We present a method for implementing optical heterodyne detection using a diffractive optic for time-resolved transient-grating experiments. This technique does not require active phase locking of pulse pairs to achieve interferometric stability. The phase stability, intrinsic time resolution, and signal amplification are demonstrated experimentally through Raman scattering in carbon disulfide.

Ligand transport through myoglobin (Mb) has been observed by using optically heterodyne-detected transient grating spectroscopy. Experimental implementation using diffractive optics has provided unprecedented sensitivity for the study of protein motions by enabling the passive phase locking of the four beams that constitute the experiment, and an unambiguous separation of the Real and Imaginary parts of the signal. Ligand photodissociation of carboxymyoglobin (MbCO) induces a sequence of events involving the relaxation of the protein structure to accommodate ligand escape. These motions show up in the Real part of the signal. The ligand (CO) transport process involves an initial, small amplitude, change in volume, reflecting the transit time of the ligand through the protein, followed by a significantly larger volume change with ligand escape to the surrounding water. The latter process is well described by a single exponential process of 725 ؎ 15 ns at room temperature. The overall dynamics provide a distinctive signature that can be understood in the context of segmental protein fluctuations that aid ligand escape via a few specific cavities, and they suggest the existence of discrete escape pathways.

Recent advances in transient grating spectroscopy are described in relation to extracting photoacoustic parameters. The time resolution for measurement of dynamically driven acoustics has been extended to the picosecond level. This improvement was achieved by adopting counter-propagating beam geometries and resolving the acoustic phase shift in analogy to phase modulation spectroscopy. At the other extreme, the dynamic range of this technique has been extended to milliseconds in order to follow dynamical processes central to biological functions. In addition, diffractive optics were used for generating the necessary excitation and probe beam geometries. A novel optical setup was developed which permits both the rapid exploration of fringe spacing dependencies in separating thermal from nonthermal contributions to the observed signal as well as heterodyne detection without active feedback. The latter capability significantly increases the signal-tonoise ratio and permits separation of the real and imaginary components from the nonlinear four-wave-mixing signal. Applications of these new methods are demonstrated by following the functionally relevant structural relaxation processes of heme proteins over 10 decades in time.

The dynamics of ligand escape from carboxymyoglobin are studied via Q-band transient absorption and diffractive optics-based four-wave mixing. The latter approach provides an interferometric method for following protein motions and energetics and allows unambiguous assignment of different signal components to specific dynamical processes, a problem that has hindered all photothermal, photoacoustic, and grating methods in the past. In particular, the real part of the four-wave mixing signal is isolated, and it is demonstrated that the excited-state population contribution to the signal can be identified and removed by tuning the probe to wavelengths where this contribution vanishes ("zero-crossings"). At these probe wavelengths, changes in the real part of the index of refraction are small compared to those of the imaginary part, making the heterodyne measurement sensitive to errors in setting the phase of the reference field. We solve this problem with a new balanced detection method that isolates the real part of the signal and is extremely robust against phase errors. The location of the zero-crossings in the population contribution to the index of refraction is found to be complicated by the presence of spectral shifts in the transient absorption that can be characterized and removed with a time-dependent Kramers-Kronig analysis. The spectral shifts are most apparent near the isosbestic points and show similar dynamics to the four-wave mixing signal, suggesting that both are sensitive to the same dynamical processes. These dynamics were observed previously by using four-wave mixing with an off-resonant probe and were assigned to CO migration out of the protein via a number of discrete channels. It appears that both the photoinduced protein conformational relaxation and CO migration through the protein contribute to the transient absorption signal, making the two effects difficult to separate. The direct coupling of the protein motions to the index of refraction changes in the four-wave mixing experiments aids in distinguishing the two processes.