Template-directed 2D nanopatterning of S = 1/2 molecular spins.

Molecular spins are emerging platforms for quantum information processing. By chemically tuning their molecular structure, it is possible to prepare a robust environment for electron spins and drive the assembly of a large number of qubits in atomically precise spin-architectures. The main challenges in the integration of molecular qubits into solid-state devices are (i) minimizing the interaction with the supporting substrate to suppress quantum decoherence and (ii) controlling the spatial distribution of the spins at the nanometer scale to tailor the coupling among qubits. Herein, we provide a nanofabrication method for the realization of a 2D patterned array of individually addressable Vanadyl Phthalocyanine (VOPc) spin qubits. The molecular nanoarchitecture is crafted on top of a diamagnetic monolayer of Titanyl Phthalocyanine (TiOPc) that electronically decouples the electronic spin of VOPc from the underlying Ag(100) substrate. The isostructural TiOPc interlayer also serves as a template to regulate the spacing between VOPc spin qubits on a scale of a few nanometers, as demonstrated using scanning tunneling microscopy, X-ray circular dichroism, and density functional theory. The long-range molecular ordering is due to a combination of charge transfer from the metallic substrate and strain in the TiOPc interlayer, which is attained without altering the pristine VOPc spin characteristics. Our results pave a viable route towards the future integration of molecular spin qubits into solid-state devices.

Orienting dilute thin films of non-planar spin-1/2 vanadyl-phthalocyanine complexes.

The molecular orientation as well as the electronic and magnetic properties of vanadyl-phthalocyanine (VOPc) diluted into titanyl-phthalocyanine (TiOPc) thin films on Si(100) and polycrystalline aluminum substrates have been investigated by soft X-ray absorption spectroscopy (XAS), X-ray linear dichroism (XLD) and X-ray magnetic circular dichroism (XMCD). On the bare substrates the films grow with a standing-up geometry. By contrast, on template layers of 3,4,9,10-perylene-tetracarboxylic dianhydride (PTCDA), they assume a lying-down orientation. Moreover, a theoretical model based on the normalized intensity of the nitrogen K-edge XLD is established in order to extract the molecular orientation angle quantitatively without the need for crystallinity and with the sub-monolayer sensitivity of soft-XAS. XMCD reveals that the vanadium magnetic properties are preserved in both non-diluted and diluted films. The results pave the way toward the use of VOPc as nanometer-sized spin quantum bits.

Hyperfine spectroscopy in a quantum-limited spectrometer.

We report measurements of electron-spin-echo envelope modulation (ESEEM) performed at millikelvin temperatures in a custom-built high-sensitivity spectrometer based on superconducting micro-resonators. The high quality factor and small mode volume (down to 0.2 pL) of the resonator allow us to probe a small number of spins, down to 5×102. We measure two-pulse ESEEM on two systems: erbium ions coupled to 183W nuclei in a natural-abundance CaWO4 crystal and bismuth donors coupled to residual 29Si nuclei in a silicon substrate that was isotopically enriched in the 28Si isotope. We also measure three- and five-pulse ESEEM for the bismuth donors in silicon. Quantitative agreement is obtained for both the hyperfine coupling strength of proximal nuclei and the nuclear-spin concentration.

Wideband frequency-swept excitation in pulsed EPR spectroscopy.

Excitation of electron spins with monochromatic rectangular pulses is limited to bandwidths that are smaller than the spectral widths of most organic radicals and much smaller than the spectral widths of transition and rare earth metal ions. With frequency-swept pulses, bandwidths of up to 800MHz have previously been attained for excitation and detection of spin packets at frequencies of about 9.6GHz and bandwidths of up to 2.5GHz in a polarization transfer experiment at frequencies of about 34GHz. The remaining limitations, mainly due to resonator bandwidth and due to pulse length restrictions are discussed. Flip angles for state-space rotations on passage of a transition can generally be computed from the critical adiabaticity by the Landau-Zener-Stückelberg-Majorana expression. For hyperbolic secant pulses, the Demkov-Kunike model describes excitation for spin packets within and outside the sweep range. Well within the sweep range, the Bloch-Siegert phase shift is proportional to critical adiabaticity to a very good approximation. Because of the dependence of both flip angle and coherence phase on critical adiabaticity, it is advantageous to use pairs of amplitude and frequency modulation functions that provide such offset-independent adiabaticity. Compensation for the resonator response function should restore offset-independent adiabaticity. Whereas resonance offsets and Bloch-Siegert phase can be refocused at certain pulse length ratios, phase dispersion in coupled spin systems cannot generally be refocused. Based on the bandwidth limitations that arise from spin dynamics, requirements are derived for a spectrometer that achieves precise spin control over wide bands. The design of such a spectrometer and hardware characterization by EPR experiments are discussed.

Artefact suppression in 5-pulse double electron electron resonance for distance distribution measurements.

A 5-pulse version of the Double Electron Electron Resonance (DEER) experiment with Carr-Purcell delays and an additional pump pulse has been shown to significantly extend the experimentally accessible distance range in cases where nuclear spin diffusion dominates electron spin phase memory loss [Borbat et al., J. Phys. Chem. Lett., 2013, 4, 170]. We show that the sequence also prolongs coherence decay for spin labels in or near lipid bilayers, where this decay is mono-exponential. Compared to 4-pulse DEER, 5-pulse DEER suffers from additional artefacts that stem from pulse imperfection and excitation band overlap. Only some of these artefacts can be suppressed by phase cycling and the remaining ones have hindered widespread utilization of the method. Here, we report previously unknown additional artefact contributions stemming from overlap between the excitation bands of the microwave pulses that introduce additional dipolar evolution pathways. Experimental conditions are analyzed in detail that suppress these as well as the already known artefacts. Such suppression results in data that contain at most the partial excitation artefact, which can be deliberately shifted in time by a change in pulse timing without affecting the wanted contribution.

Double electron-electron resonance with multiple non-selective chirp refocusing.

A new approach to double electron-electron resonance (DEER) for distance determination involving nitroxide spin labels at dilute concentrations is presented. In general, DEER pulse sequences rely on double resonance between pump and observer spins excited by selective pulses at two distinct microwave frequencies. In the new approach abbreviated as nDEER, non-selective chirp pulses that refocus all relevant spin pairs are combined with DEER. This non-selective refocusing results in suppression of unmodulated contributions, such as the constant contribution as well as the background curvature due to inter-molecular spin partners in ordinary DEER data. Due to this dipolar attenuation effect, primary nDEER data are closer to the dipolar modulation of primary interest than ordinary DEER data. Restrictions of nDEER are that secondary information related to these unmodulated contributions becomes difficult to retrieve. Accordingly, incomplete deconvolution of the inter-molecular background prevents the application of nDEER to rigid spin pairs at high concentrations. A key advantage of nDEER is the high fidelity of the chirp refocusing pulses, which is important for nDEER schemes that incorporate dynamical decoupling to access longer distances. In this context, nDEER with Carr-Purcell (CP) pulse trains having N = 2 and N = 4 refocusing pulses are demonstrated. These CP nDEER sequences require a total of N + 2 pulses, which is less than the 2N + 1 pulses required for CP DEER schemes. The pump pulse position is incremented throughout the refocusing pulses, which restricts the minimum time increment to 96 ns on our spectrometer and therefore complicates application to distances below 3 nm. At Q-band frequencies, unwanted modulations related to pulse imperfections contribute only 3.5% relative to the principal nDEER modulation. Accordingly, there is no need for dedicated data reconstruction methods as in CP DEER methods.

CIDME: Short distances measured with long chirp pulses.

Frequency-swept pulses have recently been introduced as pump pulses into double electron-electron resonance (DEER) experiments. A limitation of this approach is that the pump pulses need to be short in comparison to dipolar evolution periods. The "chirp-induced dipolar modulation enhancement" (CIDME) pulse sequence introduced in this work circumvents this limitation by means of longitudinal storage during the application of one single or two consecutive pump pulses. The resulting six-pulse sequence is closely related to the five-pulse "relaxation-induced dipolar modulation enhancement" (RIDME) pulse sequence: While dipolar modulation in RIDME is due to stochastic spin flips during longitudinal storage, modulation in CIDME is due to the pump pulse during longitudinal storage. Experimentally, CIDME is examined for Gd-Gd and nitroxide-nitroxide distance determination using a high-power Q-band spectrometer. Since longitudinal storage results in a 50% signal loss, comparisons between DEER using short chirp pump pulses of 64ns duration and CIDME using longer pump pulses are in favor of DEER. While the lower sensitivity restrains the applicability of CIDME for routine distance determination on high-power spectrometers, this result is not to be generalized to spectrometers having lower power and to specialized "non-routine" applications or different types of spin labels. In particular, the advantage of prolonged CIDME pump pulses is demonstrated for experiments at large frequency offset between the pumped and observed spins. At a frequency separation of 1GHz, where broadening due to dipolar pseudo-secular contributions becomes largely suppressed, a Gd-Gd modulation depth larger than 10% is achieved. Moreover, a CIDME experiment at deliberately reduced power underlines the potential of the new technique for spectrometers with lower power, as often encountered at higher microwave frequencies. With longitudinal storage times T below 10µs, however, CIDME appears rather susceptible to artifacts. For nitroxide-nitroxide experiments, these currently inhibit a faithful data analysis. To facilitate further developments, the artifacts are characterized experimentally. In addition, effects that are specific to the high spin of S=7/2 Gd-centers are examined. Herein, population transfer within the observer spin's multiplet due to the pump pulse as well as excitation of dipolar harmonics are discussed.

Transverse interference peaks in chirp FT-EPR correlated three-pulse ESEEM spectra.

Fourier transform (FT) electron paramagnetic resonance (EPR) correlation spectroscopy usually requires broader excitation bandwidth than can be achieved by monochromatic rectangular pulses. Replacement of such pulses by frequency-swept pulses affords the correlation spectra, which, however, may not look the same as those that would be obtained with sufficiently broad-banded monochromatic rectangular pulses. This was recently observed for correlating nuclear frequencies to FT-EPR spectra by a three-pulse electron spin echo envelope modulation experiment. Here we analyze the origin of the additional cross peaks, whose position depends on the direction of the frequency sweep. We find that such peaks arise if coherence or polarization is transferred to an electron spin transition already before this transition is actually passed during the frequency sweep. This happens by excitation of a chain of transitions that connect levels of the source transition, where coherence resides before mixing, and the target transition, where it resides after mixing. The correlation spectra can be simplified by combining data from frequency up and down sweeps.

Averaging of nuclear modulation artefacts in RIDME experiments.

The presence of artefacts due to Electron Spin Echo Envelope Modulation (ESEEM) complicates the analysis of dipolar evolution data in Relaxation Induced Dipolar Modulation Enhancement (RIDME) experiments. Here we demonstrate that averaging over the two delay times in the refocused RIDME experiment allows for nearly quantitative removal of the ESEEM artefacts, resulting in potentially much better performance than the so far used methods. The analytical equations are presented and analyzed for the case of electron and nuclear spins S=1/2,I=1/2. The presented analysis is also relevant for Double Electron Electron Resonance (DEER) and Chirp-Induced Dipolar Modulation Enhancement (CIDME) techniques. The applicability of the ESEEM averaging approach is demonstrated on a Gd(III)-Gd(III) rigid ruler compound in deuterated frozen solution at Q band (35GHz).

EPR-correlated dipolar spectroscopy by Q-band chirp SIFTER.

While two-dimensional correlation spectra contain more information as compared to one-dimensional spectra, typical spectral widths encountered in electron paramagnetic resonance (EPR) spectroscopy largely restrict the applicability of correlation techniques. In essence, the monochromatic excitation pulses established in pulsed EPR often cannot uniformly excite the entire spectrum. Here, this restriction is alleviated for nitroxide spin labels at Q-band microwave frequencies around 35 GHz. This is achieved by substitution of monochromatic pulses by frequency-swept chirp pulses tailored for uniform excitation. Unwanted interference effects brought by this substitution are analyzed for a pair of electron spins with secular dipolar coupling. Experimentally, the dipole-dipole interaction can be separated from other interactions by a constant-time Zeeman-compensated solid echo sequence called SIFTER. Such SIFTER experiments usually yield a one-dimensional dipolar spectrum. EPR-correlated dipolar spectra can be obtained when the four pulses are replaced by chirp pulses. These two-dimensional spectra encode additional information on the geometrical arrangement of the two spin labels. With the excitation parameters achieved by a home-built Q-band spectrometer capable of frequency-swept excitation, unwanted interference effects can be largely neglected for the examined model compound with a spin-spin distance of 4 nm. The experimentally obtained correlation pattern conforms to the expectation based on the inter-spin geometry of the investigated rigid model compound.

Water accessibility in a membrane-inserting peptide comparing Overhauser DNP and pulse EPR methods.

Water accessibility is a key parameter for the understanding of the structure of biomolecules, especially membrane proteins. Several experimental techniques based on the combination of electron paramagnetic resonance (EPR) spectroscopy with site-directed spin labeling are currently available. Among those, we compare relaxation time measurements and electron spin echo envelope modulation (ESEEM) experiments using pulse EPR with Overhauser dynamic nuclear polarization (DNP) at X-band frequency and a magnetic field of 0.33 T. Overhauser DNP transfers the electron spin polarization to nuclear spins via cross-relaxation. The change in the intensity of the (1)H NMR spectrum of H2O at a Larmor frequency of 14 MHz under a continuous-wave microwave irradiation of the nitroxide spin label contains information on the water accessibility of the labeled site. As a model system for a membrane protein, we use the hydrophobic α-helical peptide WALP23 in unilamellar liposomes of DOPC. Water accessibility measurements with all techniques are conducted for eight peptides with different spin label positions and low radical concentrations (10-20 µM). Consistently in all experiments, the water accessibility appears to be very low, even for labels positioned near the end of the helix. The best profile is obtained by Overhauser DNP, which is the only technique that succeeds in discriminating neighboring positions in WALP23. Since the concentration of the spin-labeled peptides varied, we normalized the DNP parameter ϵ, being the relative change of the NMR intensity, by the electron spin concentration, which was determined from a continuous-wave EPR spectrum.

SPIDYAN, a MATLAB library for simulating pulse EPR experiments with arbitrary waveform excitation.

Frequency-swept chirp pulses, created with arbitrary waveform generators (AWGs), can achieve inversion over a range of several hundreds of MHz. Such passage pulses provide defined flip angles and increase sensitivity. The fact that spectra are not excited at once, but single transitions are passed one after another, can cause new effects in established pulse EPR sequences. We developed a MATLAB library for simulation of pulse EPR, which is especially suited for modeling spin dynamics in ultra-wideband (UWB) EPR experiments, but can also be used for other experiments and NMR. At present the command line controlled SPin DYnamics ANalysis (SPIDYAN) package supports one-spin and two-spin systems with arbitrary spin quantum numbers. By providing the program with appropriate spin operators and Hamiltonian matrices any spin system is accessible, with limits set only by available memory and computation time. Any pulse sequence using rectangular and linearly or variable-rate frequency-swept chirp pulses, including phase cycling can be quickly created. To keep track of spin evolution the user can choose from a vast variety of detection operators, including transition selective operators. If relaxation effects can be neglected, the program solves the Liouville-von Neumann equation and propagates spin density matrices. In the other cases SPIDYAN uses the quantum mechanical master equation and Liouvillians for propagation. In order to consider the resonator response function, which on the scale of UWB excitation limits bandwidth, the program includes a simple RLC circuit model. Another subroutine can compute waveforms that, for a given resonator, maintain a constant critical adiabaticity factor over the excitation band. Computational efficiency is enhanced by precomputing propagator lookup tables for the whole set of AWG output levels. The features of the software library are discussed and demonstrated with spin-echo and population transfer simulations.

Gd(III)-Gd(III) distance measurements with chirp pump pulses.

The broad EPR spectrum of Gd(III) spin labels restricts the dipolar modulation depth in distance measurements between Gd(III) pairs to a few percent. To overcome this limitation, frequency-swept chirp pulses are utilized as pump pulses in the DEER experiment. Using a model system with 3.4 nm Gd-Gd distance, application of one single chirp pump pulse at Q-band frequencies leads to modulation depths beyond 10%. However, the larger modulation depth is counteracted by a reduction of the absolute echo intensity due to the pump pulse. As supported by spin dynamics simulations, this effect is primarily driven by signal loss to double-quantum coherence and specific to the Gd(III) high spin state of S=7/2. In order to balance modulation depth and echo intensity for optimum sensitivity, a simple experimental procedure is proposed. An additional improvement by 25% in DEER sensitivity is achieved with two consecutive chirp pump pulses. These pulses pump the Gd(III) spectrum symmetrically around the observation position, therefore mutually compensating for dynamical Bloch-Siegert phase shifts at the observer spins. The improved sensitivity of the DEER data with modulation depths on the order of 20% is due to mitigation of the echo reduction effects by the consecutive pump pulses. In particular, the second pump pulse does not lead to additional signal loss if perfect inversion is assumed. Moreover, the compensation of the dynamical Bloch-Siegert phase prevents signal loss due to spatial dependence of the dynamical phase, which is caused by inhomogeneities in the driving field. The new methodology is combined with pre-polarization techniques to measure long distances up to 8.6 nm, where signal intensity and modulation depth become attenuated by long dipolar evolution windows. In addition, the influence of the zero-field splitting parameters on the echo intensity is studied with simulations. Herein, larger sensitivity is anticipated for Gd(III) complexes with zero-field splitting that is smaller than for the employed Gd-PyMTA complex.

Copper ESEEM and HYSCORE through ultra-wideband chirp EPR spectroscopy.

The main limitation of pulse electron paramagnetic resonance (EPR) spectroscopy is its narrow excitation bandwidth. Ultra-wideband (UWB) excitation with frequency-swept chirp pulses over several hundreds of megahertz overcomes this drawback. This allows to excite electron spin echo envelope modulation (ESEEM) from paramagnetic copper centers in crystals, whereas up to now, only ESEEM of ligand nuclei like protons or nitrogens at lower frequencies could be detected. ESEEM spectra are recorded as two-dimensional correlation experiments, since the full digitization of the electron spin echo provides an additional Fourier transform EPR dimension. Thus, UWB hyperfine-sublevel correlation experiments generate a novel three-dimensional EPR-correlated nuclear modulation spectrum.

Coherence Transfer by Passage Pulses in Electron Paramagnetic Resonance Spectroscopy.

Linear passage pulses provide a simple approach to ultra-wideband electron paramagnetic resonance (EPR) spectroscopy. We show by numerical simulations that the efficiency of inversion of polarization or coherence order on a single transition by idealized passage pulses is an exponential function of critical adiabaticity during passage, which allows for defining an effective flip angle for fast passage. This result is confirmed by experiments on E' centers in Herasil glass. Deviations from the exponential law arise due to relaxation and a distribution of the adiabaticity parameter that comes from inhomogeneity of the irradiation field. Such inhomogeneity effects as well as edge effects in finite sweep bands cause a distribution of dynamic phase shifts, which can be partially refocused in echo experiments. In multilevel systems, passage of several transitions leads to generation of coherence on formally forbidden transitions that can also be described by the concept of an effective flip angle. On the one hand, such transfer to coherence on forbidden transitions is a significant magnetization loss mechanism for dipole-dipole coupled electron spin pairs at distances below about 2 nm. On the other hand, it can potentially be harnessed for electron spin echo envelope modulation (ESEEM) experiments, where matching of the irradiation field strength to the nuclear Zeeman frequency leads to efficient generation of nuclear coherence and efficient back transfer to electron coherence on allowed transitions at high adiabaticity.

Sensitivity enhancement by population transfer in Gd(III) spin labels.

In order to enhance echo signals observed with selective pulses, equilibrium populations of the energy levels of S = 7/2 Gd(III) spin labels are rearranged with frequency-swept passage pulses. To transfer population from as many energy levels as possible, the 2 µs long passage pulses range over more than 1 GHz. Application of this technique at Q-band frequencies to three different Gd(III) complexes and spin dynamics simulations reveal large signal enhancements beyond 100% for Gd(III) complexes with zero-field splitting parameters below 1 GHz. For complexes with larger splittings, experimental enhancements are on the order of 90%. Moreover, population transfer is combined with distance measurements on a model system with a pair of Gd(III) ions. As a result, a signal enhancement of 85% is achieved without inducing changes in the obtained distance information. Besides this enhancement by population transfer, a dipolar modulation depth of 9% is demonstrated, which results in a total enhancement of 3.3 with respect to data obtained with monochromatic rectangular pulses. The limitations of the population transfer technique are discussed. In particular, the extraordinary broad pulse bandwidths caused heating effects and pulse distortions, which constrain the pulse length and thus the achievable signal enhancement.

Fourier-transform electron spin resonance with bandwidth-compensated chirp pulses.

Electron spin echo experiments using chirp pulses at X-band around 9GHz have been performed with a home-built spectrometer based on an arbitrary waveform generator. Primary echoes without phase dispersion were obtained by employing the Böhlen-Bodenhausen scheme with the refocusing pulse being half as long as the coherence-generating pulse. To account for physical bandwidth limitation by the resonator, the instantaneous sweep rate of the chirps was adapted to the spectrometer's frequency response function, which can be recorded from the sample under study within a few minutes. Such bandwidth-compensated chirp pulses are experimentally shown to achieve an almost uniform excitation bandwidth that exceeds the resonator bandwidth. This uniform excitation allows for computing frequency-domain spectra by Fourier-transformation (FT) of the echo signal. For a nitroxide in dilute solid solution with a spectral width of 200MHz, the FT EPR spectrum agrees remarkably well with a field-swept echo-detected EPR spectrum. The overall spectral perturbation for operation far beyond the resonator bandwidth was characterized by acquiring a 700MHz wide spectral range of a copper (II) EPR spectrum with nearly uniform amplitude with excitation and refocusing pulses of 200 and 100ns, respectively. Furthermore, peculiarities were observed in solid-state FT EPR spectra of disordered systems. To understand these peculiarities two-dimensional data sets were acquired that correlate the FT EPR spectrum to inversion recovery or nuclear modulation. The echo envelope modulation experiments reveal echo decay rates increased by enhanced instantaneous diffusion and passage-specific effects in the nuclear modulations. The latter effect can be suppressed by nuclear modulation averaging. Apparent longitudinal relaxation times for a given subset of orientations are influenced by nuclear modulation effects. Proper extraction of orientation-dependent relaxation times thus requires an experimental setup that minimizes the modulations.

Adiabatic and fast passage ultra-wideband inversion in pulsed EPR.

We demonstrate that adiabatic and fast passage ultra-wideband (UWB) pulses can achieve inversion over several hundreds of MHz and thus enhance the measurement sensitivity, as shown by two selected experiments. Technically, frequency-swept pulses are generated by a 12 GS/s arbitrary waveform generator and upconverted to X-band frequencies. This pulsed UWB source is utilized as an incoherent channel in an ordinary pulsed EPR spectrometer. We discuss experimental methodologies and modeling techniques to account for the response of the resonator, which can strongly limit the excitation bandwidth of the entire non-linear excitation chain. Aided by these procedures, pulses compensated for bandwidth or variations in group delay reveal enhanced inversion efficiency. The degree of bandwidth compensation is shown to depend critically on the time available for excitation. As a result, we demonstrate optimized inversion recovery and double electron electron resonance (DEER) experiments. First, virtually complete inversion of the nitroxide spectrum with an adiabatic pulse of 128ns length is achieved. Consequently, spectral diffusion between inverted and non-inverted spins is largely suppressed and the observation bandwidth can be increased to increase measurement sensitivity. Second, DEER is performed on a terpyridine-based copper (II) complex with a nitroxide-copper distance of 2.5nm. As previously demonstrated on this complex, when pumping copper spins and observing nitroxide spins, the modulation depth is severely limited by the excitation bandwidth of the pump pulse. By using fast passage UWB pulses with a maximum length of 64ns, we achieve up to threefold enhancement of the modulation depth. Associated artifacts in distance distributions when increasing the bandwidth of the pump pulse are shown to be small.

Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures.

We demonstrate the application of continuous wave dynamic nuclear polarization (DNP) at 0.35 T for site-specific water accessibility studies on spin-labeled membrane proteins at concentrations in the 10-100 µM range. The DNP effects at such low concentrations are weak and the experimentally achievable dynamic nuclear polarizations can be below the equilibrium polarization. This sensitivity problem is solved with an optimized home-built DNP probe head consisting of a dielectric microwave resonator and a saddle coil as close as possible to the sample. The performance of the probe head is demonstrated with both a modified pulsed EPR spectrometer and a dedicated CW EPR spectrometer equipped with a commercial NMR console. In comparison to a commercial pulsed ENDOR resonator, the home-built resonator has an FID detection sensitivity improvement of 2.15 and an electron spin excitation field improvement of 1.2. The reproducibility of the DNP results is tested on the water soluble maltose binding protein MalE of the ABC maltose importer, where we determine a net standard deviation of 9% in the primary DNP data in the concentration range between 10 and 100 µM. DNP parameters are measured in a spin-labeled membrane protein, namely the vitamin B(12) importer BtuCD in both detergent-solubilized and reconstituted states. The data obtained in different nucleotide states in the presence and absence of binding protein BtuF reveal the applicability of this technique to qualitatively extract water accessibility changes between different conformations by the ratio of primary DNP parameters ϵ. The ϵ-ratio unveils the physiologically relevant transmembrane communication in the transporter in terms of changes in water accessibility at the cytoplasmic gate of the protein induced by both BtuF binding at the periplasmic region of the transporter and ATP binding at the cytoplasmic nucleotide binding domains.