The ultrafast evolution of the electric field within bacteriorhodopsin was measured by monitoring the absorption changes of a tryptophan residue after excitation of retinal. The Trp absorption decreases within the first 200 femtoseconds and then recovers on time scales typical for retinal isomerization and vibrational relaxation. A model of excitonic coupling between retinal and tryptophans shows that the signal reflects a gradual rise of the retinal difference dipole moment, which precedes and probably drives isomerization. The results suggest an intimate connection between the progressive dipole moment change and the retinal skeletal changes reported over the same time scale.

No abstract

Two-photon medical imaging has found its way into dermatology as an excellent method for noninvasive skin cancer detection without need of contrast agents as well as for in situ drug screening of topically-applied cosmetical and pharmaceutical components. There is an increasing demand to apply the multiphoton technology also for deep-tissue skin imaging as well as for intracorporal imaging. We report on the first clinical use of multiphoton endoscopes, in particular of a miniaturized rigid two-photon GRIN lens endoscope. The microendoscope was attached to the multiphoton tomograph DermaInspect and employed to detect the extracellular matrix proteins collagen and elastin in the human dermis of volunteers and patients with ulcera by in vivo second harmonic generation and in vivo two-photon autofluorescence.

A visible-pump͞UV-probe transient absorption is used to characterize the ultrafast dynamics of bacteriorhodopsin with 80-fs time resolution. We identify three spectral components in the 265-to 310-nm region, related to the all-trans retinal, tryptophan (Trp)-86 and the isomerized photoproduct, allowing us to map the dynamics from reactants to products, along with the response of Trp amino acids. The signal of the photoproduct appears with a time delay of Ϸ250 fs and is characterized by a steep rise (Ϸ150 fs), followed by additional rise and decay components, with time scales characteristic of the J intermediate. The delayed onset and the steep rise point to an impulsive formation of a transition state on the way to isomerization. We argue that this impulsive formation results from a splitting of a wave packet of torsional modes on the potential surface at the branching between the all-trans and the cis forms. Parallel to these dynamics, the signal caused by Trp response rises in Ϸ200 fs, because of the translocation of charge along the conjugate chain, and possible mechanisms are presented, which trigger isomerization.ultrafast spectroscopy ͉ retinal proteins ͉ translocation of charge ͉ structural dynamics T he different biological functions of retinal proteins (vision, ion pumping, light signaling, etc.) rely on the ultrafast isomerization of the retinal chromophore (1). This process has been intensively investigated by fs pump-probe and f luorescence up-conversion spectroscopy in the visible (VIS) and͞or near-IR (2-13), revealing the retinal excited-state dynamics before isomerization and the build-up of the isomerized form. Time-resolved VIS-pump͞mid-IR probe experiments on bacteriorhodopsin (bR) established that the isomerization time is Ն400 fs (14). Indeed, the excited-state lifetime of 300 -500 fs (12, 15) goes hand-in-hand with the rise times observed for the 13-cis photoproduct (3,4,14), whereas a longer time scale of 3-4 ps has been attributed to vibrational relaxation of the photoproduct (14).The mechanism leading to the isomer is still subject to debate. Using Ͻ5-fs pulses, Kobayashi et al. (11) were able to monitor changes in the high-frequency vibrational spectrum of the excited state, suggesting that retinal is in a twisted, nonplanar configuration during the initial 200 fs. They also showed that the high-frequency modes were modulated with a period of Ϸ200 fs, corresponding to the low-frequency torsional modes observed in transient absorption experiments at 800-nm probe wavelength (16). That the system is all-trans-like during the first 200 fs is in line with Zhong et al. (17), who carried out transient absorption studies on wild-type bR and bR reconstituted with retinal analogs that cannot undergo isomerization and found identical dynamics during the initial 200 fs. In addition, McCamant et al. (18) recently showed by fs-stimulated Raman spectroscopy that the high-frequency modes (1,000-1,500 cm Ϫ1 ) decay on a time scale of Ϸ250 fs. They argued that this observation points to the excited-sta...