To gain a better understanding of the light-induced reduction of protochlorophyllide (PChlide) to chlorophyllide as a key regulatory step in chlorophyll synthesis, we performed transient infrared absorption measurements on PChlide in d4-methanol. Excitation in the Q-band at 630 nm initiates dynamics characterized by three time constants: τ₁ = 3.6 ± 0.2, τ₂ = 38 ± 2, and τ₃ = 215 ± 8 ps. As indicated by the C13'=O carbonyl stretching mode in the electronic ground state at 1686 cm⁻¹, showing partial ground-state recovery, and in the excited electronic state at 1625 cm⁻¹, showing excited-state decay, τ₂ describes the formation of a state with a strong change in electronic structure, and τ₃ represents the partial recovery of the PChlide electronic ground state. Furthermore, τ₁ corresponds with vibrational energy relaxation. The observed kinetics strongly suggest a branched reaction scheme with a branching ratio of 0.5 for the path leading to the PChlide ground state on the 200 ps timescale and the path leading to a long-lived state (>>700 ps). The results clearly support a branched reaction scheme, as proposed previously, featuring the formation of an intramolecular charge transfer state with ∼25 ps, its decay into the PChlide ground state with 200 ps, and a parallel reaction path to the long-lived PChlide triplet state.

The production of reactive oxygen species (ROS), especially superoxide anions (O2·–), is enhanced in many normal and tumor cell types in response to ionizing radiation. The influence of ionizing radiation on the regulation of ROS production is considered as an important factor in the long-term effects of irradiation (such as genomic instability) that might contribute to the development of secondary cancers. In view of the increasing application of carbon ions in radiation therapy, we aimed to study the potential impact of ionizing density on the intracellular production of ROS, comparing photons (X-rays) with carbon ions. For this purpose, we used normal human cells as a model for irradiated tissue surrounding a tumor. By quantifying the oxidization of Dihydroethidium (DHE), a fluorescent probe sensitive to superoxide anions, we assessed the intracellular ROS status after radiation exposure in normal human fibroblasts, which do not show radiation-induced chromosomal instability. After 3–5 days post exposure to X-rays and carbon ions, the level of ROS increased to a maximum that was dose dependent. The maximum ROS level reached after irradiation was specific for the fibroblast type. However, carbon ions induced this maximum level at a lower dose compared with X-rays. Within ∼1 week, ROS decreased to control levels. The time-course of decreasing ROS coincides with an increase in cell number and decreasing p21 protein levels, indicating a release from radiation-induced growth arrest. Interestingly, radiation did not act as a trigger for chronically enhanced levels of ROS months after radiation exposure.

constructed by sandwiching a polymer spacer between two infrared transparent windows, creating a closed channel system. The channels in the spacer were laser cut to establish the layout of the mixer. The mixer takes advantage of hydrodynamic focusing with two side flow channels, squeezing a middle sample channel into a thin jet and initiating fast mixing through diffusion and advection. The mixing region is probed with a laser source in the mid-infrared region, then magnified and imaged on a focal plane array detector for absorption measurements. The mixer was experimentally calibrated in order to determine the amount of time per pixel in the detector. A pD jump mixing experiment of an adenosine monophosphate solution was employed in order to establish a mixing time on the order of 350 ms. Finally, a flow study with H2O and D2O was completed in order to compare experimental results with simulation. With the established mixing time and use of vibrational spectroscopy, this system can be applied to the study of protein and enzymatic reactions. 3097-Pos Board B789Ultra Fast Raman Hyperspectral Imaging using Bragg Tunable Filters and a High Performance Emccd Camera Because of its high specificity to a variety of molecular processes and its low sensitivity to the presence of water, Raman hyperspectral imaging is regarded as a very promising technique to help pathologists improve the accuracy of medical diagnostics when compared to conventional histopathological analysis. However, since on average approximately one photon per million undergoes Raman scattering, acquisition time per hyperspectral image is very long, typically of about 6 hours. This significantly reduces the appeal of this technique for ex-vivo diagnostics and makes in-vivo applications impracticable. To increase acquisition speed, a Raman hyperspectral imager based on holographic Bragg tunable filters was used and images of carbon nanotubes could be acquired 30 times faster than with a conventional confocal microscope optimised for fast mapping. This speed gain over traditional methods was further enhanced when also using a low-noise EMCCD camera, resulting in measurements performed 150 times faster. These results thus indicate that in vivo and ex vivo applications of wide-field Raman hyperspectral imaging are now at reach, hence paving the way for real time tumor detection during surgery. The Escherichia coli protein RecA catalyzes DNA strand exchange and plays a role DNA repair and genetic recombination. Nucleotide binding influences RecA oligomerization and its affinity for DNA. Previous studies in our lab have shown buffer-specific changes in RecA stability and unfolding transitions. Past circular dichroism (CD), infrared (IR), and fluorescence studies suggest only minimal buffer dependent changes in nucleotide binding and secondary structure that did not explain the large buffer dependent differences in RecA stability and unfolding profiles. These observations led to further investigations of how the four common biological buffers Tris, HEPES, MES,...