Direct detection of the TROSY component of proton-attached 15N nuclei (15N-detected TROSY) yields high-quality spectra for large nondeuterated proteins, by taking advantage of the slow 15N transverse relaxation rate. The narrow line width of the 15N-detected TROSY resonances compensates for the inherently low 15N sensitivity, especially in high-field magnets near and above 1 GHz. In stark contrast to the 1H-detected TROSY that requires extensive deuteration, the 15N-detected TROSY resonances exhibit narrow lines even for protonated samples. This feature provides the opportunity to record 15N-detected TROSY spectra for large nondeuterated systems that can only be expressed in mammalian or insect cell systems and proteins that have incomplete back exchange of amide protons when expressed in D2O and thus have been inaccessible by conventional 1H detection strategy.

The article gives a general overview of instrumentation commonly used in electron paramagnetic resonance. It includes magnet systems, microwave bridge configurations, and sample cryostats. A special focus has been placed on the discussion of various resonator and sample probe designs used in CW as well as pulsed EPR. Specialized EPR applications such as very high frequency EPR, electrochemistry, stopped flow, and the application to volume limited samples are briefly discussed.

This is a short introduction to the principles of pulse EPR spectroscopy, intended for the reader who is already familiar with continuous-wave (CW) EPR. After an initial comparison of pulse EPR to CW EPR, the basics of pulses and their effects on electron spins are discussed. Then, the basic pulse sequences (one, two, and three pulses) and the associated signals (free induction decay, primary echo, and stimulated echo) are introduced. The effects of spectral, pulse, and resonator bandwidths are considered together with the resulting orientation and transition selection. Non-coherent effects including longitudinal and transverse spin relaxation, phase memory time, spectral diffusion, and instantaneous diffusion are discussed. After a brief explanation of the workings of a pulse EPR spectrometer based on a block diagram and discussion of resonator ringdown, deadtime, and phase cycling, this introduction concludes with some practical aspects relevant to performing pulse EPR experiments.

In the past decades, electron spin echo envelope modulation (ESEEM) techniques have become an important asset of the EPR toolbox. A variety of pulse sequences applied at different microwave frequencies now offers the EPR spectroscopist many possibilities to interrogate the local electronic and geometric structure of paramagnetic centers. In this way, unique chemical and physical information can be obtained to tackle questions on structure–function relationships in very diverse research fields, such as medicine, biology, (bio)chemistry, physics, and material sciences. In this article, the theoretical background of the most common ESEEM experiments will be outlined as well as their interpretation and use. Emphasis will be put on the potential of these methods and on possible pitfalls. Furthermore, different strategies to optimize the outcome of ESEEM experiments will be discussed.

EPR imaging is a well-established method for the study of the spatial distribution and the local environment of electron paramagnetic centers (PC) and spin probes. Although conceptually similar to nuclear MRI, EPR imaging did not receive widespread use due to relatively low availability of endogenous electron PC, especially in vivo. However, the development of injectable spin probes sensitive to various physiological parameters allowed EPR imaging to become a valuable tool for preclinical studies. One of the most important applications of modern EPR imaging is in vivo oximetry. Partial oxygen pressure (pO2) levels in tumors are major determinants of the response to cancer therapy and thus need thorough investigation. Other areas of EPR imaging application are imaging pH, thio-redox potentials, and polymer degradation.

In this article we review methods for the study of perfused cells, tissues, and organs using magnetic resonance spectroscopy (MRS). Biologically relevant nuclei and the different but often complementary information that they make available are presented. Pulse sequence implementation is discussed in the context of tailoring the desired metabolite information and suppressing unwanted resonances. Metabolites observable with MRS are generally limited to those present in relatively high (millimolar) concentrations. The importance of these metabolites and how flux through metabolic pathways can be observed using isotopic enhancement with 13C is reviewed. Cell preparation and immobilization methods employing agarose threads, matrigel and alginate beads; and the use of porous and nonporous microcarriers, hollow fibers and bioreactors for perfused cell culture are discussed. Tissue preparation techniques for the study of perfused organs include the Langendorff heart, perfused liver, and lung preparations. Maintaining careful control of the perfusate composition, pH, oxygenation, and temperature all critically affect cell and organ viability as well as the measured levels of cell metabolites. The requisite apparatus for achieving this is reviewed. Perfusion model systems allow the acquisition of real-time kinetic metabolic information by MRS, providing unique insights into the effects of drugs and inhibitors on metabolite levels. They thus provide a valuable intermediate for translational research from cells to animals to humans and back again.

Interaction with ordered biological tissues causes the motion of molecules and ions to be anisotropic. As a result, their dipolar and quadrupolar interactions do not average to zero. Multiple-quantum-filtered (MQF) NMR of 1H, 2H, and 23Na is sensitive to these interactions and can single out macromolecules, compartments, and fiber orientations in accordance with the size of the residual interactions and thus filter out the signals of molecules and ions that do not interact with the ordered structures. The increased effect of gradients during the MQ evolution facilitates diffusion measurement of nuclei such 2H and 23Na. Among the applications of MQF NMR are (i) the identification of various compartments such as the extracellular matrix, axons, and myelin in nerves and brain; (ii) measurements of the effect of strain in blood vessel walls; (iii) changes of the collagen fiber architecture in cartilage upon maturation, depletion of proteoglycans, decalcification, and with mechanical vs osmotic pressure; (iv) changes of cartilage obtained from osteoarthritic and osteoporotic patients; (v) the monitoring of healing of injured tendons; (vi) the measurement of increases in a substantially intracellular sodium pool in ischemic heart; and (vii) MQF magnetization transfer imaging that reflects macromolecular structure and content.

Molecular diffusion has a significant impact on the outcomes of NMR experiments featuring magnetic field gradients. This impact can be due to either imperfect static magnetic field homogeneity or the use of pulsed field gradients. In both cases, diffusion will result in an attenuation of the measured signal. Although this phenomenon has been extensively exploited and has led to numerous applications, in many cases it is undesirable. Moreover, diffusional attenuation can also interfere in more complex ways than just affecting sensitivity, such as introducing additional line broadening, or affecting the accuracy of experiments intended to measure relaxation time constants. Even in experiments intended for the measurement of diffusion, additional diffusional attenuation caused by imperfect magnetic field homogeneity can affect accuracy. In this article, a general procedure is introduced that allows the calculation of diffusional attenuation for nearly any pulse sequence. After a general introduction to the theoretical background, this procedure will be demonstrated on various case studies, exploring how diffusional attenuation can be determined for a pulse sequence. This will also serve to illustrate how pulse sequences can be designed to avoid, as far as possible, the undesirable effects of diffusion.

This article reviews spectral fitting topics that go beyond the time- or frequency-domain fitting methods described in Quantifying spectra in the frequency domain and Time-Domain Methods for Quantifying MR Spectra . First, a detailed review on different approaches for nonparametric modeling of line-shape and baseline distortions as well as unknown spectral components by pattern fitting and principle component analysis, is provided. Next, spectral fitting approaches are extended to multidimensional spectroscopy data that result from two-dimensional MRS (magnetic resonance spectroscopy), spectroscopic imaging, dynamic parameter studies, and functional spectroscopy experiments. In addition, principal component analysis and Bayesian approaches to spectral fitting are introduced and compared to the more established least-squares optimization methods. Finally, validation methods for testing the accuracy and precision of spectral fitting and quantification algorithms are described.