Dipolar Spectroscopy - Double-Resonance Methods

Pulsed electron double-resonance (PELDOR) techniques, also termed double electron-electron resonance (DEER), are the main source of distance distribution information on nanometer length scales in disordered systems. Such techniques are mostly applied to native paramagnetic centers and, in particular, spin labels, in biological and synthetic macromolecules and complexes thereof. The use of two excitation frequencies simplifies the description of spin dynamics and often enables good separation of the distance distribution within the system of interest from background contributions due to neighboring systems in the sample. Furthermore, partitioning of the spin system into observer spins A and pumped spins B allows for spectroscopic selection in systems with multiple labels - an approach that can be combined with chemically orthogonal labeling techniques. This article first describes the basics of DEER experiments and data processing for the simplest system of an isolated spin pair with fixed distance and spectroscopically distinct A and B spins and then discusses complications that arise with increasing complexity of the system. Best practices in sample preparation, measurement, and data analysis are presented.