Refocused Out-Of-Phase (ROOPh) DEER: A pulse scheme for suppressing an unmodulated background in double electron-electron resonance experiments.

EPR pulsed dipolar spectroscopy (PDS) is indispensable for measurements of nm-scale distances between electronic spins in biological and other systems. While several useful modifications and pulse sequences for PDS have been developed in recent years, DEER experiments utilizing pump and observer pulses at two different frequencies remain the most popular for practical applications. One of the major drawbacks of all the available DEER approaches is the presence of a significant unmodulated fraction in the detected signal that arises from an incomplete inversion of the coupled spins by the pump pulse. The latter fraction is perceived as one of the major sources of error for the reconstructed distance distributions. We describe an alternative detection scheme - a Refocused Out-Of-Phase DEER (ROOPh-DEER) - to acquire only the modulated fraction of the dipolar DEER signal. When Zeeman splitting is small compared to the temperature, the out-of-phase magnetization components cancel each other and are not observed in 4-pulse DEER experiment. In ROOPh-DEER these components are refocused by an additional pump pulse while the in-phase component containing an unmodulated background is filtered out by a pulse at the observed frequency applied right at the position of the refocused echo. Experimental implementation of the ROOPh-DEER detection scheme requires at least three additional pulses as was demonstrated on an example of a 7-pulse sequence. The application of 7-pulse ROOPh-DEER sequence to a model biradical yielded the interspin distance of 1.94 ± 0.07 nm identical to the one obtained with the conventional 4-pulse DEER, however, without the unmodulated background present as a dominant fraction in the latter signal.