MICROTOME METHODS FOR ALIGNED SAMPLES FOR HIGH FIELD ESR

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A combination of isopotential spin-dry centrifugation (ISDU) and microtome techniques is used to record high field/high frequency (HF/HF) ESR spectra corresponding to different orientations of the membrane normal relative to the magnetic field. At X-band (~ 9.5 GHz) one can get ESR spectra for different orientations of the membrane normal relative to the magnetic field merely by rotating the sample in the resonator. The method is not applicable at higher frequencies. Due to unavoidable dielectric losses, a successful HF experiment requires thin ( 100 mkm) flat samples placed at a node of the millimeter wave E-field. For the standard fixed resonator geometry, the sample normal is parallel to the applied magnetic field B0. However, applying microtome techniques to ISDU aligned samples offers an opportunity to combine excellent orientational resolution, one of the main virtues of high-field ESR, with the ability to study aligned membrane samples in various orientations. The technique is used to prepare thin slices of various, controllable membrane orientations, suitable for HF/HF ESR. The X-band spectra of the slices are shown identical to the spectra obtained by rotating corresponding ISDU samples in the resonator. Examples of spin-labeled Gramicidin A and cholestane spin label in the aligned DPPC membrane illustrate applications of the technique and some virtues of HFHF ESR on aligned membranes vs. X-band.