We describe a simple methodology for the effective retrieval of Raman spectra of subsurface layers in diffusely scattering media. The technique is based on the collection of Raman scattered light from surface regions that are laterally offset away from the excitation laser spot on the sample. The Raman spectra obtained in this way exhibit a variation in relative spectral intensities of the surface and subsurface layers of the sample being investigated. The data set is processed using a multivariate data analysis to yield pure Raman spectra of the individual sample layers, providing a method for the effective elimination of surface Raman scatter. The methodology is applicable to the retrieval of pure Raman spectra from depths well in excess of those accessible with conventional confocal microscopy. In this first feasibility study we have differentiated between surface and subsurface Raman signals within a diffusely scattering sample composed of two layers: trans-stilbene powder beneath a 1 mm thick over-layer of PMMA (poly(methyl methacrylate)) powder. The improvement in contrast of the subsurface trans-stilbene layer without numerical processing was 19 times. The potential applications include biomedical subsurface probing of specific tissues through different overlying tissues such as assessment of bone quality through skin, providing an effective noninvasive means of screening for bone degeneration, other skeletal disease diagnosis, and dermatology studies, as well as materials and catalyst research.

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The photodynamics of wtGFP have been studied by ultrafast time-resolved infrared spectroscopy (TIR). In addition to the expected bleaching and transient infrared absorption of bands associated with the chromophore, we observe the dynamics of the proton relay reaction in the protein. Protonation of a protein carboxylate group occurs on the tens of picoseconds time scale following photoexcitation. Comparison with data for mutant GFPs, in which excited-state proton transfer has been disabled, supports the assignment of the carboxylate to the side chain of E222, a component of the hydrogen bonding network that links the two ends of the chromophore. The TIR data show that the rate-limiting step in the proton relay is deprotonation of the chromophore.

Ultrafast pump−probe studies of room-temperature solutions of 3-hydroxyflavone (3-HF) and some 4‘-substituted derivatives have been undertaken. Transient absorption attributable to the two zwitterionic forms of the excited tautomer arising from excited-state intramolecular proton transfer (ESIPT) was observed across most of the visible spectral region. For 3-HF in methylcyclohexane and acetonitrile, the ESIPT was found to be so rapid that it was only possible to assign a time constant of 35 fs to the process. In ethanol, however, a time constant of 60 fs was determined. The slower ESIPT in this solvent was attributed to the greater strength of the solute−solvent interactions. For the derivatives of 3-HF in all three solvents, the ESIPT step was also found to be instrument-limited. In addition to the femtosecond kinetics, there was also a picosecond component of the kinetics that is attributed to ESPT in molecules that are intermolecularly hydrogen bonded to the solvent.