe spectroscopy and its application to heme proteins

Abstract

We demonstrate novel lock-in detection techniques, using wavelength selective modulation of ultrafast pump and probe laser pulses, to discriminate between vibrational coherence and electronic population decay signals. The technique is particularly useful in extracting low frequency oscillations from the monotonically decaying background, which often dominates the signal in resonant samples. The central idea behind the technique involves modulating the red and/or blue wings of the laser light spectrum at different frequencies, ΩR and ΩB, followed by a lock-in detection at the sum or difference frequency, ΩR±ΩB. The wavelength selective modulation and detection discriminates against contributions to the pump–probe signal that arise from degenerate electric field interventions (i.e., only field interactions involving different optical frequencies are detected). This technique can be applied to either the pump or the probe pulse to enhance the off-diagonal terms of the pump induced density matrix, or to select the coherent components of the two-frequency polarizability. We apply this technique to a variety of heme-protein samples to reveal the presence of very low-frequency modes (∼20 cm−1). Such low-frequency modes are not observed in standard pump–probe experiments due to the dominant signals from electronic population decay associated with resonant conditions. Studies of the diatomic dissociation reaction of myoglobin (MbNO→Mb+NO), using wavelength selective modulation of the pump pulse, reveal the presence of an oscillatory signal corresponding to the 220 cm−1 Fe–His mode. This observation suggests that the spin selection rules involving the ferrous iron atom of the heme group may be relaxed in the NO complex. Mixed iron spin states associated with adiabatic coupling in the MbNO sample could explain the fast time scales and large amplitude that characterize the NO geminate recombination.