Heavy ion radiography and tomography

In the latest years, radiation therapy with ion beams has been rapidly spreading worldwide. This is mainly due to the favourable interaction properties of ion beams with matter, offering the possibility of more conformal dose deposition with superior sparing of healthy tissue in comparison to conventional photon radiation. Moreover, heavier ions like carbon offer a selective increase of biological effectiveness which can be advantageous for the treatment of tumours being resistant to sparsely ionizing radiation. However, full clinical exploitation of the advantages offered by ion beams is still challenged by the lack of exact knowledge of the beam range within the patient. Therefore, increasing research efforts are being devoted to the goal of reducing range uncertainties in ion beam therapy. In this context, ion transmission imaging is being recognized as a promising modality capable of providing valuable pre- (or even “in-between”) treatment information on the patient-specific stopping properties for indirect in-vivo range verification and low dose image guidance at the treatment site. The more recent availability of energetic ion beam sources at therapeutic treatment facilities, in combination with the advances in detector technologies and computational power, have considerably renewed the interest in this imaging technique. Nowadays, many research efforts are being devoted to the development of novel detector prototypes for heavy ion radiography and tomography, as will be reviewed in this contribution.     

Design of a new tracking device for on-line beam range monitor in carbon therapy

Charged particle therapy is a technique for cancer treatment that exploits hadron beams, mostly protons and carbon ions. A critical issue is the monitoring of the beam range so to check the correct dose deposition to the tumor and surrounding tissues. The design of a new tracking device for beam range real-time monitoring in pencil beam carbon ion therapy is presented. The proposed device tracks secondary charged particles produced by beam interactions in the patient tissue and exploits the correlation of the charged particle emission profile with the spatial dose deposition and the Bragg peak position. The detector, currently under construction, uses the information provided by 12 layers of scintillating fibers followed by a plastic scintillator and a pixelated Lutetium Fine Silicate (LFS) crystal calorimeter. An algorithm to account and correct for emission profile distortion due to charged secondaries absorption inside the patient tissue is also proposed. Finally detector reconstruction efficiency for charged particle emission profile is evaluated using a Monte Carlo simulation considering a quasi-realistic case of a non-homogenous phantom.     

Visualization of air and metal inhomogeneities in phantoms irradiated by carbon ion beams using prompt secondary ions

PurposeNon-invasive methods for monitoring of the therapeutic ion beam extension in the patient are desired in order to handle deteriorations of the dose distribution related to changes of the patient geometry. In carbon ion radiotherapy, secondary light ions represent one of potential sources of information about the dose distribution in the irradiated target. The capability to detect range-changing inhomogeneities inside of an otherwise homogeneous phantom, based on single track measurements, is addressed in this paper.MethodsAir and stainless steel inhomogeneities, with PMMA equivalent thickness of 10 mm and 4.8 mm respectively, were inserted into a PMMA-phantom at different positions in depth. Irradiations of the phantom with therapeutic carbon ion pencil beams were performed at the Heidelberg Ion Beam Therapy Center. Tracks of single secondary ions escaping the phantom under irradiation were detected with a pixelized semiconductor detector Timepix. The statistical relevance of the found differences between the track distributions with and without inhomogeneities was evaluated.ResultsMeasured shifts of the distal edge and changes in the fragmentation probability make the presence of inhomogeneities inserted into the traversed medium detectable for both, 10 mm air cavities and 1 mm thick stainless steel. Moreover, the method was shown to be sensitive also on their position in the observed body, even when localized behind the Bragg-peak.ConclusionsThe presented results demonstrate experimentally, that the method using distributions of single secondary ion tracks is sensitive to the changes of homogeneity of the traversed material for the studied geometries of the target.     

Assignment of the protonated 13C resonances of apo-neocarzinostatin by 2D heteronuclear NMR spectroscopy at natural abundance

Summary Nearly complete assignment of the protonated carbon resonances of apo-neocarzinostatin, 113-amino acid antitumor antibiotic carrier protein, has been achieved at natural ¹³C abundance using heteronuclear 2D experiments. Most of the cross peaks in the proton-carbon correlation map were identified by the combined use of HMQC, HMQC-RELAY and HMQC-NOESY spectra, using already published proton chemical shifts. However, double-DEPT and triple-quantum experiments had to be performed for the edition of CH and CH₂ side-chain groups, respectively, which were hardly visible on HMQC-type maps. The triple-quantum pulse sequence was adapted from its original scheme to be applicable to a natural abundance sample. The correlation between carbon chemical shifts and the apo-neocarzinostatin structure is discussed. In particular, ¹³C alpha secondary shifts correlate well with the backbone conformation. These shifts also yield information about the main-chain flexibility of the protein. Assignments reported herein will be used further for interpretation of carbon relaxation times in a study of the internal dynamics of apo-neocarzinostatin.     

Fabrication of Silica Ultra High Quality Factor Microresonators

Whispering gallery resonant cavities confine light in circular orbits at their periphery.^1-2 The photon storage lifetime in the cavity, quantified by the quality factor (Q) of the cavity, can be in excess of 500ns for cavities with Q factors above 100 million. As a result of their low material losses, silica microcavities have demonstrated some of the longest photon lifetimes to date^1-2. Since a portion of the circulating light extends outside the resonator, these devices can also be used to probe the surroundings. This interaction has enabled numerous experiments in biology, such as single molecule biodetection and antibody-antigen kinetics, as well as discoveries in other fields, such as development of ultra-low-threshold microlasers, characterization of thin films, and cavity quantum electrodynamics studies.^3-7The two primary silica resonant cavity geometries are the microsphere and the microtoroid. Both devices rely on a carbon dioxide laser reflow step to achieve their ultra-high-Q factors (Q>100 million).^1-2,8-9 However, there are several notable differences between the two structures. Silica microspheres are free-standing, supported by a single optical fiber, whereas silica microtoroids can be fabricated on a silicon wafer in large arrays using a combination of lithography and etching steps. These differences influence which device is optimal for a given experiment.Here, we present detailed fabrication protocols for both types of resonant cavities. While the fabrication of microsphere resonant cavities is fairly straightforward, the fabrication of microtoroid resonant cavities requires additional specialized equipment and facilities (cleanroom). Therefore, this additional requirement may also influence which device is selected for a given experiment.

Introduction

An optical resonator efficiently confines light at specific wavelengths, known as the resonant wavelengths of the device. ^1-2 The common figure of merit for these optical resonators is the quality factor or Q. This term describes the photon lifetime (τ_o) within the resonator, which is directly related to the resonator''s optical losses. Therefore, an optical resonator with a high Q factor has low optical losses, long photon lifetimes, and very low photon decay rates (1/τ_o). As a result of the long photon lifetimes, it is possible to build-up extremely large circulating optical field intensities in these devices. This very unique property has allowed these devices to be used as laser sources and integrated biosensors.¹⁰A unique sub-class of resonators is the whispering gallery mode optical microcavity. In these devices, the light is confined in circular orbits at the periphery. Therefore, the field is not completely confined within the device, but evanesces into the environment. Whispering gallery mode optical cavities have demonstrated some of the highest quality factors of any optical resonant cavity to date.^9,11 Therefore, these devices are used throughout science and engineering, including in fundamental physics studies and in telecommunications as well as in biodetection experiments. ^3-7,12Optical microcavities can be fabricated from a wide range of materials and in a wide variety of geometries. A few examples include silica and silicon microtoroids, silicon, silicon nitride, and silica microdisks, micropillars, and silica and polymer microrings.^13-17 The range in quality factor (Q) varies as dramatically as the geometry. Although both geometry and high Q are important considerations in any field, in many applications, there is far greater leverage in boosting device performance through Q enhancement. Among the numerous options detailed previously, the silica microsphere and the silica microtoroid resonator have achieved some of the highest Q factors to date.^1,9 Additionally, as a result of the extremely low optical loss of silica from the visible through the near-IR, both microspheres and microtoroids are able to maintain their Q factors over a wide range of testing wavelengths.¹⁸ Finally, because silica is inherently biocompatible, it is routinely used in biodetection experiments.In addition to high material absorption, there are several other potential loss mechanisms, including surface roughness, radiation loss, and contamination loss.² Through an optimization of the device size, it is possible to eliminate radiation losses, which arise from poor optical field confinement within the device. Similarly, by storing a device in an appropriately clean environment, contamination of the surface can be minimized. Therefore, in addition to material loss, surface scattering is the primary loss mechanism of concern.^2,8In silica devices, surface scattering is minimized by using a laser reflow technique, which melts the silica through surface tension induced reflow. While spherical optical resonators have been studied for many years, it is only with recent advances in fabrication technologies that researchers been able to fabricate high quality silica optical toroidal microresonators (Q>100 million) on a silicon substrate, thus paving the way for integration with microfluidics.¹The present series of protocols details how to fabricate both silica microsphere and microtoroid resonant cavities. While silica microsphere resonant cavities are well-established, microtoroid resonant cavities were only recently invented.¹ As many of the fundamental methods used to fabricate the microsphere are also used in the more complex microtoroid fabrication procedure, by including both in a single protocol it will enable researchers to more easily trouble-shoot their experiments.     

Fluorescence correlation spectroscopy in the nanosecond time range: rotational diffusion of bovine carbonic anhydrase B

A fluorescence correlation experiment for measurement of rotational diffusion in the nanosecond time scale is described. Using this method, the rotational diffusion coefficient of bovine carbonic anhydrase B labelled with tetramethylrhodamine isothiocyanate was estimated to be D _r=(1.14±0.15)×10⁷ s^-1 at 22°C. The experiment is based on a cw argon ion laser, a microfluorimeter with local solution flow inside the sample cell, and two photon detectors. The fluorescence intensity autocorrelation function in the nanosecond time range is computed with the help of a time-to-amplitude converter and a multichannel pulse-amplitude analyser.     

Effects of flashing light-emitting diodes on algal biomass productivity

To reduce power consumption and enhance algal biomass productivity in a thin flat-plate bioreactor (called a sliver tank bioreactor), flashing (pulsing) light was used. Biomass productivity and power consumption were monitored in controlled experiments using various photon flux levels, including a constant (non-flashing) flux of 75 μmol photons m^?2 s^?1 and three flashing experiments with photon fluxes of 375, 275, and 175 μmol photons m^?2 s^?1. Flashing experiments were performed at 10 kHz and a duty cycle of 20 %. A sliver tank bioreactor with a chamber width of 6.4 mm was used for its short optical path. Data from the experiments where light was flashed with a photon flux of 375 μmol photons m^?2 s^?1 indicated 9.6 % less power and 2.86 times the biomass productivity compared to the constant photon flux experiments. Similar results were obtained for the other flashing light regimes, which had lower biomass yields but also less input power per unit biomass produced, indicating that a large fraction of the continuously applied photons are shed or wasted, even at levels approximately 1/30th the intensity of full sun.     

Measurement of diffusion coefficients of meningococcal polysaccharide by optical mixing spectroscopy. I. A preliminary characterization on the aggregation of the group C polysaccharide

The angular distribution of scattered intensity and decay times of concentration fluctuations have been measured by means of digital photon counting and single-clipped photon correlation for solutions of Group C meningococcal polysaccharides at 31°C. The z-average diffusion coefficient 〈D〉_z and its second moment 〈D²〉_z have been determined from the time-dependent correlation function using the cumulant expansion technique. Very low observed values of 〈D〉_z and the tremendous width of the polydispersity index, which is the z-average normalized variance, suggest a higher degree of aggregation than the monomer–dimer type self-association at finite concentrations.     

Carboxyl-Dominant Oxygen Rich Carbon for Improved Sodium Ion Storage: Synergistic Enhancement of Adsorption and Intercalation Mechanisms

Oxygen-containing groups in carbon materials have been shown to affect the carbon anode performance of sodium ion batteries; however, precise identification of the correlation between specific oxygen specie and Na⁺ storage behavior still remains challenging as various oxygen groups coexist in the carbon framework. Herein, a postengineering method via a mechanochemistry process is developed to achieve accurate doping of (20.12 at%) carboxyl groups in a carbon framework. The constructed carbon anode delivers all-round improvements in Na⁺ storage properties in terms of a large reversible capacity (382 mAg⁻¹ at 30 mA g⁻¹), an excellent rate capability (153 mAg⁻¹ at 2 A g⁻¹) as well as good cycling stability (141 mAg⁻¹ after 2000 cycles at 1.5 A g⁻¹). Control experiments, kinetic analysis, density functional theory calculations, and operando measurements collectively demonstrate that carboxyl groups not only act as active sites for Na⁺ capacitive adsorption through suitable electrostatic interactions, but also gradually expand d-spacing by inducing a repulsive force between carbon layers with Na⁺ preadsorbed, and hence facilitate diffusion-controlled Na⁺ insertion process. This work provides a new insight in the rational tunning of oxygen-containing groups in carbon for boosting reversible Na⁺ storage through a synergy of adsorption and intercalation processes.     

An improved ultrafast 2D NMR experiment: Towards atom-resolved real-time studies of protein kinetics at multi-Hz rates

Multidimensional NMR spectroscopy is a well-established technique for the characterization of structure and fast-time-scale dynamics of highly populated ground states of biological macromolecules. The investigation of short-lived excited states that are important for molecular folding, misfolding and function, however, remains a challenge for modern biomolecular NMR techniques. Off-equilibrium real-time kinetic NMR methods allow direct observation of conformational or chemical changes by following peak positions and intensities in a series of spectra recorded during a kinetic event. Because standard multidimensional NMR methods required to yield sufficient atom-resolution are intrinsically time-consuming, many interesting phenomena are excluded from real-time NMR analysis. Recently, spatially encoded ultrafast 2D NMR techniques have been proposed that allow one to acquire a 2D NMR experiment within a single transient. In addition, when combined with the SOFAST technique, such ultrafast experiments can be repeated at high rates. One of the problems detected for such ultrafast protein NMR experiments is related to the heteronuclear decoupling during detection with interferences between the pulses and the oscillatory magnetic field gradients arising in this scheme. Here we present a method for improved ultrafast data acquisition yielding higher signal to noise and sharper lines in single-scan 2D NMR spectra. In combination with a fast-mixing device, the recording of ¹H–¹⁵N correlation spectra with repetition rates of up to a few Hertz becomes feasible, enabling real-time studies of protein kinetics occurring on time scales down to a few seconds.     

Heamolysis during in vitro CO2 removal from human blood using a membrane lung

Haemolysis of human blood has been examined in vitro as a function of pH in the range 7.2–8.0. The hydrogen ion concentration of freshly donated blood from 11 donors was manipulated in 42 experiments, entirely by altering the carbon dioxide fraction of air with which the blood was equilibrated using a membrane lung. In contrast to the known alkalaemic haemolysis which occurs in canine blood, we observed no correlation between plasma haemoglobin concentrations and blood pH. We conclude that alkalaemic haemolysis is unlikely to complicate the clinical application of extracorporeal carbon dioxide removal in the management of acute respiratory failure.     

Protein dynamics detected in a membrane-embedded potassium channel using two-dimensional solid-state NMR spectroscopy

We report longitudinal ¹⁵N relaxation rates derived from two-dimensional (¹⁵N, ¹³C) chemical shift correlation experiments obtained under magic angle spinning for the potassium channel KcsA-Kv1.3 reconstituted in multilamellar vesicles. Thus, we demonstrate that solid-state NMR can be used to probe residue-specific backbone dynamics in a membrane-embedded protein. Enhanced backbone mobility was detected for two glycine residues within the selectivity filter that are highly conserved in potassium channels and that are of core relevance to the filter structure and ion selectivity.     

Diagnostics of soft X-ray emission from the plasma of a fast capillary discharge

Results are presented from time-resolved measurements of the soft X-ray emission in the 10-to 40-eV spectral range from the plasma of a pulsed capillary discharge in argon at current pulse amplitudes of up to 26 kA and a current rise time of ～10¹² A/s. The experiments were carried out with 0.3-cm-diameter 15.7-cm-long ceramic capillaries filled with argon at a pressure of 0.25–0.5 Torr in the SIGNAL electrophysical facility. The experimental data are interpreted via computer simulations of the magnetohydrodynamics and level-by-level ion kinetics of an argon plasma. The results obtained indicate that soft X-ray laser pulses with a photon energy of 26.4 eV and duration of 1–2 ns are generated ≈33 ns after the beginning of the discharge current pulse.     

Real-time tumor tracking with an artificial neural networks-based method: A feasibility study

The purpose of this study was to develop and assess the performance of a tumor tracking method designed for application in radiation therapy. This motion compensation strategy is currently applied clinically only in conventional photon radiotherapy but not in particle therapy, as greater accuracy in dose delivery is required.We proposed a tracking method that exploits artificial neural networks to estimate the internal tumor trajectory as a function of external surrogate signals. The developed algorithm was tested by means of a retrospective clinical data analysis in 20 patients, who were treated with state of the art infra-red motion tracking for photon radiotherapy, which is used as a benchmark. Integration into a hardware platform for motion tracking in particle therapy was performed and then tested on a moving phantom, specifically developed for this purpose.Clinical data show that a median tracking error reduction up to 0.7 mm can be achieved with respect to state of the art technologies. The phantom study demonstrates that a real-time tumor position estimation is feasible when the external signals are acquired at 60 Hz.The results of this work show that neural networks can be considered a valuable tool for the implementation of high accuracy real-time tumor tracking methodologies.     

Flame Experiments at the Advanced Light Source: New Insights into Soot Formation Processes

The following experimental protocols and the accompanying video are concerned with the flame experiments that are performed at the Chemical Dynamics Beamline of the Advanced Light Source (ALS) of the Lawrence Berkeley National Laboratory^1-4. This video demonstrates how the complex chemical structures of laboratory-based model flames are analyzed using flame-sampling mass spectrometry with tunable synchrotron-generated vacuum-ultraviolet (VUV) radiation. This experimental approach combines isomer-resolving capabilities with high sensitivity and a large dynamic range^5,6. The first part of the video describes experiments involving burner-stabilized, reduced-pressure (20-80 mbar) laminar premixed flames. A small hydrocarbon fuel was used for the selected flame to demonstrate the general experimental approach. It is shown how species’ profiles are acquired as a function of distance from the burner surface and how the tunability of the VUV photon energy is used advantageously to identify many combustion intermediates based on their ionization energies. For example, this technique has been used to study gas-phase aspects of the soot-formation processes, and the video shows how the resonance-stabilized radicals, such as C₃H₃, C₃H₅, and i-C₄H₅, are identified as important intermediates⁷. The work has been focused on soot formation processes, and, from the chemical point of view, this process is very intriguing because chemical structures containing millions of carbon atoms are assembled from a fuel molecule possessing only a few carbon atoms in just milliseconds. The second part of the video highlights a new experiment, in which an opposed-flow diffusion flame and synchrotron-based aerosol mass spectrometry are used to study the chemical composition of the combustion-generated soot particles⁴. The experimental results indicate that the widely accepted H-abstraction-C₂H₂-addition (HACA) mechanism is not the sole molecular growth process responsible for the formation of the observed large polycyclic aromatic hydrocarbons (PAHs).     

Picosecond laser study of fluorescence lifetimes in spinach chloroplast Photosystem I and Photosystem II preparations

Fractions enriched in either Photosystem I or Photosystem II have been prepared from chloroplasts with digitonin. A more detailed analysis of the decay kinetics of fluorescence excited by a picosecond laser pulse has been possible compared to experiments with unfractionated systems. The Photosystem I fractions show a very short component (? 100 ps) at room temperature which is apparently independent of pulse intensity over the range of photon densities used (5 · 10¹³–1 · 10¹⁶ photons cm^?2). The Photosystem II fraction has a short initial lifetime at room temperature which is strongly intensity-dependent approaching 500 ps at low photon densities, but decreasing to close to 150 ps at the highest photon densities. All of these room temperature decays appear to be non-exponential, and may possibly be fitted by at $\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ expression, expected from a random diffusion of excitations via Förster energy transfer. On cooling to 77 K, lifetimes of both Photosystem I and Photosystem II increase, the lengthening with Photosystem I being more striking. The Photosystem I decays become intensity dependent like the Photosystem II, and at the lowest photon densities decays which are more nearly exponential within the experimental error give initial lifetimes of about 2 ns. The non-exponential decays seen at high photon densities appear to fit a $\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ expression.     

An enhanced sensitivity methyl <Superscript>1</Superscript>H triple-quantum pulse scheme for measuring diffusion constants of macromolecules

We present a pulse scheme that exploits methyl ¹H triple-quantum (TQ) coherences for the measurement of diffusion rates of slowly diffusing molecules in solution. It is based on the well-known stimulated echo experiment, with encoding and decoding of TQ coherences. The size of quantifiable diffusion coefficients is thus lowered by an order of magnitude with respect to single-quantum (SQ) approaches. Notably, the sensitivity of the scheme is high, approximately ¾ that of the corresponding single quantum experiment, neglecting relaxation losses, and on the order of a factor of 4 more sensitive than a previously published sequence for AX₃ spin systems (Zheng et al. in JMR 198:271–274, 2009) for molecules that are only ¹³C labeled at the methyl carbon position. Diffusion coefficients measured from TQ- and SQ-based experiments recorded on a range of protein samples are in excellent agreement. We present an application of this technique to the study of phase-separated proteins where protein concentrations in the condensed phase can exceed 400 mg/mL, diffusion coefficients can be as low as ~10^?9 cm²s^?1 and traditional SQ experiments fail.     

Effect of photon flux density on inorganic carbon accumulation and net CO2 exchange in a high-CO2-requiring mutant of Chlamydomonas reinhardtii

The effect of photon flux density on inorganic carbon accumulation and photosynthetic CO₂ assimilation was determined by CO₂ exchange studies at three, limiting CO₂ concentrations with a ca-1 mutant of Chlamydomonas reinhardiii. This mutant accumulates a large internal inorganic carbon pool in the light which apparently is unavailable for photosynthetic assimilation. Although steady-state photosynthetic CO₂ assimilation did not respond to the varying photon flux densities because of CO₂ limitation, components of inorganic-carbon accumulation were not clearly light saturated even at 1100 mol photons m^-2 s^-1, indicating a substantial energy requirement for inorganic carbon transport and accumulation. Steady-state photosynthetic CO₂ assimilation responded to external CO₂ concentrations but not to changing internal inorganic carbon concentrations, confirming that diffusion of CO₂ into the cells supplies most of the CO₂ for photosynthetic assimilation and that the internal inorganic carbon pool is essentially unavailable for photosynthetic assimilation. The estimated concentration of the internal inorganic carbon pool was found to be relatively insensitive to the external CO₂ concentration over the small range tested, as would be expected if the concentration of this pool is limited by the internal to external inorganic carbon gradient. An attempt to use this CO₂ exchange method to determine whether inorganic carbon accumulation and photosynthetic CO₂ assimilation compete for energy at low photon flux densities proved inconclusive.     

Phosphorus nuclear magnetic resonance studies of phosphoglucomutase and its metal ion complexes.

The ³¹P nuclear magnetic resonance of the covalently bound phosphate group at the active site of phosphoglucomutase has been examined by means of Fourier transform nuclear magnetic resonance spectroscopy. At a pD of 7.9, the chemical shift of the ³¹P nucleus is 3.8 ± 0.1 ppm downfield from 85% H₃PO₄; this shift is close to that of phosphoserine (dianionic form). Proton decoupling experiments suggest that the phosphorus of the enzymic phosphate group is coupled to protons with chemical shifts similar to those of phosphoserine. In D₂O, with proton decoupling, the ratio of the longitudinal and transverse diamagnetic relaxation times in solutions of 1.6 mm phosphoenzyme yields an approximate correlation time of 10^?7s for the ³¹P nucleus of the enzyme. This is within the range of values expected for tumbling of the entire protein molecule and suggests that the covalently attached phosphate group is immobilized or “frozen” at the active site of the enzyme by means of noncovalent interactions with adjacent groups. Consistent with this, the pK_a of the enzymic phosphate is significantly lower than that of phosphoserine. Binding of the diamagnetic activator, Mg²⁺, causes little or no change in the chemical shift of the resonance of the enzymic phosphorus from pD = 5.3 to 7.6, a downfield shift (?0.5 ± 0.1 ppm) at pD = 8.6, but an upfield shift (0.8 ±0.1 ppm) for that of phosphoserine, suggesting that bound Mg²⁺ is not coordinated to the enzymic phosphate. Independent evidence against direct coordination is provided by the paramagnetic effects of Ni²⁺ bound at the active site on the relaxation rates of the enzymic phosphorus. By assessing the paramagnetic effect of bound Ni²⁺ on both the longitudinal and transverse relaxation rates of the observed resonance, and by using correlation times determined for water proton relaxation induced by the Ni²⁺ complex, a range of Ni²⁺ to phosphorus distances of 4 to 6 Å is calculated. These distances suggest a second sphere interaction between the enzyme-bound metal and the enzymic phosphate group. Bound Ni²⁺ also markedly decreases the integrated intensity of the ³¹P resonance. Although the reason for this intensity decrease is incompletely explained, the present data establish the close proximity of the bound metal ion and the active site phosphoserine on phosphoglucomutase.