For cochlear INS it has been debated whether the radiation directly stimulates the SGNs or evokes a photoacoustic effect. The results support the view that a direct interaction between neurons and radiation dominates the response to INS.Infrared neural stimulation (INS) has been proposed as an alternative method to electrical stimulation because of its spatial selective stimulation. Independent of the mechanism for INS, to translate the method into a device it is important to determine the energy for stimulation required at the target structure. Custom-designed, flat and angle polished fibers, were used to deliver the photons. By rotating the angle polished fibers, the orientation of the radiation beam in the cochlea could be changed. INS-evoked compound action potentials and single unit responses in the central nucleus of the inferior colliculus (ICC) were recorded. X-ray computed tomography was used to determine the orientation of the optical fiber. Maximum responses were observed when the radiation beam was directed towards the spiral ganglion neurons (SGNs), whereas little responses were seen when the beam was directed towards the basilar membrane. The radiant exposure required at the SGNs to evoke compound action potentials (CAPs) or ICC responses was on average 18.9 ± 12.2 or 10.3 ± 4.9 mJ/cm The spot size has been either reported as the core diameter of the optical fiber51113Infrared radiation (IR) can be used to directly stimulate nerves, neurons, cardiomyocytes and vestibular hair cells  and neural responses from single units in the central nucleus of the inferior colliculus (ICC). The distances between the optical source and the possible target structures and the orientation of the radiation beam towards the target structure were determined at the conclusion of the experiments from X-ray micro computed tomography (microCT). The spot size at the target structure was calculated from the numerical aperture of the optical fiber and from direct measurements using the knife-edge technique. The radiant energy was measured at the tip of the optical fiber in air and the corresponding energy at the target structure was calculated accounting for the distance and the optical properties of the matter between the fiber and the target structure.In this study, flat and angle polished optical fibers were used to determine the radiant energy for INS at the SGNs. In The radiant energy was measured at the tip of the optical fiber in air and evoked responses were recorded for selected energies between 0 and 127 μJ/pulse. From the microCT image, the distance between the tip of the optical fiber and the modiolus could be measured, and the orientation of the beam path could be determined with the help of the tungsten wire.CAP frequency tuning curves to pure tone acoustic stimuli and laser evoked CAP input/output functions were measured to determine cochlear function and the responses to INS. For all stimuli, CAP threshold was defined as the lowest stimulus level at which a response was observed. Representative recordings are shown in The angle polished fiber was inserted into the cochlea until contact was made with the modiolus and was rotated until a maximum CAP amplitude was obtained. While maintaining the same orientation, the optical fiber was retracted using a micromanipulator and CAP amplitude was measured at different locations along the track. A typical sequence of measurements, which was obtained in one animal, is shown in At marked locations in Since the CAP is the summation of the responses from many neurons along the cochlea it is not clear whether the response comes right from the target structure. For example, photoacoustic events caused by the infrared irradiation may activate large areas of the cochlea. To study local responses to INS along the cochlea, recordings in the ICC were conducted with multichannel electrodes, which were inserted in the ICC across frequency laminae. Multi-unit responses might be recorded at each electrode contact of the multichannel electrode arrayTo further identify the target structure for INS, single unit recordings in the ICC were combined with synchrotron microCT imaging in 3 normal hearing animals. The possible involvement of hair cells in the response to INS should be more obvious in normal hearing animals. The selective stimulation of INS in the cochlea is well-represented in the spatial activation profile in ICC. An example is shown in The radiant energy required for stimulation was determined from the experiments in which CAP amplitudes or ICC single unit responses were measured. Threshold for INS of single units was defined as the radiant energy required to evoke significant phase-locked firing. The energy at the tip of the optical fiber was on average 14.1 ± 8.1 μJ/pulse for ICC for single unit responses and 17.2 ± 13.9 μJ/pulse for CAP response, respectively . The var2 = 0.67). The radiant energy at the target structure is influenced by the radiation spot size of the laser, which can be calculated by the numerical aperture of the fiber and by the distance between the tip of the optical fiber and the neural structure. Therefore, we measured this distance using the reconstructed microCT images. This distance varied between 150 and 950 μm . The spot size at the target structure was calculated accordingly as has been presented in a previous publication2, was then used to estimate the energy at the target structure to reach response threshold. After correcting for the distance between the tip of the optical fiber and the modiolus, the radiant energy on target was between 1.4 and 16.4 μJ/pulse, on average 4.1 ± 1.9 μJ/pulse for ICC single units and 7.2 ± 4.7 μJ/pulse for CAP responses, respectively :025002. doi: 10.1117/1.NPh.2.2.025002. Epub 2015 May 18. Transient heating of the neural tissue through the absorption of photons constitutes the one step of the mechanism of INS everybody can agree to1172931323335363839The mechanism of INS, in particular the contributions of the pressure which is generated through local heating through the absorption of the laser pulses, has recently been discussed in detail in a separate paper by Young et al. suggest that a pressure wave is the sole mechanism by which the cochlea is stimulated with INS pulses in nanosecondsIn particular, the experiments by Schultz et al. showed a photomechanical vibration of basilar membrane when it was irradiated directly with a 930 nm laserRecent work by Ren While parts of the mechanism for INS have been identified173539Pigmented guinea pigs (200–800 g) of either sex were used in the experiments. Care and use of animals were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Northwestern University.Surgery and data acquisition were similar to those described in detail elsewhere2Cochlear stimulation was achieved with diode lasers  coupled to the optical fibers  with core diameters of 200 μm . The radE at the modiolus was calculated with the following equation: E = Eo * ed−μ, where E0 denotes the energy per pulse at the tip of the optical fiber, μ the extinction coefficient, and d the distance between the optical fiber and the modiolus that was determined from the microCT scans (see below). From the radiant energy the peak power was calculated by dividing the E by the pulse duration. The radiant exposure was calculated by dividing E by the spot size. The spot size was calculated by using the core diameter of the optical fiber dfiber, and the spread of the radiation in water as determined in previous experiments r = d × tan αα is the angle by which the radiation beams spreads. The radius R for the spot was calculated as The energy CAPs were measured with a silver ball electrode at the round window and a reference electrode in the tissue near the neck. Threshold was defined as a CAP that was visible above the noise floor of the recordings, typically between 3–10 μV. CAPs to acoustic stimuli were measured before and after creating the cochleostomy and after neomycin application. For the angle polished fiber, threshold energy and response amplitude was determined for the initial placements of the optical fiber. An energy-versus-CAP amplitude contour (laser input/output curve) was recorded for at least 10 different radiant energy levels. Next, the fiber was rotated by 45° counterclockwise and the procedure was repeated until a full rotation (360°) was completed. The optical fiber was then rotated to the pointing angle at which the largest CAP amplitude was measured and was retracted in steps of 50 μm. CAP amplitudes in response to a fixed radiant energy  was measured at each step.2/site) along a single shank at center-to-center intervals of 100 μm. To access the ICC, a 5 × 5-mm opening was made in the right parietal bone. The multichannel electrode mounted on a 3D-micromanipulator  was advanced through an incision in the dura mater and through the occipital cortex into the ICC. The electrode was inserted into the ICC on a dorsolateral to ventromedial trajectory at approximately 45° off the parasagittal plane, approximately orthogonal to its isofrequency laminae242A multichannel penetrating electrode array  was used for ICC recordingsIn most cases, the ICC recording electrode array was placed prior to creating the cochleostomy because the procedure could result in an elevation of the CAP thresholds and lead to a poor correlation of the ICC location to the best acoustic frequency.Measurements were conducted in normal hearing and deaf animals . Acute dThe orientation specific changes of CAPs induced by INS were also verified in a chronic deaf animal. For chronic deafening, at least 4 weeks before the experiments, neomycin  was injected in the bulla through tympanic membrane. The pure tone CAP thresholds were over 80 dB SPL  and the ®) and followed by cardiac perfusion with 2.5% glutaraldehyde and 1.5% paraformaldehyde (pH 7.4) in Ringer’s Lactate (RL). After fixation, the animals were decapitated and the bullae were harvested with the optical fiber in place. The specimens were imaged using microCT  and synchrotron radiation at 2-BM at the Advanced Photon Source (APS), Argonne National Laboratory. From the tomographic reconstruction, a plane across the center of the optical fiber and the tungsten wire and the target structure was selected. The distance between the optical fiber and the target structure was then measured in this plan.Before insertion, a 25 μm tungsten wire was attached at the tip of the optical fiber with Loctite super glue , opposite to the site where the radiation beam exited the optical fiber . The tunThe phase-locked firing of the ICC single units was evaluated by calculating the vector strength4445How to cite this article: Tan, X. et al. Radiant energy required for infrared neural stimulation. Sci. Rep. 5, 13273; doi: 10.1038/srep13273 (2015).


There is a lack of understanding of the morphological characteristics of the cartilage-bone interface. Materials that are currently being used in tissue engineering do not adequately support the regeneration of bone and cartilage tissues. The present study aimed to explore the morphological characteristics of cartilage-bone transitional structures in the human knee joint and to design a biomimetic osteochondral scaffold based on morphological data.Histology, micro-computed tomography (micro-CT), and scanning electron microscopy (SEM) were used to investigate the microstructure of the cartilage-bone transitional structures. Morphological characteristics and their distribution were obtained and summarized into a biomimetic design. A three-dimensional model of a biomimetic osteochondral scaffold was CAD designed. A prototype of the resulting subchondral bone scaffold was constructed by stereolithography using resin.Micro-CT revealed that subchondral bone presented a gradually changing structure from the subchondral to spongy bone tissue. The subchondral bone plate was more compact with ~20 % porosity compared with ~60 % porosity for the spongy bone. Histology and SEM showed that cartilage was stabilized on the subchondral bone plate by conjunctions, imbedding, interlocking, and binding forces generated by collagen fibers. Some scattered defects allow blood vessel invasion and nutritional supply.The subchondral bone plate is not an intact plate between the cartilage and bone cavity, and some scattered defects exist that allow blood vessel invasion and nutritional supply. This characteristic was used to design an osteochondral scaffold. This could be used to construct an osteochondral complex that is similar to native bones. Over the last decade, many three-dimensional 3D) osteochondral scaffolds have been designed using a number of heterogeneous materials. These osteochondral scaffolds have the potential to be used for repairs of the knee and for the treatment of osteoarthritis , 2, but D osteochTherefore, the solution could be to design and build an osteochondral scaffold with a true biomimetic microarchitecture reflecting the morphology of the natural osteochondral complex, which is made of bone, cartilage, and their transitional structures. The transitional structures including the calcified cartilage zone (CCZ) and the subchondral bone plate provide the attachment of cartilage to bone, allow force transmission across the joint, and limit the diffusion from the subchondral bone , 13.Wang et al.  used hisMany studies have focused on osteochondral complex structures and their pathological changes in joint abnormalities –11, but Therefore, the present study aimed to explore the morphological characteristics of cartilage-bone transitional structures, specifically the micro-architectures and conjunctions of the osteochondral complex in the human knee joint, and to design a biomimetic osteochondral scaffold based on these morphological data.Eight human knees were extracted from four cadavers ; they were fixed and preserved in formalin. These cadavers were never frozen. The specimens were investigated at the Department of Anatomy of Xi’an Jiao Tong University. Samples were from individuals who donated their corpse to science after their death. This study was approved by the Anatomic Gift Program of the Xi’an Jiao Tong University No. 2012-211). All specimens had no cartilage defect or pathological change indicative of osteoarthritis  Fig. a. The boThe images were analyzed in terms of gray values. The gray value of water was calibrated as 0, the gray value of air was −1000, and the gray value of cortical bone was 2000 . A line 2 for 24 h. The samples were placed in an ultrasonic bath for 1 h. The oxidation and washing process was repeated four times to remove cartilage. Two samples were excised from the middle of the medial condyle position of two cadaveric right knees as cubes (10 × 10 × 10 mm)  from the regions shown in Fig. Four samples were excised from the middle of the lateral condyle position of four cadaveric right knees as cubes (10 × 10 × 5 mm)  according to the morphological characteristics acquired from the observation of the natural osteochondral complex.To determine if the resulting scaffold could be manufactured, a prototype of the resulting subchondral bone scaffold was constructed by stereolithography using resin, based on a previously published method .The slow increase in gray value observed in the plot of the 3D cartilage model suggests that the cartilage closer to the bone had a high gray value Fig. b. The plThe BMD, BMC, and TMC values were the highest around the subchondral bone region; the distance from the surface of cartilage to the subchondral plate below was about 2.3 mm , 9.61 % pore area of the total surface area , and pore diameter ratio of 35 % (pore diameter over the entire slice length) (Fig. The design of the cancellous bone area was derived from the data portion of cancellous bone, but cell growth and the manufacturing process were considered in this design , 27, resThe aim and ultimate usefulness of tissue engineering is to create engineered grafts that can interact with their environment in order to provide functionalities that are comparable to the original replaced tissue; the graft must also be integrated by the host . Based oIt has been suggested that the CCZ represents a zone of transitional stiffness and decreases the workload stress at the cartilage-bone interface , 38. IndLane et al.  discoverBased on the cognition of natural osteochondral complexes and previous studies , 22–26, The present study is not without limitations. First, results were not analyzed in terms of weight-bearing/non-weight-bearing regions of the knees, and future studies should address this issue. Results were obtained from cadavers and samples may have suffered from some deterioration. However, it might be impossible to obtain intact fresh non-cadaver knees since patients undergoing knee resection usually does so for good pathological reasons that would affect the results. The aim of the present study was only to provide a morphological analysis of the transitional zone in order to build a biomimetic scaffold, and no quantitative analysis was performed. Finally, the constructed scaffold was not tested in vivo. This will be the focus of our future studies.The present study is the first part of a systematic research on osteochondral tissue regeneration. This study was only a preliminary study that observed the microarchitecture of the transition zone and that examined if a suitable material could be engineered from these observations. Nevertheless, this material needs to be tested in vivo and will probably require a number of adjustments before being used for treating patients. In addition, only one manufacturing process was used to create the prototype, and the optimal process needs to be defined. Efforts will be made to use these results to generate materials that could be used to repair damaged knees and to treat osteoarthritis.Results suggest that the subchondral bone plate is not an intact plate between the cartilage and bone cavity, and that some scattered defects exist that allow blood vessel invasion and nutritional supply. This characteristic was used to design an osteochondral scaffold. This could be implemented to construct an osteochondral complex that is similar to native bones.


To meet high expectations concerning precision and accuracy of reference materials, preparation of matrix-free reference materials using thermal decomposition-gas chromatography-mass spectrometry (TD-GC-MS) was proposed in this study. In the paper, the results obtained in preparation of the new reference materials for benzene and toluene are presented, based on the thermal decomposition technique of compounds chemically bound to the surface of optical fibre segments. The results obtained at various stages of the research procedure  confirmed the possibility of using prepared laboratory samples of materials as reference materials for benzene and toluene. For the prepared batch of materials, reference values 1.26 ± 0.91 (ng/fibre) for benzene and 11.3 ± 7.4 (ng/fibre) for toluene were determined. Homogeneity of the material. The reference material should be sufficiently uniform in the various parts of the material so that the value(s) refers to the properties of any other batch of reference material.Stability of the material. A reference material and the value(s) characterizing its properties should be stable at a certain time and under storage, transport and usage conditions.Precision and accuracy of determining the value of a reference material property. The property value should be determined using a method burdened with a slight systematic error and using measuring instruments or measures of material that can be linked to national standards of measurement units and thus also consistent with similar units in other countries.Documentation of reference material. There should be a clear description of the material’s characteristics, the methods of determining the values of the reference material, and the value of the measurement’s uncertainty of the property value. Certification and possession of a certificate prepared in accordance with ISO Guide 31 recommendations are an additional advantage of a reference material.Reference materials play a role of property value carriers determined for the applied specific material. Thus, by evaluating the suitability of a reference material, the following criteria should be taken into consideration –5:HomogeGiven the above guidelines, it can be concluded that the ability to collect and transmit information about the material properties results from the requirements of reference materials.IRMM i IAEA (Europe)National Institute of Standards and Technology (NIST) (USA)NRCC (Canada)IChTJ (Poland)Many scientific-technical institutions whose activities relate to metrological issues are involved in the production and certification of various kinds of reference materials. The most important institutions involved in the manufacture of gaseous reference materials include the following:Linde Industrial Gases and Messer Group (Germany). The choice of a suitable reference material depends on the type of analytical task, the characteristics of the test material, the applied analytical procedure and the expected analytical information obtained in the course of the measurements.Also, in other institutions, attempts are made to manufacture various types of reference materials. They are often focused on a specific type of material. The largest producers of certified gaseous mixtures are The formation of photochemical smog (ozone + oxidants)Intensification of the greenhouse effectDue to the fact that one-ring aromatic hydrocarbons play a significant role in the chemistry of the atmosphere and have a harmful effect on living organisms, they are a common element of laboratory research conducted worldwide –11. BenzThe danger associated with benzene and its alkylated derivatives in the environment is subject to their toxic, mutagenic and carcinogenic effects on living organisms. Additionally, benzene, toluene, ethyl benzene and xylene (BTEX) group compounds constitute a risk not only to the environment but also to the public health. Therefore, they are the most important pollutants considered by environment protection organizations.Intensive development of analytical techniques for measurement of gaseous media and the negative effects  of BTX and oxygenated organic compounds make it particularly important to monitoring and determination of these compounds in the air. The problem is the availability of gaseous reference materials.As a standard gaseous mixtures in cylindersCRM 112), the reference material for BTEX compoundsTenax  have conducted the research on the prototype of reference material that emits a single volatile organic compound such as toluene, analogously to the emissions of a diffusion-controlled building product .On the other hand, the Brazilian Metrology Institute (Inmetro) has developed a certified reference material for BTEX compounds in methanol .The worldwide research projects are focused on searching for new solutions which enable to prepare homogenous, stable and handy reference materials for volatile organic compounds.The principle of preparation of standard gas mixture using thermal decomposition (TD) process is based on controlled decomposition of surface compound in strictly defined temperature during the specified period of time. TD process results in the formation of the analyte stream which is mixed with the stream of diluting gas forming the stream of standard gas mixture .Thermal decomposition of surface compounds is characterized by many advantages due to the preparation of VOC reference materials. The possibility of application in case of toxic, volatile and reactive analytes and the production of single component and multi components of standard mixtures with a broad and controlled range of concentrations give possibility to apply a TD-GC-MS technique for preparation of reference materials for benzene and toluene.The research procedure is shown in Fig. The process of chemical modification of glass fibres has been developed and carried out by a team from the Department of Chemical Technology, Faculty of Chemistry, University of Gdańsk. The starting material for the process of glass fibre chemical modification was coated with a thin layer of aluminium (inner fibre diameter/outer fibre diameter ratio 660:680 μm).In the first stage of the study, an attempt was made to chemically modify the glass fibres in order to obtain the surface compound, acting as a source of benzene in the process of thermal decomposition. A diagram of the chemical modification of the fibre surface process is shown in Fig. 2 in the presence of hydrochloric acid. The reaction proceeded in an aqueous solution at a temperature of 0–5 °C. The formed diazonium salt was coupled with pre-modified fibres. The reaction proceeded at 0–5 °C, and the diazonium salt was added dropwise for 30 min to the fibres in a solution of pyridine. The prepared fibres were left for 2 h in a solution and then filtered off; washed successively with methanol, acetone and water; and dried in a vacuum evaporator.The first step was to bind a suitable silane to fibres, in this case 3-chloropropyl–trimethoxysilane in order to introduce onto the surface of the fibres the connecting arm of suitable length and the –Cl functional group. The reaction was carried out in toluene at 80 °C. After finishing the reaction, fibres were washed sequentially with toluene, methanol and acetone solution and then dried in a vacuum evaporator. Then, the Schiff base  was added to the prepared fibres. The reaction was conducted in dimethylformamide (DMF) at 80 °C for 12 h. After finishing the reaction, the fibres were again washed with solutions: DMF, methanol and acetone and then dried. The next step was the diazotization reaction of aniline by nitrate(III) sodium NaNOIn the next stage of the research, a procedure for modifying the surface of the carrier material to obtain a suitable surface compound acting as a source of toluene in the thermal decomposition process was developed.The process of chemical modification proceeded in several stages, analogous to the process of chemical modification, to obtain a suitable surface compound acting as a source of benzene. The change occurred at the stage of diazotization. In the case of benzene, it was aniline, and the toluene phase was used for diazotization by using p-toluidine (p-methylaniline).The information on the analytical procedure is summarized in Table y = 2.90°105 × + 6.75°105; R2 = 0.999From 3.1 to 15.6 ng benzene: y = 2.46°105 × + 1.57°106; R2 = 0.995From 15.6 to 62.5 ng benzene: y = 3.06°105 × + 6.59°105; R2 = 0.999From 3.6 to 17.9 ng toluene: y = 2.58°105 × + 1.85°106; R2 = 0.993From 17.9 to 71.4 ng toluene: The calibration process of the TD-GC-MS system was performed using a reference gas mixture defined by the manufacturer , a concentration of the analyte from the BTX group, which was 9.80 ± 0.2 ppm for benzene and 9.50 ± 0.2 ppm for toluene. For each analyte (benzene and toluene), two four-point calibration curves were created for two ranges of masses of analytes: from 3.1 to 15.6 ng and from 15.6 to 62.5 ng for benzene and from 3.6 to 17.9 ng and from 17.9 to 71.4 ng for toluene. The equations for the calibration curves were as follows:y is the MSD response expressed as a peak area , x is the mass of analyte (ng), and R2 is the coefficient of determination.where A homogeneity study was performed for randomly selected samples of chemically modified fibres coated with a surface compound, from which thermal decomposition was obtained: (a) benzene and (b) toluene.One package of each material containing 20 optical fibres was modified with appropriate surface compound. Determination was made for 15 fibres (five fibres from three different packages), separately for benzene and toluene samples.Stability studies were carried out for fibres stored at room temperature. The obtained results were compared with the results obtained for fibres stored under refrigeration (−18 °C) taken as a reference condition.The results obtained at the assessment stage of homogeneity and stability are shown in Fig. In order to verify the accuracy of values obtained in the course of measurements, it is necessary to check the insignificance of the differences between the results. For this purpose, the method of calculating the ratio of the obtained results and the uncertainty of the determination was applied.Fvalue) and the critical value (Fcritic).In order to verify the homogeneity of the material’s within-unit and between-unit homogeneity and to calculate the uncertainty in heterogeneity of materials, one-factor analysis of variance (one-way ANOVA) was conducted –21. ThisThe information on the parameters characterizing ANOVA analysis applied to evaluate a set of obtained measurement results is summarized in Table Fvalue) is smaller than the critical value (Fcritic). The prepared candidates are characterized by good agreement among the units and packages. It was found that prepared candidates for reference materials for benzene and toluene are homogenous. It is proved by low values of such parameters as the coefficient of variation and standard deviation. It is also shown that the main element of uncertainty related to heterogeneity of prepared materials is connected with within-unit homogeneity of materials (83.5 % for benzene and 97.9 % for toluene).On the basis of the obtained result, it is shown that the test criterion value ))3))2\docuh is the combined standard uncertainty of homogeneity stage and Sr is the repeatability of measurement method.where The statistical analysis of the results obtained to assess the stability of the prepared reference materials was carried out using the method of calculating the ratio of the obtained results and the uncertainty of its determination.R) was determined for the fibres stored over the 12 months. The parameter R was calculated for each of the periods in which the measurements were performed as a basis to assess the material’s stability.The ratio of the average quantities of released benzene and toluene  has a value of 1. That is why it can be concluded that the prepared material is stable in room temperature and its stability period is at least 12 months.The calculated means for the compared series of results obtained for benzene and toluene do not differ in a statistically significant manner. This is due to the fact that in the range specified, value ± uncertainty of the calculated ratio of the appointment .where The contributions of uncertainties obtained at the stages of homogeneity and stability are presented in Table Benzene:Toluene:Prepared candidates meet all the requirements for reference materials. Accordingly, Fig. Analysis of uncertainty sources resulting from the applied analytical procedure can be stated that they do not affect the results obtained during the thermal decomposition of the prepared candidate reference materials.The obtained results confirmed the homogeneity and stability of the prepared batch of candidates for the title of matrix-free reference materials and resulted in the determination of the indicative value and estimation of the expanded uncertainty of the indicative value.Prepared candidates for benzene and toluene reference materials are characterized by relatively high uncertainty values (72 % for benzene and 66 % for toluene). However, for microtrace and ultratrace concentration levels, the precision of the determination can be in the range of tens of percents due to the empirical equation of Horwitz .As the result of this study, the possibility of applying glass fibres as a carrier for preparation of matrix-free benzene and toluene reference materials was confirmed.Taking into account the commercially available types and forms of gaseous preparation reference materials, it should be noted that the proven solution is an alternative to the commonly used techniques for obtaining the reference gaseous mixtures in bottles.The limited nature of the quantitative preparation of the mixtureTime-consuming and inaccurate proceduresLosses related to the processes of adsorption and condensation on the walls of the ventricular system for preparing gaseous calibration mixturesFailure to store an amount for reuse.The possibility of applying benzene and toluene reference materials in analytical studies in the form of glass fibres coated with a suitable carrier material allows the elimination of most of the disadvantages arising from the traditionally used static techniques for the preparation of reference materials, which include the following:The simplicity of preparation and the much higher stability of the above-described candidate reference materials should be underlined as an advantage over bottled standard gaseous calibration mixtures.


Microsphere concentrations are needed to assign equivalent reference fluorophores (ERF) units to microspheres used in quantitative flow cytometry. A flow cytometer with a syringe based sample delivery system was evaluated for the measurement of the concentration of microspheres contained in a vial of lyophilized microspheres certified by BD Biosciences to contain 50,600 microspheres. The concentration was measured by counting the number of microspheres contained in the volume delivered by the flow cytometer and dividing the number by the volume. The syringe volume was calibrated both in the delivery and draw modes, and the results of the volume calibration were summarized by two calibration lines. The delivered volume was obtained by dividing the number of recorded events by the concentration of microsphere count standard in the sample tube. The draw volume was obtained by weighting the sample tube before and after the draw. The slope of the draw volume calibration line was equal to 1.00 with an offset of −13 µL. The slope of the delivered volume calibration was 0.93 suggesting a systematic volume-dependent bias, which can be rationalized as an effect of suspension flow in capillaries. When the sample volume was set to values between 150 µL and 300 µL, both calibration curves gave similar results suggesting that a good estimate of the true delivered volume can be obtained by subtracting 13 µL from the delivered volume indicated by the syringe settings. The number of microspheres in the volume was obtained by passing the suspension contained in the volume through a laser beam and counting the number of events in which the signals from the scattering and fluorescence detectors exceeded threshold values.Measurements were performed with the lyophilized microspheres made by BD Biosciences and fluorescein microspheres  in three buffers: a phosphate buffer saline (PBS), a buffer containing PBS and 0.05 % BSA  by mass, and a buffer containing PBS and 0.05 % TWEEN 20 detergent solution (P1379 Sigma-Aldrich) by mass. It was found that the concentration of count standard was significantly higher in the PBS+BSA buffer relative to the value obtained in PBS buffer. Values for PBS+0.05 % TWEEN 20 buffer were intermediate. The effect of buffer on the measured microsphere concentration was reported previously.The suggested procedure for the measurement of the concentration of microspheres with the flow cytometer is to use PBS+0.05 % BSA buffer, accumulate data for a delivered volume of 150 µL to 300 µL, and reduce the indicated delivered volume by 13 µL when performing the concentration calculation. The procedure was tested on a mixture of lyophilized microspheres and RM 8640 microspheres. The resulting lyophilized microsphere concentration was consistent with the certified value. The RM 8640 concentration determined using the suggested procedure was consistent with the concentration value determined using the relative method with the lyophilized microspheres as the reference. The uncertainties, obtained from one standard deviation of repeated measurements, were about 4 %. The accurate measurement of the concentration of specific cell types in peripheral blood is an important goal , 2. In aIn practice, the measurements are preceded by a “performance check” of the Attune flow cytometer. The performance check is carried out by placing a suspension of special microspheres (Performance beads) in the sample tube. The performance beads are run in the cytometer as a normal sample, and the Attune software adjusts the PMT voltages and the delay between the signals associated with the two lasers, so that these values are within the expected design limits. If the performance check is passed successfully, the measurement of the analyte suspension can be initiated. The user has no control over the procedure followed in the “performance check”.Samples were prepared by pipetting either 1 mL or 2 mL of PBS (or another buffer) into a tube  containing the Trucount polystyrene microspheres with a diameter of 4.2 µm. The tube was gently mixed (Daigger Vortex Genie 2) and placed into the SIP. In most cases, the Attune was set to deliver a volume of 200 µL from the sample syringe at a flow rate of 100 µL/min. The measurement was started by drawing 314 µL of sample suspension from the tube in the SIP. The value 314 µL was determined by the Attune software and was not under control of the user . The measurement was always started in the “run” mode, and switched over to the “record mode” after about 10 µL of the sample suspension have passed. This procedure was used to minimize transient effects. The threshold of the forward scattering (FS) detector was placed in the “or” mode while the side scattering and fluorescence detectors were placed in the “ignore” mode. This required that the FS signal in a recorded event was above threshold. The amplitudes of the side scattering and fluorescence signals were localized near well-defined peak values so that ignoring a threshold, which is well below the peak value, did not bias the acquisition of “true” (near the peak) signals. The threshold settings were not expected to introduce significant uncertainty since with all thresholds set to “and” mode the number of events decreased by only 2 %, and could be rationalized as a decrease in detected electronic noise pulses. The measurements described below consisted of a sequence of approximately 10,000 scattering and fluorescence events associated with individual microspheres which passed sequentially through the laser interrogation plane.In the following, we describe procedures to determine the number of events, and to estimate the volume associated with these events. The concentration is defined as the ratio of events and the volume. Several buffers were tried as well as a more detailed examination of the distribution of events in a gate. The objective was to find conditions which minimize systematic uncertainties.events. Note that the number of events in gates 1, 2, 3, 4, and 5 are almost identical as shown by the values of Nevents associated with each plot. This means that whenever FS triggered, signals were also present in all of the other detectors. The number of events in each gate can be made larger by increasing the size of the gate. However, there is minimal change in the number of events once the gate size is as shown in the events in An event occurred whenever the signal in the forward scattering detector (associated with the 405 nm laser) exceeded a threshold value. When this happened, signals from all detectors were digitized for a preset period of time. The digitized signals were analyzed, and the area, amplitude, and width of the digitized signals (assumed to be pulses) were recorded in real time. The sequence of such recordings, taken for a preset sample volume, constituted a measurement. The analysis was performed using FCS Express V3™  softwareThe sample syringe delivered a measured volume of the suspension determined by the inner diameter of the syringe and the travel of the piston. The value of the delivered volume was used to calculate the concentration. The same syringe was used to draw a volume of the sample suspension contained in the tube placed in the SIP. In order to account for the volume of the tubing in the sample path, the instrument was configured to draw approximately 114 µL more than it delivers. Since the same syringe was used to draw the sample and deliver the sample, it was possible to calibrate the volume of the syringe using the capability to draw a specified volume of the sample. The calibration was performed by measuring the mass of the sample tube before the draw and after the draw. The difference of the two masses was divided by the density of water (0.99777 g/mL at 22.5 °C) to yield the drawn volume. The open circles in V = eventsN/C where eventsN is the number of recorded events, and C is the purported concentration of the microspheres in the tube. The solid spheres in An alternate volume calibration was performed using a tube of Trucount microspheres (TC) which was purported to contain 50,600 microspheres. The TC microspheres were resuspended in 2 mL of PBS+0.5 % BSA buffer. A preset (indicated by software) volume of the suspension was delivered to the laser interrogation region and the number of scattering and fluorescence events was counted. The measured volume of the suspension was calculated using the expression The motion of the sample syringe provides a volumetric flow rate of the sample suspension, and the motion of the sheath syringe provides a volumetric flow rate of the sheath fluid. Assuming laminar flow, there is no mixing between the sample suspension and the sheath fluid. Mass conservation gives a relation between the volumetric flow rate at the laser interrogation region and the volumetric flow rates of the sample and sheath fluids in the respective syringes. It was assumed that the counting of events was coordinated by the software with the start and stop positions of the syringe piston.−1 = 1000 µL/mL * 8426/(196 µL − 13 µL), where the indicated volume of 196 µL was obtained from the TOTALVOLUME keyword in the FCS file. Note that the indicated volume given by the keyword is slightly smaller than the sample volume (200 µL) set in the experiment control window. The difference is due to the small amount of volume which was delivered by the syringe between the time the “run” and “record” buttons were pushed. The “run” button started the motion of the syringe, and the “record” button started the recording of events in the FCS file. The samples used in Examples of concentration measurements are shown in −1). The sample volume was set to 300 µL, and the flow rate was set to 25 µL/min. A single measurement took about 10 min which was sufficiently long to lead to biases due to settling of microspheres. Settling appears to have a discernible but minor effect since the expected settling velocity of the TC microspheres is only 5·10−7 m/s [The six points in −1 obtained from the measurements shown in Measurements of biological samples in microfluidic devices are biased by adsorption of components of the sample suspension on surfaces of the microfluidic device. It has been demonstrated that including surfactants in the suspension minimizes the adsorption . Five me−1. When the events in the major peak were combined with twice the number of events in the minor peak the microsphere concentration was 49,000 mL−1. Therefore the splitting of the total dot plot into singlets and doublets leads to an increase of concentration by about 2 %. However, the identification of the minor peak as doublets is not consistent with scattering signals shown in the FS-SS dot plot in The difference in the slope of the two volume calibration curves shown in −1 which compares favorably with the expected value of 23,000 mL−1. The RM concentration was 56,600 mL−1 using the syringe volume calibration method described above. The RM concentration was also evaluated relative to the purported concentration of TC and given by the relation RMC = (RMN /TCN)23000 where RMN and TCN are given by the averaged event numbers in the table in −1. All uncertainties were 2.5 %.The suggested procedure for the measurement of the concentration of microspheres is to use a buffer PBS+0.05 % BSA, accumulate data for a set volume of 200 µL to 300 µL, and reduce the indicated volume by 13 µL when performing the concentration calculation. . −1. Therefore the measurements with the Attune flow cytometer can provide reliable values of the microsphere concentration. Possible source of uncertainty were adsorption, aggregation, settling, and volume calibration. The adsorption of microspheres was reduced by including an adsorption blocking agent (BSA) in the suspension buffer. Settling was reduced by mixing the suspension, however random fluctuations in microsphere concentration near the sampling tube in the SIP are inevitable. Volume delivered by syringe was calibrated by either directly weighing the mass drawn by the syringe or using a suspension of known concentration. Finally aggregation was minimized by gentle mixing. Overall an uncertainty in the measured concentration of 4 % could be rationalized. Since a calibration is needed to validate the indicated volume given by the Attune software, it would be useful if the Attune provided a capability to calibrate the volume delivered by the sample syringe (rather than volume drawn by the syringe). In addition, a gentle stirrer in the SIP would insure better mixing and minimize settling. With proper attention given to the buffer and the volume calibration, the flow cytometer with a volumetric sample delivery is well adapted to the measurement of microsphere concentration.The Trucount tubes contain about 50,600 microspheres while the measurements with the Attune flow cytometer  gave about 50,800 ± 1500 mL


Surfactants are important ingredients of personal care products and household products. The main characteristic of these compounds is to decrease the surface tension of solvent and resulting many properties such as contact angle, foam properties etc. The coexistence of other ingredients in the product may affect the properties of surfactants. One of the main components contained in almost every personal care and household product is sodium chloride. The main aim of this work was to determine the effect of this salt on some surface and usage properties of cocamidopropyl betaine (CAPB). From our experiments it was shown that the effect of added sodium chloride in the aqueous solutions of CAPB on the properties is the opposite to the one described in the literature for cationic and anionic surfactants, i.e., CMC increases with increasing ionic strength, foam height decreases with increasing salt concentration. Our investigation showed that sodium chloride makes worse the properties of the CAPB solutions examined in this work. Surfactants are one of the most important groups of organic chemicals, and are used in vast amounts in domestic and industrial applications , 2. They3. It is easily soluble in water and its 10 % solution has an acidic reaction with a pH of about 5–6 [Probably the most important class of amphoteric surfactants is amidopropyl betaines, in particular Cocamidopropyl betaine (CAPB) . It is about 5–6 . The genbout 5–6 .Fig. 1GeA literature survey shows that chaotropic ions influence some properties like solubilization, micellization and rheology. The addition of salts to aqueous surfactant solutions may result in a modification of both intramicellar and intermicellar interactions . AccordiSodium chloride is used as a thickener in shampoos and conditioners containing sodium lauryl sulfate. It is a contributing factor to the eye irritation experienced with most shampoos, and it may also cause dry and itchy scalp. Salt may also cause some hair loss . The addThe aim of this study was to check the effect of sodium chloride on the surface and wetting properties of aqueous solutions of CAPB.In the presence of sodium chloride, cocamidopropyl betaine may be described as each the inner salt or as the respective sodium salt .The sample of cocobetaine was purchased from one of the cosmetics companies making it available on the Polish market. It was stored in a refrigerator and not used after the expiration date specified by the producer. According to the manufacturer's information, the commercial CAPB consists of 30 % cocamidopropyl betaine and 70 % water, without sodium chloride .3. The surface tension measurement was made for stock aqueous and salt solutions and for the following 18 solutions obtained by a serial dilutions method. The measurement was carried out for aqueous and sodium chloride solutions with four concentrations: 0.01, 0.05, 0.2 and 2 M using a pendent drop tensiometer TRACKER . The tensiometer captures the image of an air drop immersed in the aqueous solution of surfactant and records the drop shape as a function of time. The drop shape is determined by the surface tension of the liquid, gravity, and the density difference between the air and surrounding medium. The drop image is analysed with a profile fitting method in order to determine the contact angle and the surface tension. Measurements of surface tension were performed at constant temperature for aqueous solutions at 297 K. Measurement duration depends on concentration of surfactants in the sample and was conducted to reach equilibrium. Additional physicochemical analyses on the basis of the surface tension data were carried out using the Szyszkowski and Frumkin equation [3. Surfaces used for measurements of contact angle were: aluminium, paraffin, PVC (poly(1-chloroethylene)), PTFE ), glass, PE (polyethene), PMMA (poly(methyl 2-methylpropenoate)).The samples of cocobetaine used to measure surface tension were prepared as aqueous and salt solutions  of surfactant. The initial concentration of cocobetaine solution was 10 g/dmequation . ContactThe foaming properties of the water and aqueous salt solutions of cocobetaine were investigated in a glass apparatus using the Ross-Miles method in accordance with the standard ASTM00A51E47 . After aversus time was observed. Because the adsorption process of surfactants is dynamic, exact equilibrium values of the surface tension are necessary to determine the adsorption isotherms. For surfactants with low surface activity the adsorption reaches its equilibrium in time on the order of seconds to hours. It was found that the time to reach the adsorption equilibrium decreases with the increase in the concentration of cocobetaine in the aqueous solutions and changed from about 600 to 2,400 s at the lowest measured concentration.During the measurement of the surface tension by the drop shape method, the dependence of surface tension versus the log of concentration are presented. It was found that cocobetaine lowered the surface tension in all system investigated. The minimum values of the surface tension were similar (about 25 mN/m) regardless of the sodium chloride concentrations. From these curves critical micellar concentration (CMC) values were estimated and are listed in Table In Fig. ar about 5 mN/m reAs was mentioned above, in the presence of sodium chloride in an aqueous solution of cocamidopropyl betaine could be described as the inner salt or as the respective sodium salt, and that could be a reason for the different behavior of this compound in systems with NaCl compared to systems with water. In the commercial products, salts are often added, and this should be taken into account to explain the different surface properties of cocamidopropyl betaine.γ0 is the interfacial tension for concentration c = 0 and ASz and BSz are the adsorption coefficients. Using the Szyszkowski adsorption coefficients ASz, BSz, the surface excess at the saturated interface (Γ∞), the minimum molecular area in the adsorption layer at the saturated interface (Amin) and the Gibbs free energy of adsorption (ΔGads) could be estimated [A′ characterizes the mutual interactions between the adsorbed molecules [The measured change of the surface tension with concentration was fitted with the Szyszkowski isotherm:stimated . On the entclass1pt{minimaolecules :2\docume∞ is higher in comparison to the solution with various concentration of NaCl. In consequence, the statistical area occupied by the adsorbed molecules of cocobetaine at the saturated air/water interface is smaller in comparison to the system with salt. The same relationship was described for other zwitterionic surfactants: n-alkyl phosphocholines [The surface tension measurements showed that the cocobetaine is surface active in all aqueous solutions and depends on the salts concentration. The results obtained, presented in Table cholines . Salt adcholines . The resGads is also affected by the type of aqueous solution. The values of ΔGads calculated in systems with NaCl are more negative in comparison to the system without salt, and decrease with increasing salt concentration. Thus a less pronounced tendency for adsorption was observed in the systems with water. In other words, the results obtained suggest more spontaneous adsorption processes of molecules of cocobetaine in systems with NaCl in aqueous solutions.As indicated by the data presented in Table A′ values indicated that the interactions are insignificant and can be neglected in all the systems considered, irrespective of the concentration of NaCl in aqueous solution. The values of A′ obtained changed in the range from almost −10−2 to 10−3.The analysis of the mutual interactions between the adsorbed molecules on the ground of the magnitude of The efficiency of adsorption of cocobetaine in the various aqueous solutions is clearly different, too. This can be concluded from studies on surface excess isotherms shown in Fig. Another important attribute of surfactants is the ability to wet out solid surfaces. Wetting of solid surfaces by liquids depends on both the surface and liquid .The wetting properties are the ability to test product to dissolve in water, to lower the surface tension between liquid and solid, and to remove air from liquids and solids by aqueous solutions , 34. OneSolid surfaces with high surface energy are completely wetted by the liquids with high surface tension, respectively. As it can be seen in Fig. γ × cos(Θ)) and surface tension (γ) of aqueous solutions of surfactants:a and b are constants. They have also proposed that for nonpolar solids, such as a Teflon and surfactant system, the value of constant a is −1. The results of adhesional tension vs surface tension of aqueous solutions of cocobetaine for Teflon plate are presented in Fig. Bargeman and van Voorst Vader  found th3 of foam. This is much lower amount of foam than for the same solutions of cocobetaine [Foaming ability is a typical property of surfactant solutions. The ability to produce foam is of great importance in cosmetics and household products. In the process of cleaning and washing the important role played by the quantity and durability of the produced foam . The maiobetaine .Fig. 6VoStability of foam was assessed by calculating the ratio of the initial foam volume to its value after 5 and 10 min. Main indicators applied to estimate the stability of the foam are: h5/h1index, h10/h1 index and constant speed of falling foam (k) . Table 2Based on the research and analysis carried out, it can be concluded that properties of cocamidopropyl betaine depend on the concentration of salt in the surfactant solutions. Experimental data showed that cocobetaine exhibits good surface properties and can be used as a substance which reduce surface tension even at low initial concentrations. The presence of salt in the solutions lessens their ability to reduce surface tension, decreases the CMC and increases the adsorption parameters and structure of the adsorbed monolayer. The experimental data showed that salt solutions of cocobetaine create a much smaller column of foam than aqueous solutions.


We present theoretical proposals for two-dimensional nuclear magnetic resonance spectroscopy protocols based on Nitrogen-vacancy (NV) centers in diamond that are strongly coupled to the target nuclei. Continuous microwave and radio-frequency driving fields together with magnetic field gradients achieve Hartmann-Hahn resonances between NV spin sensor and selected nuclei for control of nuclear spins and subsequent measurement of their polarization dynamics. The strong coupling between the NV sensor and the nuclei facilitates coherence control of nuclear spins and relaxes the requirement of nuclear spin polarization to achieve strong signals and therefore reduced measurement times. Additionally, we employ a singular value thresholding matrix completion algorithm to further reduce the amount of data required to permit the identification of key features in the spectra of strongly sub-sampled data. We illustrate the potential of this combined approach by applying the protocol to a shallowly implanted NV center addressing a small amino acid, alanine, to target specific hydrogen nuclei and to identify the corresponding peaks in their spectra. Thus NMR is susceptible to inhomogenous broadening and can only deliver ensemble information while the structure and dynamics of individual specimens remain hidden from observation45Nuclear magnetic resonance spectroscopy (NMR) allows for the structure determination of molecules and proteins and therefore contributes fundamentally to the advancement of the biological sciences. This structural information is obtained by probing the target of investigation, typically a large molecular ensemble, by means of multiple radio frequency pulses and measuring their response. This response is then mapped to multi-dimensional spectra which encode the dynamical properties of the system and therefore of the interactions between its constituent nuclear spins12The recent progress in the control of a single electron spin in Nitrogen-vacancy (NV) centers in diamond offers a new perspective here as it becomes possible to use optically detected magnetic resonance79101113151718192122Beyond this remarkable enhancement of the NMR signal, it is important to realize that this new detection regime offers further opportunities beyond the capabilities of standard NMR. In addition to the manipulation and probing of nuclear spins by means of external radio-frequency fields we are now in a position to control the properties of the NV center such as to tailor its response and, crucially, via its strong interaction with nuclei also to obtain an additional handle for the manipulation of the nuclear spins. In this work we take advantage of this potential and demonstrate the usefulness of single molecule NMR by means of strongly coupled NV centers.The large amount of required data and the associated long measurement times represent a challenge that is common to both ensemble NMR and single molecule NMR measurements. However, the spectral information that is being obtained in any NMR experiment possesses underlying structure, determined by the Hamiltonian that describes the mutual interactions of the nuclear spins in the molecule, which makes the system sparse in a suitable basis. This fact can be exploited by non-linear reconstruction methods from signal processing that are designed to unravel such structures without prior knowledge with the minimal possible number of measurements. Indeed, measuring only a small subset of all accessible data points can be shown to allow for the reliable reconstruction of spectral information as it is required in NMR protocols.In this work we are combining one such approach, matrix completion25This article proceeds as follows: The first section begins with a numerical discussion of single molecule two-dimensional-NMR (COSY) by means of strongly coupled NV centers in diamond. It also introduces techniques of selective polarization enabled by strong coupling to enhance the contrast in the NMR spectra and presents realistic numerical simulations of the envisaged set-up. A second part of this section presents protocols that employ an NV quantum sensor that is strongly coupled to external nuclei to resolve inter-molecular couplings in 2D correlation spectroscopy at the single molecule level even without previous polarisation of the target molecule. For illustration we apply these schemes numerically to simulations of the application of our scheme to a simple bio-molecule, alanine. In a final section this article discusses and demonstrates the power of matrix completion to reduce the data taking effort and hence experiment time in single molecule NMR by means of numerical examples.In this section, we will introduce the basic principles of 2D correlation spectroscopy (COSY) which we will then use as a test case for the theoretical study of the exploitation of the strong coupling regime of an NV quantum sensor with target nuclei.Elements of COSY in the strong coupling limit – The basic variant of COSY employs two pulses separated by an incremented delay t1 where the response of the system is measured after a time interval t2 (see red and green sections of t1 and t2) to yield the frequency domain COSY spectrum. The resulting diagonal peaks of the signal in frequency space refer to the eigenfrequencies of the nuclear spin system, and the off-diagonal peaks indicate polarization exchange between pairs of nuclei in the target molecule.In COSY, as is common to standard NMR protocols executed on large ensembles or in the weak coupling regime, the measured signal will be proportional to the nuclear spin polarisation. In the strong coupling regime we can enhance the collected signal by making use of the possibility of achieving hyperpolarization, i.e. nuclear spin polarization far beyond thermal equilibrium conditions, of all or a selected few of the nuclei by means of dynamical nuclear spin polarization via the NV centers28H0 =  ωMWσz, where ħ = 1 and  ωMW is the frequency of the microwave drive, is governed by the effective HamiltonianNeglecting rapidly oscillating terms, that is under the rotating wave approximation, the dynamics of the NV center and the nuclei in an interaction picture with respect to nv is the effective Rabi frequency of the microwave field applied to the electronic σx is the Pauli operator in the corresponding subspace spanned by {|m = −1〉, |m = 0〉}, σx and p from the Larmor frequency of the nuclei and a field strength Ωp that satisfy Here ΩIf this conditions is fulfilled, electron-nuclear spin-flip-flop processes lead to polarization exchange between the NV center spin and the nuclei and result in the efficient polarization of the nuclear spins. Alternatively to the radio-frequency field decoupling, a strong magnetic gradient may be applied in which case even identical nuclear spin species will have different Larmor frequencies. While this may require a sweep across the frequency range, as implemented e.g. in the integrated solid effect29Finally, the measurement of the polarization of the nuclei is also achieved by the NV center. Indeed, we can infer the average polarization of the nuclei from the measurement signal of the NV center spin by31v〉 after being initialized in state |μ〉 and a free evolution determined by x and y direction can be measured similarly by applying an additional pulse to rotate the nuclear spins before performing the NV measurementswhere Å, which represents a lower limit to the distance between hydrogen in proteins. In our numerical simulation, we assume an applied magnetic field of 1000 G which allows us to comfortably resolve dipole-dipole energy splitting of about 100 kHz for our choice of physical simulation times for a single measurement of a few milliseconds. The NV center is assumed to be located at a distance of 2 nm to both Hydrogen nuclei. To support selective addressing of the nuclei, the applied magnetic field includes a gradient of 60 G/nm along the axis joining the Hydrogen atoms. This allows for a dominant polarization of a distinct nucleus in the sample, dominant coupling of the NV center to a specific nucleus during the read out and a maximum fidelity of applied pulses for selected nuclei. The applied π/2-pulses are implemented by radio-frequency fields with a Rabi frequency of 5 kHz.We demonstrate the basic working principle of this protocol at the hand of the simple example of two Hydrogen atoms at a distance of 1 r is the distance between two nuclei, r and θ, two independent magnetic field directions are sufficient. From the distances and orientations of the various nuclei relative to the selectively addressed nucleus in the sample, we can learn the spatial structure of its local environment within the molecule.Here Single molecule NMR of Alanine — Before moving on, we consider the NV-based COSY spectrum of an Alanine molecule in the strong coupling regime as described in the above section. Alanine is one of the smallest amino acids with chemical composition HOOCCH(NH2)CH3, which carries nuclear spins on the 7 Hydrogen atoms and the single Nitrogen atom. The molecule has a total size of about 0.45 nm and is illustrated in the upper part of Å which results in an interaction of order of around 20 kHz. While it is difficult to exceed this by means of magnetic field gradients our simulations show that the combination of a rf-field with Rabi frequency of 100 kHz with a realistic gradients of 60 G/nmWe assume a shallowly implanted NV center located at a distance of 2 nm to the surfaceIn order to achieve dominant coupling to a selected hydrogen atom we perform our selective polarization protocol and apply the COSY procedure with the NV center tuned on resonance to the hydrogen transition frequency. This yields the l.h.s of The results of this simulation will be used also for the demonstration of the application of matrix completion in the last section.Exploiting entanglement between the NV electron spin and the target nuclei — The protocols so far exploit the ability to achieve hyperpolarization of the molecule or of some of its constituents. Importantly, however, operating in the strong coupling regime, it is not strictly necessary to polarize nuclear spins, while still retaining a full signal contrast due to the quantum nature of the interaction between the NV center spin and the nuclei. Moreover, we can make use of the strong coupling for the coherent control of both nuclear spins (by means of radio-frequency fields or via the interaction with the NV center) and of the NV center spin (microwave frequency fields) thus allowing for more complex pulse sequences for the simultaneous control of sensor and target.t1 and t2 which in turn allow for an accumulation of the effect of the nuclear Hamiltonian that can subsequently be measured via the NV center. While this scheme appears similar to the well-known COSY sequence it differs from it due to the entanglement that is created between NV center and nuclear spin which in turn allows us to generate the same spectral information as in the case of COSY with fully polarized nuclear spins but now without the need for nuclear spin polarization. Although the actual detection protocol is quite different, this scheme shares sufficient parallels to the COSY scheme from As an illustrative example, we construct a novel NV-based 2D-spectroscopy scheme by exploiting the strong interaction of nuclei in a target molecule with an NV center spin, see Initialization: the NV center is initialized in a polarized state, while the nuclear spins remain unpolarized (blue bar).NV-nuclear interaction: the NV center interacts with the nuclei for a time Free evolution period 1: the interaction of the NV center with the nuclei is switched off by transfer of the NV center to the m = 0 state and if necessary the quantum information may be transferred further to the nuclear spin degree of freedom of the NV center. The nuclear spins precess freely for a time t1 during which the dynamics is governed by the HamiltonianFor definiteness, we describe the pulse sequence in detail together with the relevant Hamiltonians see also :InitialiNV-nuclei interaction: The NV center again interacts with the nuclei with the same Hamiltonian t1.Free evolution period 2: the interaction with the NV center is switched off and the nuclear spins precesses freely for a time t2 under the same Hamiltonian as in the free evolution period 1.NV-nuclear interaction: A third interaction employing the same dynamics as in the previous two NV-nuclear interaction periods prepares the final measurement.Measurement: Finally, we perform a projective measurement on the initial state of the NV center spin (green bar).n atoms allows for the generation of n independent spectra to decongest the spectra.As illustration of the above 2D NMR protocol with a strongly coupled NV center at a depth of 2 nm we consider Alanine in a magnetic field of 100 G. To preferably address selected nuclear spins we apply an additional gradient field of 60 G/nm, to support dominant entanglement between the NV and selected nuclear spins during the NV interaction periods. The simulated magnetic resonance spectrum is plotted in Assuming that the nuclei are initially not polarized, we have also verified that they will remain nearly unpolarized across the entire pulse sequence, that is we observe NMR signals without relying on any nuclear polarization, which represents a distinct feature as compared with conventional NMR techniques. As above, the application of gradient fields allows for individual coupling even for identical atoms.For larger molecules, the application of NV-based 2D-NMR suffers from a rapidly increasing experiment effort as a function of the number of nuclei in the target molecule. In this section, we explain that the experimental overhead of NV-based 2D spectroscopy can be reduced significantly by exploiting the technique of matrix completion25Background — For a given matrix A we denote with σi its descendingly ordered singular values, that is the diagonal entries of the matrix D in U and V are unitaries. If these singular values are close to zero for indices i > r, one obtains a high fidelity approximation r for the matrix A bywhere A by finding a low rank approximation i, j)} = Ω such that i, j) is in Ω or not and The idea of matrix completion is to reconstruct the matrix n × n matrix the number of required samples scales as rn ln n where i〉} (i.e. most of its entries vanish or are negligible) it is in this sense optimal to sample the matrix in the Fourier transformed basis {F|ei〉}  from a small number of sampled entries  in the time-domain signal, which are those data that would be measured in real experiments. The matrix completion algorithm, whose run time is negligible compared to the savings in measurement timeSt out of the subsampled entries. We will see that in the frequency domain, the hyperfine structure can then be identified from the reconstructed matrix.As a comparison, we apply the completion algorithm to the Alanine COSY spectra generated above and plot in The shallow implantation of NV centers in diamond allow for a new magnetometry regime in which the coupling of the NV center to nuclei is comparable or larger than the inter-nuclear coupling, in rapid departure from the standard regime in which nuclear magnetic resonance in bulk is situated. We have demonstrated that this strong coupling regime offers new opportunities for single molecule nuclear magnetic resonance spectroscopy that can reduce the experimental requirements both by relaxing the need for nuclear spin polarization while retaining a significant signal, by decongesting the NMR spectra thanks to individual addressing for polarization and readout of nuclear spins and by reducing significantly the number of measurements required to recover the 2D spectrum thanks to the application of matrix completion, a method from signal processing. In numerical studies we have applied these ideas to a specific example of a small amino acid, alanine to demonstrate the feasibility and potential of this approach. We expect that the combination of these approaches may be applied beyond this example to extend to the study of membrane proteins whose structure and dynamics are of considerable importance in biology and medicine.How to cite this article: Kost, M. et al. Resolving single molecule structures with Nitrogen-vacancy centers in diamond. Sci. Rep.5, 11007; doi: 10.1038/srep11007 (2015).