Femtosecond spectral and anisotropy study of excitation energy transfer between neighbouring α-80 and β-81 chromophores of allophycocyanin trimers

Polarization pump-probe femtosecond spectroscopy was used to investigate photoinduced optical density changes in allophycocyanin (APC) trimers at 635–690 nm after excitation with 230-fs pulses at 618 nm. The initial bleaching observed at λ < 645 nm is followed by subpicosecond absorption recovery corresponding to 430 ± 40 fs recovery kinetics measured at 615 nm with 70-fs pulses. Only the red part of the APC absorption band remains strongly bleached at 3 ps after excitation. The spectral and kinetic results can be described in terms of two different models of interaction between neighbouring α-80 and β-81 chromophores of APC trimers. According to the first one, the observed subpicosecond kinetics corresponds to relaxation between the levels of excitonically coupled, spectrally identical α-80 and β-81 chromophores. Excited state absorption to doubly excited excitonic state should in this case contribute to the measured difference spectra. According to the second one, the femtosecond excitation energy transfer in APC trimers takes place between a donor chromophore absorbing predominantly at 620 nm and an acceptor chromophore absorbing at 650 nm. The high anisotropy value observed at 615 nm during the first 1.2 ps is in good agreement with the donor-acceptor model. Anisotropy values calculated in the 635–675 nm spectral region at 3 ps after excitation are in the 0.1–0.25 range corresponding to an angle of 30°–45° between donor and acceptor transition dipole orientations. The high anisotropy obtained at 658 nm during the excitation is probably due to stimulated emission of the donor chromophore

Förster energy transfer between neighbouring chromophores in C-phycocyanin trimers

The excitation-energy transfer in C-phycocyanin (C-PC) trimers and monomers isolated from phycobilisomes of Mastigocladus laminosus has been studied by polarization femtosecond laser spectroscopy. Excitation with 70-fs pulses at 615 nm gave rise to a 500-fs energy-transfer process that was observed only in trimeric preparations. The rate of the process is in agreement with earlier calculated Förster energy transfer rates between neighbouring α-84 and β-84 chromophores of different monomeric subunits. This process is most clearly seen in the anisotropy decay kinetics. As a result of femtosecond excitation-energy transfer, the anisotropy relaxes from 0.4 to 0.23. The final anisotropy value is in fair agreement with the results of calculations based on the crystal structure and spectroscopic data of C-PC trimers. Our results support the conclusion that Förster energy transfer can occur between excitonically coupled chromophores

Determination of the temperature dependence of the dynamic nuclear polarisation enhancement of water protons at 3.4 Tesla

It is shown that the temperature dependence of the DNP enhancement of the NMR signal from water protons at 3.4 T using TEMPOL as a polarising agent can be obtained provided that the nuclear relaxation, T1I, is sufficiently fast and the resolution sufficient to measure the 1H NMR shift. For high radical concentrations (100 mM) the leakage factor is approximately 1 and, provided sufficient microwave power is available, the saturation factor is also approximately 1. In this situation the DNP enhancement is solely a product of the ratio of the electron and nuclear gyromagnetic ratios and the coupling factor enabling the latter to be directly determined. Although the use of high microwave power levels needed to ensure saturation causes rapid heating of the sample, this does not prevent maximum DNP enhancements, ε0, being obtained since T1I is very much less than the characteristic heating time at these concentrations. It is necessary, however, to know the temperature variation of T1I to allow accurate modelling of the behaviour. The DNP enhancement is found to vary linearly with temperature with ε0(T) = −2 ± 2 − (1.35 ± 0.02)T for 6 °C ≤ T ≤ 100 °C. The value determined for the coupling factor, 0.055 ± 0.003 at 25 °C, agrees very well with the molecular dynamics simulations of Sezer et al. (Phys. Chem. Chem. Phys., 2009, 11, 6626) who calculated 0.0534, however the experimental values increase much more rapidly with increasing temperature than predicted by these simulations. Large DNP enhancements (|ε0| > 100) are reported at high temperatures but it is also shown that significant enhancements (e.g. 40) can be achieved whilst maintaining the sample temperature at 40 °C by adjusting the microwave power and irradiation time. In addition, short polarisation times enable rapid data acquisition which permits further enhancement of the signal, such that useful liquid state DNP-NMR experiments could be carried out on very small samples

DNP enhanced NMR using a high-power 94 GHz microwave source: a study of the TEMPOL radical in toluene

DNP enhanced H-1 NMR at 143 MHz in toluene is investigated using an NMR spectrometer coupled with a modified EPR spectrometer operating at 94 GHz and TEMPOL as the polarisation agent. A 100 W microwave amplifier was incorporated into the output stage of the EPR instrument so that high microwave powers could be delivered to the probe in either CW or pulsed mode. The maximum enhancement for the ring protons increases from similar to-16 for a 5 mM TEMPOL solution to similar to-50 for a 20 mM solution at a microwave power of similar to-480 mW. The temperature dependence of the enhancement, the NMR relaxation rates and the ESR spectrum of TEMPOL were also studied in an effort to obtain information on the dynamics of the system

A spectrometer designed for 6.7 and 14.1T DNP-enhanced solid-state MAS NMR using quasi-optical microwave transmission

A Dynamic Nuclear Polarisation (DNP) enhanced solid-state Magic Angle Spinning (MAS) NMR spectrometer operating at 6.7 T is described and demonstrated. The 187 GHz TE13 fundamental mode of the FU CW VII gyrotron is used as the microwave source for this magnetic field strength and 284 MHz 1H DNP-NMR. The spectrometer is designed for use with microwave frequencies up to 395 GHz (the TE16 second-harmonic mode of the gyrotron) for DNP at 14.1 T (600 MHz 1H NMR). The pulsed microwave output from the gyrotron is converted to a quasi-optical Gaussian beam using a Vlasov antenna and transmitted to the NMR probe via an optical bench, with beam splitters for monitoring and adjusting the microwave power, a ferrite rotator to isolate the gyrotron from the reflected power and a Martin–Puplett interferometer for adjusting the polarisation. The Gaussian beam is reflected by curved mirrors inside the DNP-MAS-NMR probe to be incident at the sample along the MAS rotation axis. The beam is focussed to a ∼1 mm waist at the top of the rotor and then gradually diverges to give much more efficient coupling throughout the sample than designs using direct waveguide irradiation. The probe can be used in triple channel HXY mode for 600 MHz 1H and double channel HX mode for 284 MHz 1H, with MAS sample temperatures ≥85 K. Initial data at 6.7 T and ∼1 W pulsed microwave power are presented with 13C enhancements of 60 for a frozen urea solution (1H–13C CP), 16 for bacteriorhodopsin in purple membrane (1H–13C CP) and 22 for 15N in a frozen glycine solution (1H–15N CP) being obtained. In comparison with designs which irradiate perpendicular to the rotation axis the approach used here provides a highly efficient use of the incident microwave beam and an NMR-optimised coil design