Ultrahigh‐Resolution Diffusion‐Ordered Spectroscopy

Diffusion‐ordered spectroscopy (DOSY) is an effective method for the analysis of intact mixtures, but the quality of results is critically limited by resolution in the NMR dimension. A new experiment integrating diffusion weighting into the PSYCHE method for pure shift NMR spectroscopy allows DOSY spectra to be measured with ultrahigh NMR resolution at improved sensitivity.     

Two‐Dimensional NMR Spectroscopy with Temperature‐Sweep

Two‐dimensional nuclear magnetic resonance (NMR) spectroscopy is useful for studying temperature‐dependent effects on molecular structure. However, experimental time is usually long, because sampling is repeated at several temperatures. A novel solution to the problem is proposed, in which signal sampling is performed in parallel to the linear temperature‐sweep.     

Noble‐Metal‐Free Materials for Surface‐Enhanced Raman Spectroscopy Detection

Surface‐enhanced Raman spectroscopy (SERS) is an attractive tool for the sensing of molecules in the fields of chemical and biochemical analysis as it enables the sensitive detection of molecular fingerprint information even at the single‐molecule level. In addition to traditional coinage metals in SERS analysis, recent research on noble‐metal‐free materials has also yielded highly sensitive SERS activity. This Minireview presents the recent development of noble‐metal‐free materials as SERS substrates and their potential applications, especially semiconductors and emerging graphene‐based nanostructures. Rather than providing an exhaustive review of this field, possible contributions from semiconductor substrates, characteristics of graphene enhanced Raman scattering, as well as effect factors such as surface plasmon resonance, structure and defects of the nanostructures that are considered essential for SERS activity are emphasized. The intention is to illustrate, through these examples, that the promise of noble‐metal‐free materials for enhancing detection sensitivity can further fuel the development of SERS‐related applications.     

Correcting for Spectral Cross‐Talk in Dual‐Color Fluorescence Cross‐Correlation Spectroscopy

Dual‐color fluorescence cross‐correlation spectroscopy (dcFCCS) allows one to quantitatively assess the interactions of mobile molecules labeled with distinct fluorophores. The technique is widely applied to both reconstituted and live‐cell biological systems. A major drawback of dcFCCS is the risk of an artifactual false‐positive or overestimated cross‐correlation amplitude arising from spectral cross‐talk. Cross‐talk can be reduced or prevented by fast alternating excitation, but the technology is not easily implemented in standard commercial setups. An experimental strategy is devised that does not require specialized hardware and software for recognizing and correcting for cross‐talk in standard dcFCCS. The dependence of the cross‐talk on particle concentrations and brightnesses is quantitatively confirmed. Moreover, it is straightforward to quantitatively correct for cross‐talk using quickly accessible parameters, that is, the measured (apparent) fluorescence count rates and correlation amplitudes. Only the bleed‐through ratio needs to be determined in a calibration measurement. Finally, the limitations of cross‐talk correction and its influence on experimental error are explored.     

Matrix‐Free DNP‐Enhanced NMR Spectroscopy of Liposomes Using a Lipid‐Anchored Biradical

Magic‐angle spinning dynamic nuclear polarization (MAS‐DNP) has been proven to be a powerful technique to enhance the sensitivity of solid‐state NMR (SSNMR) in a wide range of systems. Here, we show that DNP can be used to polarize lipids using a lipid‐anchored polarizing agent. More specifically, we introduce a C16‐functionalized biradical, which allows localization of the polarizing agents in the lipid bilayer and DNP experiments to be performed in the absence of excess cryo‐protectant molecules (glycerol, dimethyl sulfoxide, etc.). This constitutes another original example of the matrix‐free DNP approach that we recently introduced.     

PELDOR Spectroscopy Distance Fingerprinting of the Octameric Outer‐Membrane Protein Wza from Escherichia coli.

Distance fingerprinting : Pulsed electron–electron double resonance spectroscopy (PELDOR) is applied to the octameric membrane protein complex Wza of E. coli. The data yielded a detailed distance fingerprint of its periplasmic region that compares favorably to the crystal structure. These results provide the foundation to study conformation changes from interaction with partner proteins.

     

Terahertz Time‐Domain Spectroscopy of Gases,Liquids, and Solids

The techniques and methods employed in the spectroscopic characterization of gases, liquids, and solids in the terahertz frequency range are reviewed. Terahertz time‐domain spectroscopy is applied to address a broadband frequency range between 100 GHz and 5 THz with a sub‐10 GHz frequency resolution. The unique spectral absorption features measured can be efficiently used in material identification and sensing. Possibilities and limitations of fundamental and industrial applications are discussed.     

Solid‐state NMR and EPR Spectroscopy of Mn2+‐Substituted ATP‐Fueled Protein Engines

Paramagnetic metal ions deliver structural information both in EPR and solid‐state NMR experiments, offering a profitable synergetic approach to study bio‐macromolecules. We demonstrate the spectral consequences of Mg²⁺/ Mn²⁺ substitution and the resulting information contents for two different ATP:Mg²⁺‐fueled protein engines, a DnaB helicase from Helicobacter pylori active in the bacterial replisome, and the ABC transporter BmrA, a bacterial efflux pump. We show that, while EPR spectra report on metal binding and provide information on the geometry of the metal centers in the proteins, paramagnetic relaxation enhancements identified in the NMR spectra can be used to localize residues at the binding site. Protein engines are ubiquitous and the methods described herein should be applicable in a broad context.     

Cross‐Polarization Electron‐Nuclear Double Resonance Spectroscopy

Magnetic nuclei in the proximity of a paramagnetic center can be polarized through electron‐nuclear cross‐polarization and detected in electron‐nuclear double resonance (ENDOR) spectroscopy. This principle is demonstrated in a single‐crystal model sample as well as on a protein, the β2 subunit of E.coli ribonucleotide reductase (RNR), which contains an essential tyrosyl radical. ENDOR is a fundamental technique to detect magnetic nuclei coupled to paramagnetic centers. It is widely employed in biological and materials sciences. Despite its utility, its sensitivity in real samples is about one to two orders of magnitude lower than conventional electron paramagnetic resonance, thus restricting its application potential. Herein, we report the performance of a recently introduced concept to polarize nuclear spins and detect their ENDOR spectrum, which is based on electron‐nuclear cross polarization (eNCP). A single‐crystal study permits us to disentangle eNCP conditions and CP‐ENDOR intensities, providing the experimental foundation in agreement with the theoretical prediction. The CP‐ENDOR performance on a real protein sample is best demonstrated with the spectra of the essential tyrosyl radical in the β2 subunit of E.coli RNR.     

Thin‐Film Infrared Spectroscopy of Acetonitrile

Acetonitrile and [D₃]acetonitrile in the vicinal region of a planar AgX fiber contain linear dipole–dipole linked oligomers as shown by 1) comparison of infrared band intensity ratios in the gaseous and condensed phases and 2) remarkable plots of absorbance (C? N stretch) versus time during evaporation from an AgX planar fiber element. The plots (CH₃CN 2252 cm^?1, CD₃CN 2262 cm^?1) reveal the presence of octamers, hexamers, tetramers, and dimers along with some heptamer, trimer, and monomer structures. A novel isotope effect arises from the somewhat smaller size of the CD₃CN resulting in an increase in the CN band intensity. The organized oligomers may be termed pseudocrystals and are the main components responsible for absorption intensity in the infrared spectrum of acetonitrile, on the AgX planar fiber or in an IR cell.     

Multiphase‐Separated Polyurethanes Studied by Micro‐Raman Spectroscopy

Micro‐Raman spectroscopy has been used to investigate the chemical micro‐heterogeneity of multiphase‐separated poly(ether urethanes) (PETU). Analysis of PETU cross‐sections by means of micro‐Raman spectroscopy revealed the nearly complete absence of soft segments in AI aggregates (called globules). These aggregates are in the order of a few micrometers in size. The composition of the matrix and the AII aggregates (spherulites) was comparable.

Example of an AFM image (sample 706, scan size 25 μm, converted to monochromatic image).     

Chemical‐Shift‐Resolved 19F NMR Spectroscopy between 13.5 and 135 MHz: Overhauser–DNP‐Enhanced Diagonal Suppressed Correlation Spectroscopy

Overhauser–DNP‐enhanced homonuclear 2D ¹⁹F correlation spectroscopy with diagonal suppression is presented for small molecules in the solution state at moderate fields. Multi‐frequency, multi‐radical studies demonstrate that these relatively low‐field experiments may be operated with sensitivity rivalling that of standard 200–1000 MHz NMR spectroscopy. Structural information is accessible without a sensitivity penalty, and diagonal suppressed 2D NMR correlations emerge despite the general lack of multiplet resolution in the 1D ODNP spectra. This powerful general approach avoids the rather stiff excitation, detection, and other special requirements of high‐field ¹⁹F NMR spectroscopy.     

Understanding J‐Modulation during Spatial Encoding for Sensitivity‐Optimized Ultrafast NMR Spectroscopy

Ultrafast (UF) NMR spectroscopy is an approach that yields 2D spectra in a single scan. This methodology has become a powerful analytical tool that is used in a large array of applications. However, UF NMR spectroscopy still suffers from an intrinsic low sensitivity, and from the need to compromise between sensitivity, spectral width, and resolution. In particular, the modulation of signal intensities by the spin–spin J‐coupling interaction (J‐modulation) impacts significantly on the intensities of the spectral peaks. This effect can lead to large sensitivity losses and even to missing spectral peaks, depending on the nature of the spin system. Herein, a general simulation package (Spinach) is used to describe J‐modulation effects in UF experiments. The results from simulations match with experimental data and the results of product operator calculations. Several methods are proposed to optimize the sensitivity in UF COSY spectra. The potential and drawbacks of the different strategies are also discussed. These approaches provide a way to adjust the sensitivity of UF experiments for a large range of applications.     

Recovering Invisible Signals by Two‐Field NMR Spectroscopy

Nuclear magnetic resonance (NMR) studies have benefited tremendously from the steady increase in the strength of magnetic fields. Spectacular improvements in both sensitivity and resolution have enabled the investigation of molecular systems of rising complexity. At very high fields, this progress may be jeopardized by line broadening, which is due to chemical exchange or relaxation by chemical shift anisotropy. In this work, we introduce a two‐field NMR spectrometer designed for both excitation and observation of nuclear spins in two distinct magnetic fields in a single experiment. NMR spectra of several small molecules as well as a protein were obtained, with two dimensions acquired at vastly different magnetic fields. Resonances of exchanging groups that are broadened beyond recognition at high field can be sharpened to narrow peaks in the low‐field dimension. Two‐field NMR spectroscopy enables the measurement of chemical shifts at optimal fields and the study of molecular systems that suffer from internal dynamics, and opens new avenues for NMR spectroscopy at very high magnetic fields.