The aim of this study is to find a correlation between linear measurements and the graft length required for different anterior cruciate ligament (ACL) revision techniques, to extract formulas to predict required graft length during the preoperative planning.At time 0 and 30 days later, two observers measured eight linear distances on standard 2D knee magnetic resonance imaging (MRI), and nine curved distances on 3D MRI sequences, corresponding to different techniques for ACL revision, anatomic anterolateral ligament (ALL) reconstruction, and lateral extrarticular tenodesis (LET). Intra- and interobserver reliability was tested for 2D and 3D measurements. The correlation between 2D and 3D measurements was tested. The 2D measurements with highest repeatability and reproducibility, and with strongest correlation with 3D measurements were used to extract formulas to calculate the graft length from 2D values.Fifty MRIs acquired with both 2D and 3D sequences were used. The intra- and interobserver reliability of linear 2D measurement was high, with the transepicondylar distance (TD) showing the highest reproducibility and repeatability. The intra- and interobserver reliability of 3D measurements was lower than 2D, but acceptable for all measurements except for ALL reconstruction. The TD showed the strongest correlation with 3D measurements. The formulas extracted to calculate the graft length from the TD proved to be accurate.Accurate formulas were created to calculate the graft length needed for different ACL revision techniques and ALL reconstruction/LET techniques from TD. These formulas can be used during preoperative planning of ACL revision cases. Anterior cruciate ligament reconstruction (ACLR) is one of the most common orthopedic procedures , 2. A beThe length of the graft needed to perform these procedures is significantly longer than in standard ACLR, and knowing it preoperatively can be useful to switch to another technique when the harvested hamstrings (HS) are too short, to harvest the correct length of fascia lata autograft , and toMeasuring the length of the graft needed on standard 2D magnetic resonance imaging (MRI) is not possible, but high-quality isotropic 3D MRI sequences should be obtained for a 3D reconstruction and multiplanar reformatting . HoweverThe aim of this study was to obtain formulas to calculate the length of the graft required for different ACLR techniques (useful in the revision setting), from simple and linear 2D measurements on 2D MRI. However, the following steps have to be performed before obtaining such formulas: (1) test the intra and interobserver reliability of linear 2D measurements  on standard 2D MRI sequences; (2) test the intra and interobserver reliability of complex/curved 3D measurements  on 3D MRI; and (3) investigate the correlation between linear 2D MRI measurements with 3D measurements (that are the length of the graft required for different ACL revision/reconstruction techniques) to find the 2D measurement with the higher correlation, which will be used to extract the formulas. The authors’ hypotheses are (1) there is a correlation between some 2D measurements and 3D measurements, and (2) creating formulas to calculate the length of the required graft based on 2D measurement is possible, and would be useful for ACLR revision preoperative planning.This study was approved by the local Ethical Committee .Fifty healthy volunteers younger than 55 years of age were enrolled (from January 2018 to Mar 2019), after signing an informed consent for participation in this study. Exclusion criteria included: (1) presence of arthritis; (2) congenital/hereditary diseases causing articular deformities ; (3) previous knee surgeries; (4) chronic inflammatory diseases; (5) presence of hardware in the knee; and (6) previous tibial, femoral, or patellar fractures. Patient demographics were recorded, including age, sex, height, weight, and body mass index (BMI).All images were acquired with a 1.5 T MRI scanner  and an eight-channel SENSE knee coil. Routine 2D  and 3D Turbo Spin Echo (TSE) T2 weighted images with isotropic voxel  were acquired in each patient. The acquisition time was 5–10 min for the entire 2D MRI protocol and 5–10 min for the T2 VISTA 3D sequence. The sagittal source images from the 3D TSE technique were used to create sagittal, coronal, and axial reformatted images of the knee joint. The reformatted images were used for the 3D TSE assessment of the knee. The post-processing of the 3D TSE sequence was performed by a radiologist on a Philips Achieva MRI workstation  after acquisition of the images.Two sports medicine fellowship trained orthopedic surgeons  performed the 2D and 3D measurements at time 0 (T0) and after 30 days (T1) on the Philips Achieva MRI workstation .To decide the 2D measurements to be included in the study, two sports medicine fellowship trained orthopedic surgeons  were asked the following question:Morphological measurements of the knee joint (bone or soft tissue) that can potentially affect the length of the graft required for different ACLR revision techniquesMeasurements obtainable on standard 2D MRI protocolsMeasurements easy to perform in the clinical practice (with simple or no instructions to be provided to clinicians)Include one or more measurements potentially affected by both tibial and femoral dimensionsBased on the recent literature and your experience, which 2D measurements having the following characteristics would you include in this study?Transepicondylar distance (TD): defined as the longest distance between the apices of the medial and lateral femoral epicondyles in the mediolateral (ML) axis, measured on axial cuts.Medial femoral condyle anteroposterior (AP) dimension (MFAP): defined as the longest dimension of the medial femoral condyle in the AP axis, measured on axial cuts.Lateral femoral condyle AP dimension (LFAP): defined as the longest dimension of the lateral femoral condyle in the AP axis, measured on axial cuts.Proximal tibia medio-lateral dimension (PTML): defined as the longest mediolateral dimension of the proximal tibial in the ML axis, measured on proximal axial cuts just distal to the menisci.Medial tibial plateau AP dimension (MTAP): defined as a segment drawn perpendicular to PTML and passing through the most posterior point of the medial tibial condyle in the AP axis, measured on proximal axial cuts just distal to the menisci.Lateral tibial plateau AP dimension (LTAP): defined as a segment drawn perpendicular to PTML and passing through the most posterior point of the lateral tibial condyle in the AP axis, measured on proximal axial cuts just distal to the menisciPosterior cruciate ligament (PCL) and trochlear cartilage distance (PCLTC): defined as an oblique distance between the center of the PCL tibial insertion and the most anterior and proximal point of the trochlear cartilage, measured on sagittal cuts.Patellar tendon length (PTL): defined as an oblique distance between the inferior pole of the patella and the most proximal part of the patellar tendon insertion on the tibial tuberosity, measured on sagittal cuts.After discussion, the two surgeons agreed on the following measurements to be included  Fig. 1)1):TranseThe observers were intentionally not instructed regarding the weighting of the sequence for 2D measurements, for possible subsequent application in the clinical setting.The 3D measurements were performed on the 3D MRI sequences  by the same observers at time 0 (T0) and after 30 days (T1). The 3D measurements corresponded to different ACLR revision techniques and anatomic anterolateral ligament (ALL) reconstruction or lateral extrarticular tenodesis (LET).Intrarticular length of the graft (IAGL): defined as the distance from the center of the tibial ACL footprint to the center of the femoral footprintOutside-in femoral tunnel (OIFT) length: defined as the distance from the center of the femoral ACL footprint to a point located 5 mm proximal and 5 mm posterior to the apex of the lateral femoral epicondyle.Single bundle outside-in anatomic ACL reconstruction, without tibial tunnel (SBOI) length: defined as the sum of measurements one and two. It should represent the total length of the reconstruction.Anatomic anterolateral ligament (ALL) reconstruction length according to Laprade et al.  : definedModified Lemaire lateral extrarticular tenodesis length, anatomical on the femur (ALET): defined as the distance from the center of the Gerdy’s tubercle to a point located 5 mm proximal and 5 mm posterior to the apex of the lateral femoral epicondyle, passing under the lateral collateral ligament, and representing the length of the required graft .Length of Saragaglia and Yamaguchi technique, without tibial tunnel (SYT): defined as the sum of measurements one, two, and five. In the original Saragaglia technique the hamstrings were harvested  and left attached, then passed through a standard tibial tunnel. The femoral tunnel was performed in an outside-in manner inferior to the proximal insertion of the lateral collateral ligament (LCL). The graft is fixed with absorbable screws in the femoral and tibial tunnel. A lateral skin incision is performed at the Gerdy’s tubercle and the remaining portion of the graft is passed under the fascia and fixed to the tubercle with anchor or nonabsorbable stitches with the knee at 30° of flexion and the foot in neutral position. The Yamaguchi technique is similar, but it is performed with a strip of ITB left attached to the Gerdy’s tubercle, passed under the fascia and then into a femoral and tibial tunnel performed as in the Saragaglia technique , 14. In Over the top femoral position ACL reconstruction length, without tibial tunnel (OTT): defined as the ACL graft path from a point located just distal to the proximal ridge of the distal femur at the diaphyseal-metaphyseal junction to a point located in the center of the tibial ACL footprint . It woulMarcacci technique length, without tibial tunnel (MT): defined as the ACL graft path of the Marcacci technique, passing through (a) the center of the tibial ACL footprint; (b) the over the top femoral position; (c) the distal aspect of the proximal ridge of the femur at the diaphyseal-metaphyseal junction; (d) the Gerdy’s tubercle . ParticuModified Lemaire LET procedure length, non-anatomical on the femur (NALET) according to Spencer et al.  : definedThe measurements included Figs.  and 3 wer) in case of normal distribution or the Spearman’s rank correlation coefficient (rho) in case of not normal distribution, together with the Cronbach’s alpha coefficient. The interobserver reliability was tested with the Cronbach’s alpha coefficient, the Kappa index, and the intraclass correlation coefficient (ICC). In addition, the correlation between the 2D measurements and the 3D measurements was tested with the Pearson’s correlation index and Spearman’s rank correlation coefficient . The 2D measurements with highest intra and interobserver reliability and with the strongest correlation with 3D measurements (length of the different reconstruction techniques) were used to extract formulas to calculate the 3D measurements from 2D values. The formulas were obtained with linear regression. In addition, the accuracy of the curved measurements obtained with the formulas was compared with the same measurements performed on the 3D MRI and the difference between the groups was tested with a paired t-test and Bland Altman plots. Statistical analysis was performed with MedCalc Statistical Software version 16.4.3 .Data were reported with mean, standard deviation, and/or ranges. The normality of the distribution of the measurements was tested with the D’Agostino-Pearson test. The intraobserver reliability was tested with the Pearson correlation index  were included in the study, 18 (36%) were women, 32 (64%) were men, and the mean age was 28.1 years . The mean age was 27.89 years  for women, and 28.22 years  for men. The average height was 179 cm  for men, 169 cm (SD 5.95 range 158–176 cm) for women, and 175 cm  for the total population. The right knee was studied in 50% of the study group.Table The intr-observer reliability  for the 2D and 3D measurements is summarized in Table The interobserver reliability tested with intraclass correlation coefficient (ICC), Kappa Index, and Cronbach's alpha for the 2D and 3D measurements is presented in Table The second measurement (T1) of each observer was used for every test. Among the 2D measurements, the highest interobserver reliability was found for the transepicondylar distance.As expected, 2D measurements showed higher interobserver reliability than 3D measurements. However, the reproducibility was acceptable for all 2D and 3D measurements, except for anatomic anterolateral ligament (ALL) reconstruction.The correlation between 2D measurements (independent variables) and 3D measurements (dependent variables) was analyzed with Pearson's or Spearman's correlation tests  and is presented in Table In addition, the patients’ height was tested as well as an independent variable for possible correlation with 3D measurements.All independent variables, except for patellar tendon length and patient’s height, were strongly correlated with the dependent variables (3D measurements). The transepicondylar distance (TD) showed the strongest correlation with most of the dependent variables.Simple linear regression was used to develop mathematical formulas able to calculate the 3D measurements (Y variable) from the transepicondylar distance (X variable). The transepicondylar distance (TD) was chosen among all the other 2D measurements because it had the highest repeatability, reproducibility, and strongest correlation with 3D measurements. A linear correlation was found between the transepicondylar distance and all 3D measurements.Intrarticular length of ACL graft (IAGL) = 0.13 + (0.46 × TD)Outside-in femoral tunnel length (OIFT) = 0.62 + (0.42 × TD)Outside-in anatomic ACL reconstruction (SBOI) length = 2.26 + (0.84 × TD) + tibial tunnelLength of Saragaglia/Yamaguchi techniques (SYT) = 15.09 + (1.3 × TD) + tibial tunnelLateral extrarticular tenodesis anatomical femur (ALET) length = 15.49 + (0.42 × TD)Lateral extrarticular tenodesis non-anatomical femur (NALET) length = 21.23 + (0.59 × TD)Over the top femoral position ACL reconstruction (OTT) length = 8.25 + (0.85 × TD) + tibial tunnelMarcacci technique (MT) length = 29.47 + (1.43 × TD) + tibial tunnelThe formulas obtained with the linear regression were:Since the anatomic anterolateral ligament reconstruction 3D measurement showed a poor interobserver reliability, the formula obtained with linear regression was considered inaccurate and not included here. All formulas must be intended in mm.t-test for every 3D parameter included in the study, showing no difference between the groups: intrarticular length of ACL graft p = 0.98, outside-in femoral tunnel length p = 0.97, outside-in anatomic ACL reconstruction p = 0.94, Saragaglia/Yamaguchi techniques p = 0.97, lateral extrarticular tenodesis  p = 0.98, lateral extrarticular tenodesis  p = 0.99, over the top femoral position ACL reconstruction p = 0.97, Marcacci technique p = 0.9721.To test the accuracy of the formulas, 3D measurements performed on the MRI and calculated with the formulas were compared with a paired The 3D measurements performed on the MRI and calculated with the formulas were also compared with the Bland–Altman plot Fig. . The BlaIntrarticular length of ACL graft (IAGL) = (0.46 × TD)Outside-in femoral tunnel length (OIFT) = 1 + (0.42 × TD)Outside-in anatomic ACL reconstruction (SBOI) length = 2 + (0.84 × TD) + tibial tunnelLength of Saragaglia/Yamaguchi techniques (SYT) = 22 + (1.3 × TD) + tibial tunnelLateral extrarticular tenodesis anatomical femur (ALET) length = 21 + (0.42 × TD)Lateral extrarticular tenodesis non-anatomical femur (NALET) length = 27 + (0.59 × TD)Over the top femoral position ACL reconstruction (OTT) length = 8 + (0.85 × TD) + tibial tunnelMarcacci technique (MT) length = 36 + (1.43 × TD) + tibial tunnelAnother advantage of the Bland–Altman plot in this type of study is that investigators can interpret the limits of agreement to assess whether the agreement is acceptable, based on what is clinically important or not. The limits of agreement are expected to include about 95% of the differences observed in the future. In this study, the acceptable range of agreement was defined a priori at ± 5 mm for shorter measurements , from −3 mm to +20 mm for longer measurements (SYT and MT), and from 0 mm to +15 mm for lateral extrarticular tenodesis (ALET and NALET). In fact, from the clinical point of view, an over- or under-estimation of 5 mm can be acceptable for shorter measurements . On the other hand, for longer reconstruction techniques  or lateral extrarticular tenodesis, a minimal underestimation of the graft length can be accepted from the formula, but an overestimation of the length does not represent a clinical problem in the surgical setting: i.e., a surgeon does not want a graft shorter than needed, but can always cut the remnant of a longer graft. Based on the Bland–Altman plots, the formulas for the shorter ACLR revision measurements  were considered accurate enough. On the other hand, according to the needs of the surgeons, 7 mm were added to the formulas for longer ACLR revision measurements (SYT and MT) and 6 mm to the formulas for lateral extrarticular tenodesis (ALET and NALET). In addition, the decimals of all formulas were rounded for easier clinical application. The final formulas were:Please note that TD, tibial tunnel, and the result of the formula are all expressed in millimeters (mm).The main findings of the present study were as follows (1) the intra and interobserver reliability of linear 2D measurement on standard 2D MRI sequences was high, with the transepicondylar distance (TD) showing the highest reproducibility and repeatability; (2) the intra and interobserver reliability of complex/curved 3D measurements on 3D MRI was lower than 2D measurements, but acceptable for all measurements, except for anatomic anterolateral ligament reconstruction; (3) a strong linear correlation was found between most 2D MRI measurements and 3D measurements, except for patellar tendon length; (4) the TD showed the strongest correlation with 3D measurements; and (5) the authors were able to extract accurate formulas to calculate the length of the graft required for different ACLR revision techniques from the TD measured on standard 2D MRI.The position of the present study in the existing sports medicine literature is rather unique. Some papers have studied the correlation between morphometric parameters in arthritic knees (measured preoperatively on different imaging techniques or intraoperatively) and the final total knee replacement implant, in terms of size or rotation –31.Some other papers have investigated whether anthropometric parameters or MRI measurements were able to predict the length and thickness of hamstring graft for ACLR –35. HoweAs previously mentioned, ACLR revision surgery cannot be standardized as primary ACLR, and for this reason there is an increasing interest among sports medicine surgeons regarding the preoperative planning, with the goal of avoiding intraoperative complications. Grasso et al. compared 3D MRI and 3D computed tomography (CT) scans in 24 patients diagnosed with a failed ACLR, to establish an accurate MRI protocol as an alternative to 3D-CT scans. The authors found that a high-resolution 3D turbo spin echo proton density MRI sequence could quantitatively evaluate the location and orientation of previous bone tunnels for routine postoperative assessment, presurgical planning, and outcome evaluation . With thIn addition, based on the recommendations of the Anterolateral Complex consensus group , ALL recIn the Marcacci technique, the hamstring tendons are harvested preserving the distal insertion. The graft is then passed through a tibial tunnel and “over the top” on the femoral side using a lateral incision to the distal femur. The graft is fixed just proximal to the lateral femoral condyle with two Richard’s staples, with the knee flexed at 90° and the foot externally rotated. The remnant of the graft is then passed under the iliotibial band and fixed with one staple at the level of the Gerdy’s tubercle . In 2009In 2006, Yamaguchi et al. described an iliotibial band (ITB) ACLR with combined anterolateral plasty. A 25 cm longitudinal incision is performed on the lateral femur. A 22 cm-long strip of the ITB is harvested, leaving the tibial insertion attached on the Gerdy’s tubercle. A 7.5 mm femoral tunnel is then drilled in an outside-in fashion. A 7.5 mm tibial tunnel is then drilled. The graft is passed deep to the LCL and through the femoral and tibial tunnels. With the knee at 90° of flexion and the foot externally rotated, the graft is sutured to the LCL and the periosteum of the lateral femoral condyle. Then, with the knee at 30° of flexion and the foot externally rotated, the graft is sutured to the periosteum around the outlet of the tibial tunnel. The authors described good long-term outcomes, with a mean KT-1000 side-to-side difference of 3.5 mm at 24 years follow-up .A technique very similar to Yamaguchi’s was described in 2013 by Saragaglia et al. The hamstrings are harvested and left attached distally on the proximal anteromedial tibia. At the level of the intrarticular portion, the semitendinosus is doubled. Tibial and femoral full tunnels are drilled with an outside-in technique. The graft is then passed into the knee through the tunnels, exiting the lateral femur. A second lateral skin incision is made on the proximal tibia for the anterolateral plasty. The tail of the graft is tensioned towards Gerdy’s tubercle and attached to the fascia lata with five or six large absorbable sutures and the foot in a neutral position. Again, the authors described good outcomes with this technique, with no pivot shift in 75% of the cases .The graft required for all these reconstruction techniques is much longer than standard ACLR procedures. Both Marcacci and Yamaguchi underlined in their papers the importance of having a long enough graft (20 cm according to Marcacci and 22 cm for Yamaguchi) , 41. HowRegarding lateral extrarticular tenodesis (LET), two different techniques were included: (1) an anatomical LET (ALET), with the femoral fixation located at the anatomical insertion of the ALL  and (2) This study is not without limitations. First, the data were obtained from MRI measurements and not cadaveric knees. However, the number of specimens required would have been too large for the study to be performed on cadavers, and the reliability of MRI measurements on DICOM viewers is well established. In addition, the design of the study entailed calculating the graft length with a formula from a simple 2D MRI measurement, and this would not have been possible in cadaveric specimens. However, a spin-off study comparing the data extracted from the formulas with real reconstructions (during surgeries or on cadaveric specimens) would be advisable. A second limitation is related to the ethnicity of the population studied: all patients included were Caucasian and significant morphological differences have been described among patients of different races . As a laIn conclusion, the transepicondylar distance (TD) showed the highest repeatability and reproducibility, as well as the strongest correlation with 3D measurements. Formulas were created from the TD and based on the clinical needs, for the following procedures: ACLR revision techniques  and two lateral extrarticular tenodesis (LET) techniques . The formulas extracted proved to be accurate and can be used by surgeons in ACLR revision pre-operative planning, graft harvesting and allograft selection.


There are growing demands for multimaterial three-dimensional (3D) printing to manufacture 3D object where voxels with different properties and functions are precisely arranged. Digital light processing (DLP) is a high-resolution fast-speed 3D printing technology suitable for various materials. However, multimaterial 3D printing is challenging for DLP as the current multimaterial switching methods require direct contact onto the printed part to remove residual resin. Here we report a DLP-based centrifugal multimaterial (CM) 3D printing method to generate large-volume heterogeneous 3D objects where composition, property and function are programmable at voxel scale. Centrifugal force enables non-contact, high-efficiency multimaterial switching, so that the CM 3D printer can print heterogenous 3D structures in large area (up to 180 mm × 130 mm) made of materials ranging from hydrogels to functional polymers, and even ceramics. Our CM 3D printing method exhibits excellent capability of fabricating digital materials, soft robots, and ceramic devices. Multimaterial 3D printing using digital light processing (DLP) is challenging because multimaterial switching methods require direct contact onto the printed part to remove residual resin. Here, the authors report a DLP-based centrifugal multimaterial 3D printing method to generate large-volume heterogeneous 3D objects where composition, property and function are programmable at voxel scale. Beyond the conventional techniques of 3D printing with single material, it is desired to develop multimaterial 3D printing capability to manufacture heterogeneous 3D object where the volumetric elements (“voxels”) with different properties and functions can be precisely arranged in 3D space9. Only a few multimaterial 3D printing systems offer such capability by either selectively ink-jetting multiple micro droplets that are cured through photopolymerization8 or developing a multinozzle printing head that generates continuous multimaterial filaments by high-frequency material switching9. However, the diversity of printable materials is constrained by the special rheological requirements of these techniques. The feature size and the size of multimaterial transition zoom are also limited by the manner of selectively depositing materials through printing nozzles  3D printing is a high-resolution fast-speed additive manufacturing technology that forms 3D structures through digitalized UV irradiations that convert liquid photocurable resin to solid 3D structure. DLP can print a variety of materials ranging from hydrogelHere we report a DLP-based centrifugal multimaterial (CM) 3D printing method to generate large-volume heterogeneous 3D objects with multiple properties and functions through precise control on the spatial arrangement of each material voxel. Using the CM 3D printing system, we can directly fabricate a large-volume octet truss structure (155 × 108 × 57 mm) where the white and black units are alternatively arranged in space Fig. . As show Figure 31, we develop the CM 3D printing system that removes the residual resin during multimaterial switching by spinning the printed part with a high angular speed . Compared with previously reported methods30, the method in this work avoids the direct contact between the printed part and the solid wiper26 or fluidic flow29, and thus is applicable to print multimaterial structures with much greater area. As demonstrated in Fig. ω = 6000 rpm. Moreover, the proposed approach can remove residual resins with a wide range of viscosity. In Fig. hR) of residual resin with viscosity measured at shear rate of 0.1/s , a high angular speed may lead to severe deformation or even damage of the printed part  where the bending, pressure and temperature sensors are seamlessly integrated Fig. . The ent2 and interfacial fracture is cohesive  with a weight ratio of 4:1:20. Blue ceramic resin in Fig. 2O4.Structures in Fig. x and y directions. In this printing mode, the maximum area is 180 × 130 mm, and the optical resolution is 25 μm. Detailed printing process for this mode can be found in Supplementary Movie The CM 3D printing system illustrated in Fig. We design a heterogeneous 3D structure through a commercial computer aided design software (SolidWorks), and save the design model as assembly in.STL format so that different parts are described in the same coordinate system. The STL files for the assembly were then loaded into a self-developed slicing software programmed through MATLAB . The sliced two 2D images for each layer are arranged in the order that the horizontal stage follows to deliver the resin containers for printing the corresponding parts  which allows us to accurately control the spinning speed and duration. We chose the Indium Tin Oxide (ITO) glass as the printing substrate due to its low surface roughness, and attached the ITO glass onto the center of the spinning disc of the spinning coater. Before turning on the spinning coater, we deposited a droplet of polymer resin  onto the ITO glass. The spinning removes most of the residual resin but leaves a thin layer on the ITO glass. In order to measure the thickness of such thin-layer residual resin, we photo-cured it in an oxygen-free environment to eliminate the effect of oxygen inhabitation on the thickness of the cured residual resin. Finally, we measured the thickness of cured residue resin films on a surface roughness measuring instrument .We measured the viscosity of all polymer resins on Discovery Hybrid Rheometer  with a steel plate geometry (diameter: 20 mm). The tests were conducted with shear rate ranging from 0.1 to 1000/s at room temperature. The plate gap was set as 200 μm.www.python.org, with OpenCV 4.3.0, https://opencv.org). Each raw image photographed by Nikon Z7 under the same lighting and camera parameters. The region of interest (ROI) was obtained through binarization and opening operation (corrosion before expansion). According to the ROI, the sampling lines are equally divided in the X or Y direction. Then the gray value of raw image was traversed and recorded according to each sample line. Finally, all the recorded values are normalized.The data of grayscale transition graph was obtained by customized image processing technology  to measure the interfacial toughness between elastomer and ceramic green body.ω for resin with different viscosities based on a previous work32 but the thin lay of liquid is attached to the bottom surface of the disc  with the center of the bottom surface of the disk as the origin. The disc spins with a constant angular velocity ω. The initial thickness of the liquid layer is h0  where the shear force must vanish. Thus,Equation  may be iz = 0). Thus,Equation  may be fq per unit length of circumference isThe radial flow dt is:As shown in Supplementary Fig. V:As shown in Supplementary Fig. Q equals to the volume change ΔV, we have:Because the net outflow ΔThe above equation can be further simplified as:q, Eq.  and , we can h-t relation:By integral of the reorganized form of Eq. , we can c and reorganize Eq. (Based on the initial condition nize Eq. :16\documh to be 10 μm. The initial height of the liquid h0 is set to be 1000 μm.To calculate the time (Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9Supplementary Movie 10


By analyzing the hyperfine structure of the spin defect while changing the boron isotope, we first confirm that it corresponds to the negatively charged boron-vacancy center  are promising quantum systems for the design of flexible two-dimensional quantum sensing platforms. Here we rely on hBN crystals isotopically enriched with either  Recently, coherent control of spin defects in hBN has been realized, enabling future applications in quantum sensing technologies. Here the authors perform a systematic study of isotope-dependent spin coherence properties of the negatively-charged boron-vacancy defect in monoisotopic hBN crystals. These defects, that feature localized electronic states with energy levels deeply buried inside the bandgap of the host material, behave as artificial atoms with efficient optical transitions. Importantly, the electronic spin of the defect can offer a fertile quantum resource for the development of a wide variety of applications. Besides its use as a quantum bit for the realization of advanced quantum information protocols4, the electronic spin state can also be exploited for quantum sensing purposes, by taking advantage of its extreme sensitivity to external perturbations such as magnetic and electric fields, pressure or temperature5. In this context, the nitrogen-vacancy (NV) defect in diamond is undoubtedly the most advanced solid-state quantum sensing platform, which has already found numerous applications in condensed matter physics and life sciences8. However, the search for alternative material platforms that could expand the range of quantum sensing functionalities offered by diamond remains very active worldwide9. Along this research line, a particular attention is currently being paid to layered van der Waals crystals because such materials promise the design of flexible, atomically thin quantum sensing foils13.Optically active point defects in semiconductors are currently attracting intense research attention owing to prospective applications in the growing field of quantum technologies14. During the last years, many papers have reported the isolation of individual defects in hBN, showing very bright optical emission and perfect photostability, even at ambient conditions19. However, the detection of the electron spin state associated with these point defects has long remained a missing ingredient towards the development of quantum sensors. This gap has been recently filled with the demonstration of optically detected magnetic resonance on two different types of defects, which have been tentatively assigned to negatively charged boron vacancy centers21 and carbon-related impurities24. Identifying the microscopic structure of optically active spin defects remains however a highly difficult task given the large number of potential candidates, which can involve extrinsic substitutional or interstitial impurities as well as complex assemblies of vacancies and antisite defects. Density functional theory is a powerful tool to reduce the number of possible atomic structures through ab initio calculations of defect properties such as formation energies, charge state transition levels, photoluminescence (PL) spectra or electron spin densities30. On the experimental side, valuable informations can be obtained by analysing the hyperfine structure of the spin defect23, which results from its interaction with neighboring nuclear spin species.Among the broad variety of layered van der Waals crystals, hexagonal boron nitride (hBN) is an ideal material for hosting optically active spin defects owing to its large bandgap (~6 eV)14N isotope with a nuclear spin I = 1 , while boron has two stable isotopes, 11B with a nuclear spin I = 3/2  and 10B with a nuclear spin I = 3 . To date, all the studies of spin defects in hBN were performed on crystals with the natural content of boron isotopes24, making the interpretation of hyperfine spectra a difficult task30. In this work, we circumvent this problem by employing hBN crystals containing a single boron isotope. Using such monoisotopic crystals, we focus our study on a spin defect featuring a broadband PL signal centered at a wavelength of 800 nm. The analysis of its hyperfine structure while changing the boron isotope confirms that this defect corresponds to the negatively charged boron-vacancy center  or 10B (99.2%). The resulting crystals were irradiated with thermal neutrons to produce point defects. Two different processes can lead to the creation of defects via neutron irradiation: either by damages produced by neutron scattering through the crystal or via neutron absorption leading to nuclear transmutation. The latter process strongly depends on the isotopic content of the hBN crystal. Indeed, the thermal neutron capture cross-sections of 11B ( ~0.005 barn) and 14N ( ~1.8 barn) are orders of magnitude smaller than that of 10B ( ~3890 barn)32. As a result, neutron irradiation of the h10BN crystal likely creates boron vacancy-related defects through nuclear transmutation and introduces interstitial 7Li atoms resulting from the fission of the 10B isotope33. Conversely, the h11BN crystal is almost transparent to neutrons and point defects are solely created by neutron scattering. To partially compensate for the isotope-dependent efficiency of defect creation by neutron irradiation, the h11BN crystal was irradiated with a dose of ~2.6 × 1017 n cm−2 and the h10BN crystal with a smaller dose of ~2.6 × 1016 n cm−2.Monoisotopic, millimeter-sized h10BN and h11BN crystals around the energy of the intralayer E2g phonon mode that corresponds to the in-plane vibration of boron and nitrogen atoms34. The energy of this phonon mode is at 1358 cm−1 for h11BN and is shifted to 1394 cm−1 for h10BN, as expected from the isotopic mass difference36. The full-width at half maximum of the Raman line is around 4.5 cm−1, attesting for the high crystalline quality of the hBN samples. This value is however slightly larger than that usually measured on similar crystals not subjected to neutron irradiation (~3 cm−1)35.The monoisotopic content of our neutron-irradiated hBN crystals was first verified through Raman scattering spectroscopy. Figure 10BN crystal is shown in Fig. λ ≈ 800 nm [Fig. 28. A similar PL signal was observed on the h11BN crystal [Fig. 10BN crystal. Interestingly, a confocal PL scan recorded along the depth of the crystal shows that optically active defects are tightly localized at the sample surfaces [Fig. 37. Although a precise understanding of this effect is beyond the scope of the present work, this is an important information for the future integration of exfoliated hBN layers doped with optically active spin defects into van der Waals heterostructures.The PL properties were then analyzed with a scanning confocal microscope operating at ambient conditions. A laser excitation at 532 nm was focused onto the sample with a high numerical aperture microscope objective (NA = 0.95) mounted on a three-axis piezoelectric scanner. The PL signal was collected by the same objective, focused in a 50-μm-diameter pinhole and finally directed either to a spectrometer or to a silicon avalanche photodiode operating in the single-photon counting regime. A lateral PL raster scan of the hλ ≈ 800 nm exhibits very similar magneto-optical properties to those of the NV defect in diamond21. It features a spin triplet ground state S = 1, that can be polarized through optical pumping, coherently manipulated with a microwave excitation, and readout by recording its spin-dependent PL signal. These properties enable the detection of electron spin resonances (ESR) by optical means21. Optically detected ESR spectra were recorded by monitoring the PL signal while sweeping the frequency of a microwave field applied through a copper microwire directly spanned on the sample surface. When the microwave frequency crosses a transition between the ground state electron spin sublevels, the magnetic resonance is evidenced as a drop of the PL signal. At zero external magnetic field, the ESR frequencies are given by ν± = D ± E, where D and E denote the longitudinal and transverse zero-field splitting parameters, respectively. A typical ESR spectrum recorded at zero field on the h10BN crystal is displayed in Fig. D = 3.47 GHz and E = 61 ± 1 MHz, in good agreement with previous works21.The point defect associated with the emission line at c axis in order to shift the electron spin sublevels via the Zeeman effect and thus isolate a single ESR line in the spectrum [Fig. 11BN crystal is displayed in Fig. 11B21. The same experiment was performed for spin defects hosted in the monoisotopic h10BN crystal, leading to a very similar hyperfine spectrum [Fig. 14N nucleus (I = 1). This situation can be obtained either for a boron-vacancy center or for a substitutional impurity with zero nuclear spin localized at a boron site. Substitutional carbon CB could be a plausible candidate, since carbon is a well-identified contaminant in hBN with a dominant spinless isotope . However CB defects exhibit a very different hyperfine structure, as discussed in ref. 30. We thus conclude that the broad emission line centered at a wavelength of 800 nm in hBN corresponds to the 21 and supported by ab initio theoretical calculations28.To investigate the hyperfine structure of the spin defect, a static magnetic field was applied along the rum Fig. a. The hyentclass1pt{minima11BN and h10BN crystals, because the 10B isotope has a higher nuclear spin (I = 3) than 11B (I = 3/2). This is due to the difference in nuclear gyromagnetic ratio between the two isotopes, γn = 4.6 MHz/T for 10B and γn = 13.7 MHz/T for 11B. Since the hyperfine coupling strength scales linearly with γn, it is reduced by a factor of three for 10B, which compensates for the impact of a higher nuclear spin in the broadening of the ESR line. To illustrate further this effect, simple simulations of the ESR spectra were performed. An exact calculation using a direct diagonalisation of the spin Hamiltonian cannot be done with conventional computer ressources given the large number of nuclear spins involved. We therefore rely on a simplified model for which we only consider the longitudinal component z denotes the quantization axis of c-axis of the hBN crystal20. The hyperfine coupling constants were taken from the ab initio calculations reported in ref. 28. The simulation includes (i) the three equivalent 14N first-neighbors with 11B (resp. 10B), and (iii) six equivalent 14N third-neighbors with s of the Gaussian function. In Fig. s = 30 MHz for which a fair agreement with experimental data is obtained. These simulations illustrate how the weaker hyperfine interaction with 10B leads to a similar width of the hyperfine lines despite a higher nuclear spin.The hyperfine interaction of the entclass1pt{minimaπ and π/2) employed in pulsed ESR measurements. We note that the decay of the Rabi oscillation cannot be used to directly infer the electron spin dephasing time 39  nuclear spin flip-flops driven by the hyperfine interaction with the central electron spin, (ii) nuclear spin flip-flops induced by nuclear spin-nuclear spin dipolar interaction, and (iii) many-body correlation effects41. We find that all these effects up to three-nuclear spin correlation contribute to the decoherence of the 28, which include the Fermi contact interaction. While this term is often neglected owing to the highly localized nature of the electron spin density associated with point defects in semiconductors42, it plays an important role in the decoherence of the The coherence time of spin defects in solids is commonly limited by the magnetic noise produced by a bath of fluctuating paramagnetic impurities, which includes electron spin impurities in the host material, dangling bonds at the sample surface and nuclear spins. To better understand the origin of the short spin coherence time observed for mentclass2pt{minimtion gCCE in our s11BN and h10BN are shown in Fig. T2 = 92 ns and T2 = 115 ns for h11BN and h10BN, respectively, with an exponent n ~1.35. The dependence of the coherence time with the isotopic content is depicted in Fig. 10B abundance. This effect results from the reduced nuclear gyromagnetic ratio of 10B, that weakens the hyperfine interaction and the boron nuclear spin flip-flop rate, both of which has a positive impact on the coherence time of the central spin. The convergence of the simulation with respect to the size of the nuclear spin bath provides important additional informations on the decoherence processes. Indeed, our results indicate that the coherence time is mostly limited by the fluctuations of nuclear spins that are not farther than 0.8 nm from the central spin  measurements45. Some of these defects were recently assigned to NBVN and CNVB centers45. To probe the presence of such S = 1/2 paramagnetic impurities in the vicinity of T1 as a function of a static magnetic field applied along the c axis. When the ESR transition of the T1 [Fig. 48. Considering a paramagnetic center with S = 1/2, the resonance condition is obtained for a magnetic field Bc = D/2γe, where γe = 28 GHz/T is the electron spin gyromagnetic ratio. For the Bc ~62 mT. The experimental results are displayed in Fig. S = 1/2 in the close vicinity of the T2 time in our experiments compared to theoretical predictions is attributed to fluctuations of these paramagnetic centers.Even though our experimental results reproduce the slight increase of the spin coherence time in hons Fig. c. This d49, a coherence time around T2 ~80 ns was measured in a neutron-irradiated hBN samples with a natural content of boron isotopes. This value is analogous to that obtained in the present work and is supported by our theoretical predictions. On the other hand, refs. 50 have reported the observation of a few μs-long coherence time in similar hBN samples. In these studies, the contrast of the echo signal was however very weak and the experimental data at short time time scale were not shown. Such long T2 values, which are not captured by theoretical simulations using CCE methods, could be alternatively attributed to nuclear spin coherence.We note that the few studies found in the literature on the spin coherence properties of 10B or 11B can be used to identify the structure of spin defects through the analysis of their hyperfine structure. Beyond the 30. An in-depth study of the spin coherence properties of T2 time in 10B-enriched crystals, which is supported by numerical simulations using cluster correlation expansion methods. These simulations highlight the importance of the spin density distribution, the corresponding Fermi-contact hyperfine fields, and first order correlation effect for determining the coherence time of solid-state spin qubits interacting with a dense nuclear spin bath. This finding, which generally applies to any defect hosted in a solid with a high nuclear spin density, constrains the validity of prediction of spin coherence times using hypothetical defects51. Using cross-relaxation spectroscopy, we finally identified dark electron spin impurities as an additional source of decoherence in hBN. Although such paramagnetic impurities have a detrimental effect of the spin coherence time, they could also be turned into a useful resource for quantum sensing applications52. This work provides key insights into the spin relaxation dynamics of To conclude, we have illustrated how hBN crystals isotopically enriched with either Supplementary InformationPeer Review File


In recent years, there has been an intense debate in literature regarding the definition of the individual variants of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), their possible pathogenetic mechanisms, and impact in the diagnosis of CIDP.The 2021 European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS) guidelines revised the definition of the individual CIDP variants and implemented their diagnostic criteria. Diagnosis of atypical CIDP is challenging and misdiagnosis is frequent, leading to diagnostic delay and consequent greater accumulation of disability and treatment dependency. Recent studies suggest that patients with typical CIDP have an antibody-mediated mechanism of neuropathy whereas in those with Lewis--Sumner syndrome (LSS) neuropathy is preferentially mediated by macrophages and T cells.Although the validity of the 2021 EFNS/PNS diagnostic criteria for atypical CIDP is unknown, they will hopefully lead to greater uniformity in the selection of patients to be enrolled in future studies and to a greater diagnostic accuracy. New data are emerging on the possible pathological mechanisms of individual variants and this could result in the discovery of specific diagnostic biomarkers and new therapies. Along with a typical clinical phenotype , defined as symmetric sensorimotor neuropathy involving proximal and distal segments of the four limbs with a relapsing or progressive course of at least 2 months, a few atypical variants have been described  7▪▪,▪▪,9–40. ▪▪]. The application of these criteria in a large cohort of patients with atypical CIDP has surprisingly shown that 53% of the patients with atypical CIDP at onset progressed to typical CIDP during the course of the disease [7▪▪]. Progression to typical CIDP, however, was not absolute and a considerable proportion of patients maintained their atypical CIDP phenotype even after several years from symptoms onset [7▪▪]. Transition from atypical to typical CIDP has been questioned by some authors . According to some studies, DADS is the most common variant of CIDP, with a frequency that ranges from 2 to 15%  in DADS was lower compared with typical CIDP [7▪▪]. Most studies report DADS as a mild form of CIDP . These figures are interesting as they show that the asymmetric form of CIDP is much more frequent than expected from a clinical entity that is considered ‘atypical’. It cannot, however, be excluded that, as in vasculitic neuropathies, a certain number of patients with multineuropathic CIDP evolve over time towards an asymmetrical form. Finally, the 2021 EFNS/PNS criteria has defined LSS as a sensory or sensorimotor multineuropathy specifying that its clinical presentation is usually asymmetric [▪▪].LSS was initially defined as a sensory or sensorimotor multineuropathy with persistent motor nerve conduction blocks –13. Acco8ymmetric ▪▪.et al.[et al.[▪▪]. The reported response to therapy and particularly to high-dose IVIg in LSS vary among studies, although in most of them is reported to be unsatisfactory .Pure motor CIDP was initially defined as a pure motor symmetric polyneuropathy ,31. Subs49▪▪,7▪▪], whereas another study reported a response of 80% [7▪▪]. This finding, which was later confirmed by others [▪▪].Some of the initial reports of this clinical entity reported unresponsiveness or worsening with steroids while having an excellent response to IVIg ,31,32 T. This ea7e of 80% . It has y others , suggesty others ▪▪.▪▪]. In this study, patients with sensory DADS but not those with pure sensory CIDP had a lower response to treatment compared with typical CIDP [7▪▪]. This figure has not yet been confirmed by other studies.The clinical boundaries between pure sensory CIDP and sensory DADS are not well clear, and this may possibly explain why in some studies, patients with a pure sensory neuropathy with a ‘stocking-and-glove distribution’ are included under DADS while in others under pure sensory CIDP ,34,50. T7▪▪]. No studies have yet compared the clinical and immunological characteristics of these two CIDP subforms.Although defined as a pure sensory neuropathy, most of the pure sensory CIDP cases so far described had subclinical electrophysiological involvement of the motor fibers but this is likely to be explained by the fact that signs of demyelination in the motor nerves were required by the 2010 EFNS/PNS diagnostic criteria for the diagnosis of CIDP . Some de49▪▪,7▪▪,8▪▪].Response to treatment in pure sensory CIDP is reported to be similar to that of typical CIDP 7▪▪,,36,39, w▪▪]. In the first description of 15 patients, all the patients had ataxia, nine had frequent falls, and six were severely disabled [▪▪].CISP is generally considered pure sensory CIDP because of its similar clinical presentation characterized by only sensory symptoms without weakness . Its pec7disabled . All of disabled . CISP-pldisabled ▪. Its sydisabled ▪. CIMP idisabled . Imagingdisabled . To our disabled ,45▪. Eledisabled ,45▪. Altdisabled ▪▪.▪▪]. Validity of these diagnostic criteria remains, however, to be established. Furthermore, these criteria still leave some areas of uncertainty. For instance, it is not clear how to classify patients with asymmetric but not multineuropathic CIDP or patients with a sensorimotor polyneuropathic CIDP only involving the proximal and distal segments of the lower limbs.As for typical CIDP, no diagnostic biomarker exist for atypical CIDP making clinical and electrophysiological criteria essential for diagnosis. The 2021 EFNS/PNS criteria refined the diagnostic criteria for the CIDP variants and expanded the 2010 EFNS/PNS criteria by including sensory nerve conduction studies as a mandatory diagnostic criterion and by defining specific clinical and electrophysiological criteria for each CIDP variant ▪▪. Valid▪▪].Atypical CIDP is a challenging diagnosis and its diagnostic workflow and differential diagnosis may differ compared with typical CIDP ▪. In one49Although the cause of CIDP and its pathogenesis are still unknown, in the last years some progress has been made in deciphering the pathogenetic mechanisms underlying the disease. Several recent lines of evidence suggest that typical CIDP and its variants potentially have heterogeneous pathogenetic mechanisms.▪▪,8▪▪,8▪▪,8▪▪]. The hypothesis, raised by some authors on the basis of these findings, is that in typical CIDP, the damage occurs in the proximal and distal portions of the nerve where the blood--nerve barrier is most deficient, and therefore, is likely mediated mainly by antibodies and humoral factors [8▪▪,8▪▪,8▪▪,8▪▪,Electrophysiological studies have shown that the distribution of lesions in the peripheral nervous system is different among the individual CIDP forms ▪▪,10,56▪ 8▪▪,8▪▪,▪. DADS aAlthough the validity of the 2021 EFNS/PNS criteria for CIDP has not yet been assessed, they will lead to greater uniformity in the selection of patients to be enrolled in future studies and this, hopefully, will result in greater comparability of studies. Their implementation should also lead to an improvement in diagnostic accuracy. There are still, however, areas of uncertainty in the definition of the individual variants. Recent studies are starting to bring to light the pathogenetic mechanisms of the individual CIDP variants and this could result in the discovery of specific diagnostic biomarkers and new therapies.None.None.E.N.-O. reports personal fees for Advisory or Scientific Board from Argenx, Belgium, Baxter/Takeda, Italy and USA, CSL-Behring, Italy and Switzerland Janssen, USA, Kedrion, Italy, Novartis, Switzerland, Roche, Switzeland, Sanofi, USA, outside the submitted work and travel grants to attend Scientific Meeting from Baxter, Grifols, Kedrion, and Novartis, Italy. P.E.D. has received travel grants to attend scientific meetings from CSL Behring and Kedrion. M.D. declared no conflicts of interest.


The spins of atoms and atom-like systems are among the most coherent objects in which to store quantum information. However, the need to address them using oscillating magnetic fields hinders their integration with quantum electronic devices. Here, we circumvent this hurdle by operating a single-atom “flip-flop” qubit in silicon, where quantum information is encoded in the electron-nuclear states of a phosphorus donor. The qubit is controlled using local electric fields at microwave frequencies, produced within a metal-oxide-semiconductor device. The electrical drive is mediated by the modulation of the electron-nuclear hyperfine coupling, a method that can be extended to many other atomic and molecular systems and to the hyperpolarization of nuclear spin ensembles. These results pave the way to the construction of solid-state quantum processors where dense arrays of atoms can be controlled using only local electric fields. The combined electron-nuclear spins of a single donor atom in silicon are coherently controlled using electric fields. A century ago, understanding the electronic structure and optical spectra of atoms was one of the first successes of the emerging theory of quantum mechanics. Today, atoms and atom-like systems constitute the backbone of coherent quantum technologies  with e1 = γeB0 − A/2, and ESR2, ∣↓⇑〉 ↔ ∣↑⇑〉 at frequency νe2 = γeB0 + A/2), and nuclear magnetic resonance (NMR) . In our setup, the oscillating magnetic field B1 is delivered by an on-chip broadband MW antenna  at the donor location modulates A via the hyperfine Stark effect. Microscopically, the electric field distorts the electron wave function and time-dependently alters its overlap with the 31P nucleus, on which A depends. Distorting the electron wave function from the spherically symmetric 1s orbital ground state results in the formation of an electric dipole  gate, which is terminated by an open circuit to maximize the electric component of the field at its tip . By applEdc is the static electric field at the donor location. The flip-flop Rabi frequency A(E)/∂E, yielding ∂A(E)/∂E = 0 /∂E and the associated electric dipole are greatly enhanced by pulling the electron halfway between the donor and an interface quantum dot. Assuming that the donor is placed at a depth d ≈ 10 nm below the Si/SiO2 interface , this results in a large electric dipole e · d ≈ 15 debye (e is the electron charge) and flip-flop Rabi frequencies up to A ≈ 100 MHz). The same large electric dipole can be used to mediate strong (∼10 MHz), long-distance (∼200 nm) coupling between flip-flop qubits.The resonance frequency is simply xzSzIxA. Such terms are necessary when attempting to drive the nuclear spin at its own resonance frequency, while leaving the electron unchanged. Here, the electron flip-flops with the nucleus, making the flip-flop transition possible even in systems where the hyperfine interaction is purely isotropic.Unlike earlier examples of electrical drive in electron-nuclear systems  is used to read out the state of the electron spin via energy-dependent tunneling . Repeating the above for varying frequencies of the MW drive, we find the two frequencies νe1 and νe2 at which the electron spin is resonantly excited to the ∣↑〉 state. Each resonance corresponds to one of the possible nuclear states, and their frequency separation directly yields the value of the electron-nuclear hyperfine coupling A = 114.1 MHz. This differs slightly from the value A = 117.53 MHz observed in bulk experiments  and leaves it unchanged (in the ∣↓⇑〉 state) otherwise. We find a high nuclear spin flip probability at fEDSR = 28.0966 GHz  sequence comprising an aESR2 pulse, followed by an aNMR1 pulse and a subsequent electron readout (see P(↑) and nuclear P(⇑) spin-up proportions for each EDSR pulse duration. Once initialized, we apply a resonant EDSR tone, which drives the transition from the ∣↓⇑〉 to the ∣↑⇓〉 state, and first read out the electron spin . The seqBy detuning the drive frequency of the EDSR pulse around the resonance, we map out the Rabi chevron pattern of the nuclear spin see . Here, w31P donors, the relaxation process within the flip-flop qubit subspace (∣↑⇓〉 → ∣↓⇑〉) is known to be extremely slow, T1ff ≈ 5 hours (T1e = 6.45(39) s in this device , measuring T1ff requires saturating the ESR1 transition while monitoring the decay of the system from the ∣⇓〉 subspace via the flip-flop transition. We adopted the pumping scheme depicted in a∣↓⇓〉 + b∣↑⇓〉, with ∣a∣2 ≈ ∣b∣2 ≈ 0.5, using a slow frequency sweep (labeled 1/2aESR1) calibrated to yield a 50% probability of inverting the electron spin (see section S1 for details)  s, much longer than T1e. We also independently verified that, without repopulating the ↑⇓ state, the rate of nuclear spin flip ∣↓⇓〉 → ∣↓⇑〉 is immeasurably slow, T1n ≫ 500 s.Having demonstrated the coherent operation and readout of the flip-flop qubit, we proceed to measure its key performance metrics for quantum information processing, i.e., relaxation, coherence, and gate fidelities. From bulk experiments on To investigate the coherence of the flip-flop qubit, we performed an on-resonance Ramsey experiment, where we initialized the system in the flip-flop ground state ∣↓⇑〉 using the ENDOR sequence and apply two consecutive EDSR π/2-pulses separated by a varying time delay see . We obtaelectron .A Hahn echo experiment is performed by applying a π-pulse halfway through the free evolution time, which decouples the qubit from slow noise and extends the coherence time to 4(24) μs . This va6(10) μs .The microscopic origin of the flip-flop decoherence mechanisms, and their relation to the electron spin decoherence, is still under investigation. A key observation is that the EDSR pulses induce a transient shift of the resonance frequencies of up to 80 kHz, chiefly by affecting the electron gyromagnetic ratio (see section S2 for details). The pulse-induced resonance shift (PIRS) depends on the power and duration of the pulse in a nontrivial way and decays slowly after the pulse is turned off. Similar effects were reported earlier in the literature  residual 29Si concentration, we found the flip-flop and ESR resonances to be split into six well-resolved clusters of frequencies, indicating at least three 29Si nuclei coupled to the electron by ∼100 kHz (see section S3 for details). These nuclei flip as often as once per minute.Another key decoherence mechanism is the presence of residual 29Si nuclear spin flips is mitigated by sandwiching each gate sequence between spectrum scans to monitor the instantaneous resonance frequency and only accept the measurement if the frequency remains constant (see section S4 for details). Despite this precaution, the GST analysis reveals a strong deviation from a Markovian model, possibly due to the effect of PIRS. Therefore, the GST one-qubit average fidelities ℱ1Q = 97.5 to 98.5% are additionally verified by RB.We measured the average one-qubit gate fidelities of the flip-flop qubit using the well-established methods of gate set tomography (GST) %, which corresponds to an average native gate fidelity ℱ1Q = 98.4(2)% (RB determines the average gate fidelity by applying to the qubit a random sequence of Clifford gates with varying length 98.4(2)% . CompariA(E)/∂E. If the driving mechanism is the electrical modulation of the hyperfine coupling, the two should be linked by the simple relation pp (peak-to-peak voltage) amplitude), we reach a flip-flop Rabi frequency We proceed to investigate the physical origin of the electric drive of the flip-flop qubit by comparing Rabi frequencies VFD to the FD gate (the same used for EDSR) and measure the hyperfine Stark shift ΔA(ΔVFD) through the shift of the NMR1 resonance, fNMR1 = A/2 + γnB0 (∂A/∂VFD = 512(26) kHz/V. We have performed a capacitance-based triangulation of the donor position (see section S5 for details) and found that it is located next to one of the side confining gates  = ηE2 , i.e., a quality factor Q ≈ 150, would allow simultaneously addressing the flip-flop transition for dynamic nuclear polarization and the ESR transition for readout to verify the hyperpolarization.Bulk donors only have a quadratic Stark shift of the hyperfine coupling, Future experiments will focus on operating the flip-flop qubit in the regime of large electric dipole, where the wave function of the electron is equally shared between the donor and an interface quantum dot  would provide a nuclear spin with I > 1/2. The presence of an electric quadrupole moment in I > 1/2 nuclei enables direct control of the nuclear spin through the recently demonstrated method of nuclear electric resonance (NER)  on top of a lightly p-doped natural Si handle wafer. Using optical lithography and thermal diffusion of phosphorus (boron), n+ (p) regions are defined on the sample. The n+-type regions serve as electron reservoirs for the qubit device and are connected to aluminum ohmic contacts. The p-doped regions are added to prevent leakage between ohmic contacts. Using wet thermal oxidation, a 200-nm SiO2 field oxide is grown on top of the substrate. The active device region is defined by etching a 30 μm–by–60 μm area in the center of the field oxide using HF (hydrofluoric) acid that is subsequently covered by an 8-nm high-quality SiO2 gate oxide in a dry thermal oxidation step. We define a 100 nm–by–90 nm window  . After each deposition, the sample is exposed to a pure 100-mtorr oxygen gas for 3 min to oxidize an insulating Al2O3 layer between the gates. We connect all gate electrodes from all layers electrically to avoid electrostatic discharge damage (the shorts are broken after wire bonding using a diamond scriber). Last, we anneal the qubit devices at 400°C in a forming gas (95% N2/5% H2) atmosphere for 15 min to passivate interface traps and repair EBL damage.The fabrication procedure of the donor qubit device follows the recipe outlined in  antenna, parallel to the surface of the chip and to the [110] Si crystal direction. A schematic of the experimental setup is shown in The sample is wire-bonded to a gold-plated printed circuit board inside a copper enclosure. The enclosure is mounted onto a cold finger attached to the mixing chamber of a Bluefors BF-LD400 dilution refrigerator and is cooled down to ≈17 mK. The sample is placed in the center of a superconducting magnet, operated in persistent mode at a magnetic field between BThe MW signal for EDSR control is generated by a Keysight E8267D 44-GHz vector source. For single-sideband IQ modulation, rf pulses from the LeCroy ArbStudio 1104 AWG are fed to the in-phase (I) and quadrature-phase (Q) ports of the vector source. The high-frequency signal is attenuated by 10 dB at the 4 K stage and combined to the baseband control pulses at the mixing chamber using a Marki Microwave DPX-1721 diplexer. The combined signal is routed to the FD gate (electric/EDSR antenna).7 VA−1 gain, 50-kHz bandwidth) and an SRS SIM910 JFET amplifier (100 VV−1 gain). The amplified signal is filtered using an SRS SIM965 analog 50-kHz low-pass Bessel filter and digitized by an AlazarTech ATS9440 PCI card. The above instruments are triggered by a SpinCore PulseBlasterESR-Pro. Software control of the measurement hardware and the generation of pulse sequences is done in Python using the QCoDeS /2 are aligned, i.e., μSET = μP.The white dotted line in B0 (≈1 T), the Zeeman interaction splits the electrochemical potential of the donor into two energy levels, μ↓ and μ↑, for the two electron spin ∣↓〉 and ∣↑〉 states, respectively. In this case, μ↑ > μSET > μ↓, and only the electron in the ∣↑〉 state is energetically allowed to tunnel from the donor to the SET, as there are no available states below μSET at the SET island  is then calculated by averaging over multiple repetitions. Further details of donor electron spin readout and initialization can be found in (The electron spin-up proportion The donor-bound electron can be used as an ancilla qubit to read out the state of the nuclear spin via QND measurement with fidelities exceeding 99.99% (see later section for more details).The hyperfine interaction between the electron and the nucleus results in two electron resonance frequencies Reading out the electron spin after an inversion pulse determines the nuclear state in a single shot . The fidelity of this readout process is the product of the single-shot electron readout fidelity and the fidelity of inverting the electron via an adiabatic pulse.eB0 ≫ A, the electron-nuclear hyperfine coupling is well approximated by zIzAS, meaning that the interaction commutes with the Hamiltonian of the nuclear spin. This is the quintessential requirement of a QND measurement  and calculate how many times the nuclear spin flipped (Nflip) in two consecutive measurements. The flip probability is then determined as Pflip = Nflip/(Nsamples − 1).For nuclear spin qubit manipulations that do not depend on the initial state , measuring the nuclear spin flip probability instead of the actual spin state is sufficient. For this, we read out the nuclear state For benchmarking and quantum logic experiments, we need to initialize the flip-flop qubit into the ∣↓⇑〉 state. We have seen that reading out the electron state also initializes it into the ∣↓〉 state. To deterministically initialize the nucleus into the ∣⇑〉 state, and hence the flip-flop qubit into the ∣↓⇑〉, we make use of an ENDOR sequence. The ENDOR sequence comprises an aESR2 pulse, an aNMR1 pulse, and an electron readout see . We use P(⇑), we perform nuclear readout and determine the nuclear spin state by comparing the electron spin-up proportion  to a threshold value. By averaging over Nsamples ≥ 20 repetitions, we derive P(⇑).To quantify the nuclear spin-up probability P(↑). We repeat this experiment 2500 times and apply multiple adiabatic NMR1 pulses after every 25 measurements to scramble the nuclear spin state. We determine the ENDOR fidelity to be ℱ = 90.88(55)% by calculating how many times we end up in the desired ∣⇑〉 state. The confidence interval shown in brackets represents the SD, which is calculated by dividing 2500 experiments into 100 independent measurements (25 experiments each) of the nuclear spin-up state probability P(⇑). The histogram of nuclear readouts is shown in P(⇑) ≈ 0.1 that we see in EDSR spectrum scans.The fidelity of the ENDOR sequence is mostly limited by the errors of electron spin initialization in the ∣↓〉 state because the fidelity of ESR/NMR inversion pulses is typically >98%. To measure these errors, we apply the ENDOR sequence and determine the nuclear state by measuring the electron spin-up proportion


In yeast, mutations affecting the same region loosen coupling between the proton motive force and ATP synthesis, resulting in high rates of mitochondrial respiration. Expression of the mutant allele in human cell lines recapitulates this phenotype. These data support an autosomal dominant mitochondrial uncoupling syndrome with hypermetabolism.We describe the case of identical twin boys who presented with low body weight despite excessive caloric intake. An evaluation of their fibroblasts showed elevated oxygen consumption and decreased mitochondrial membrane potential. Exome analysis revealed a  Mitochondrial oxidative phosphorylation (OXPHOS) is the major pathway for ATP production in eukaryotic cells 1. These disorders result from pathogenic variants in either the nuclear or mitochondrial DNA (mtDNA). Most are recessive conditions with decreased respiration. In contrast, the first described mitochondrial disease, Luft Syndrome, was a euthyroid, hypermetabolic state characterized by high rates of mitochondrial respiration “uncoupled” from ATP synthesis.3 Only one related case has been reported,6 but the molecular bases are unknown.Over 300 molecular genetic forms of mitochondrial disease have been reportedO, which serves as a proton pore, and the catalytic F1.8 The catalytic core of F1 contains three α and three β subunits arranged around a γ subunit of the central stalk. Protons passing through FO (the pore) cause the γ stalk to rotate, inducing conformational transitions in the β subunit that are required for the synthesis of ATP from ADP.10 The synthesis of ATP is thus dependent on (coupled to) the rotation of the γ stalk which, in turn, is dependent on (coupled to) the passage of protons through FO (the pore); see Two domains comprise CV: the transmembrane Fde novo pathogenic variant affecting CV that loosens coupling between the passage of protons  through FO and the synthesis of ATP by Complex V. Here, we report the case of monozygotic twin boys who underwent their first biochemical genetics evaluation in the neonatal intensive care unit at the age of 2 months after presenting with a euthyroid hypermetabolism, failure to thrive despite excessive caloric intake, intermittent hyperthermia, and developmental delay. Genetic and biochemical studies in patient fibroblast and engineered cellular models implicate a dominant-acting, 11). Their body-weight z-score was −2.5 and −3.2, respectively , Twin A was consuming 1280 kcal/day  and Twin B was consuming 1480 kcal/day  /piericidin+antimycin injection sequence. Mitochondrial membrane potential in intact cells was measured using tetramethylrhodamine methyl ester perchlorate (TMRM) with a BioTek Cytation5 (Agilent) plate reader. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Blue Native PAGE and western blotting were performed using standard methods employing commercially available antibodies.Full methods are described in the 12 to model the Leu335Pro variant of human ATP5B. Positions of human Leu335Pro and yeast mitochondrial genome integrity (mgi) mutants were identified both by multiple sequence alignment and by structural alignment of the yeast (PDB:6CP6)13 and ovine CV. Structures were rendered with Pymol14.We used the cryo-EM structure of ovine CV (PDB: 6TT7)We performed Mann–Whitney testing on the results of pulse oximetry  and 2G ade novo heterozygous variant NM_001686.3:c.1004 T>C in ATP5F1B, predicting a p.(Leu335Pro) change in the β subunit of CV. Both Leu335 and the surrounding region are highly conserved  in the probands revealed an apparently onserved . Based o3β3 assembly that holds the tip of the γ subunit of the central stalk  to engineer two independent clones with the heterozygous Leu335Pro mutation and compared them to an isogenic control . As in tmgi mutations can cause mtDNA depletion, we did not observe changes in mtDNA copy number in our cell lines  and dominant negativity (variant protein suppressing function of the wildtype protein), we heterologously expressed either an epitope-tagged wildtype (WT) or Leu335Pro variant in HeLa cells using lentiviral transduction. Cells tolerated expression of either allele and although the expression of the Leu335Pro variant was consistently lower than that of the WT , it assell lines . The datde novo variant in the affected twin boys, absent from controls and affects an amino acid residue that is evolutionarily conserved. Leu335 is in the critical hydrophobic sleeve region; mutations in yeast affecting nearby residues cause an uncoupling phenotype. Oxygen consumption and membrane potential studies in both patient fibroblasts and cells with genetic introduction of the Leu335 variant show loosened coupling between the proton motive force  and ATP synthesis due to intrinsic dysfunction of Complex V. Furthermore, heterologous expression of p.Leu335Pro on a wild-type background recapitulates these biochemical findings, consistent with a dominant-negative effect on chemiosmotic coupling. Based on these findings, the variant is classified as “pathogenic” by American College of Medical Genetics and Genomics criteria16  in oduction . They al1 domain of CV is comprised of a trimer of α–β dimers and a central γδε stalk17. The nucleotide-binding sites are located at the interface between each α and β subunit. Protons pass through the pore of FO, which is connected to the central stalk, driving its rotation within the α3β3 assembly. This rotation alters the affinity of the α–β interface for nucleotides, driving the catalytic cycle for ATP formation. Normally, every 360° rotation 8 protons are translocated into the matrix and 3 molecules of ATP are produced. Tight contacts between the γ subunit and the α3β3 assembly maximize efficient “powering” of ATP production by the translocation of protons.17 Importantly, the C-terminal tip of the γ subunit fits snugly in a hydrophobic sleeve made of amino acids 287–294 of the α subunits and amino acids 274–281 of the β subunits.17.CV couples the proton motive force generated by the respiratory chain to the synthesis of ATP. The catalytic F13 identified two categories of uncoupling mgi mutations in yeast CV. Group 1 mutations affect amino-acids that make up the hydrophobic sleeve of the α–β hexamer and allow the γ subunit to rotate without tightly engaging the α–β regions. Group 2 mutations alter the matrix-facing interface of the α and β subunits. Given the proximity of Leu335 to the hydrophobic sleeve and effect of certain yeast group 1 mutations, we propose that Leu335Pro is a group 1 mutation, because it probably impairs engagement of the γ subunit by the α–β hexamer by disrupting contact between the β and γ subunits. It thereby disrupts ATP synthesis, even with normal translocation of protons through FO.Our measurements of oxygen consumption and membrane potential show that if we chemically eliminate CV activity or, alternatively activate it maximally (saturating ADP), there are no residual differences in “mitochondrial leak,” demonstrating that uncoupling arises from a defect intrinsic to CV. Arsenieva and colleagues3 Both conditions are characterized by high caloric intake, low body mass and recurrent hyperthermia.We propose that the current condition and Luft Syndrome belong to a category of “mitochondrial uncoupling syndromes” characterized by elevated mitochondrial respiration uncoupled from mitochondrial ATP synthesis. Biochemically, however, Luft syndrome appears to be due to uncoupled respiration that originates outside of CV.4 the latter of which is consistent with the observation that uncoupling respiration from ATP synthesis results in heat generation in a variety of systems.19 While it is reasonable to assume that the hypermetabolism and hyperthermia are directly attributable to the effect of Leu335Pro on CV, it remains unclear why hyperthermia is episodic. Future challenges lie in delineating how the uncoupling defect within CV leads to secondary changes within mitochondria, the cell, and the patient.Distinct clinical features in the twin brothers are presentation in infancy, developmental delay and episodic hyperthermia,20. In the future, it will be interesting to determine whether inherited variation in these pathways may contribute to differences in energy metabolism in the broader population.We anticipate that mitochondrial uncoupling syndromes may have diverse molecular and biochemical etiologies. For example, uncoupling may be mediated by genetic variants that make the mitochondrial inner membrane leaky to protons. Alternatively, variants in the genes encoding the α, β, or γ subunits of CV may loosen coupling between the proton motive force and ATP synthesis Supplement


Defining the stroke etiology directly impacts management. Most patients are treated with antiplatelet therapy alone in the absence of AF because empirical initiation of anticoagulant therapy has not been shown to improve outcomes in several studies.,,Although up to one-third of ischemic strokes do not have a clearly defined mechanism and are labeled as Heart Rhythm O2, Yaghi et al2DS2-VASc score (∼2.6 vs ∼3.0).,In this issue of ,The past decade has seen tremendous advancements in ICM technology, including improved electrogram acquisition, AF detection algorithms, and remote monitoring capabilities.,First, the burden of subclinical AF that constitutes an elevated stroke risk remains elusive. Studies involving nonstroke populations with implanted pacemakers or defibrillators suggested that, beyond a certain threshold, a dose–response relationship between subclinical AF burden and stroke risk exists. However, the minimum burden of AF that defines an elevated risk remains unclear.In summary, this thought-provoking study by Yaghi et al


Microsecond melting and revitrification of cryo samples preserves the structure of embedded particles. The beam-induced motion of revitrified samples is comparable to that of conventional cryo samples. A novel approach to time-resolved cryo-electron microscopy (cryo-EM) has recently been introduced that involves melting a cryo sample with a laser beam to allow protein dynamics to briefly occur in the liquid, before trapping the particles in their transient configurations by rapidly revitrifying the sample. With a time resolution of just a few microseconds, this approach is notably fast enough to study the domain motions that are typically associated with the activity of proteins but which have previously remained inaccessible. Here, crucial details are added to the characterization of the method. It is shown that single-particle reconstructions of apoferritin and Cowpea chlorotic mottle virus from revitrified samples are indistinguishable from those from conventional samples, demonstrating that melting and revitrification leaves the particles intact and that they do not undergo structural changes within the spatial resolution afforded by the instrument. How rapid revitrification affects the properties of the ice is also characterized, showing that revitrified samples exhibit comparable amounts of beam-induced motion. The results pave the way for microsecond time-resolved studies of the conformational dynamics of proteins and open up new avenues to study the vitrification process and to address beam-induced specimen movement. Improved sample-preparation methods  in 0.1 M sodium acetate buffer with 1 mM Na2EDTA and 1 mM sodium azide at pH 5.0 . The grids were rendered hydrophilic through 10 min of plasma cleaning in an ELMO glow-discharge system operating with negative polarity, 0.8 mA plasma current and 0.2 mbar residual air pressure. Approximately 3–3.5 µl of sample solution was applied to the foil side of the grids: either 1.70 mg ml.5 Fig. 1, 0.33 mg4 Figs. 2 and 3 ▸ et al., 2020in situ melting and revitrification of the sample are obtained by chopping the output of a continuous laser (532 nm) with an acousto-optic modulator. The laser is reflected from a mirror located above the upper pole piece of the objective lens and strikes the sample at close-to-normal incidence. The laser beam is focused to a spot size of 24 ± 1 µm FWHM as measured by a knife-edge scan in the sample plane.Experiments were performed with a Jeol 2200FS transmission electron microscope that we have modified for time-resolved experiments as described previously  were carried out with cryoSPARC version 3.2.0  and heterogeneous refinement (O symmetry) into two classes, the high-resolution class (49 885 particles) was homogeneously refined using O symmetry, resulting in a 4.57 Å resolution map.All images were corrected for the magnification distortion  and heterogeneous refinement (O symmetry) into two classes, the high-resolution class (45 953 particles) was homogenously refined using O symmetry, resulting in a 4.25 Å resolution map.The revitrified apoferritin data set was comprised of 100 micrographs taken from six melted and revitrified areas on one grid. After patch motion correction and patch CTF estimation, 70 images with CTF fits under 5.5 Å were chosen. Following blob picking and inspection, particles were sorted into 30 classes, and 27 were used as templates to pick 93 800 particles from the micrographs. Next, 58 792 particles were selected and sorted into 50 classes with a 160 Å circular mask. After removal of junk, 53 783 particles from 33 classes were chosen and subjected to another round of 2D classification . A total of 40 classes, corresponding to 49 476 particles, were selected. After ab initio reconstruction (I symmetry) and heterogeneous refinement (I symmetry) into two classes, the high-resolution class (3560 particles) was homogeneously refined using I symmetry, resulting in a 4.98 Å resolution map.The conventional CCMV data set consisted of 269 images. After patch motion correction and patch CTF estimation, 97 micrographs with CTF fits below 6.0 Å were selected. Following blob picking and inspection, particles were sorted into 30 classes, and 27 were used as templates to pick 36 783 particles from the micrographs. Of the picked particles, 6616 were selected and sorted into 50 classes with a 320 Å circular mask. After removal of junk, 6554 particles from 21 classes were selected. After ab initio reconstruction (C1 symmetry) and heterogeneous refinement (I symmetry) into two classes, the high-resolution class (5029 particles) was homogeneously refined using I symmetry, resulting in a 5.20 Å resolution map.The revitrified CCMV data set consisted of 391 images taken from 22 melted and revitrified areas on seven grids. After patch motion correction and patch CTF estimation, 120 images with CTF fits below 6.5 Å were chosen. After blob picking and inspection, particles were sorted into 30 classes, and ten were used as templates to pick 42 201 particles from the micrographs. Of the picked particles, 7866 particles were selected and sorted into 50 classes with a 320 Å circular mask. After removal of junk, 7240 particles from 16 classes were selected. After ChimeraX 6v21 1cwp  and 1b) illustrate the sample geometry and experimental approach. Cryo samples are prepared on UltrAuFoil R1.2/1.3 300 mesh gold grids . Under illumination with a 20 µs laser pulse, the sample rapidly melts in the vicinity of the laser focus and subsequently revitrifies, arresting the motions of the embedded particles .Figs. 1es Fig. 1b.c)–1f) display micrographs of a typical experiment with a cryo sample of mouse apoferritin. Fig. 1d) shows a low-magnification view of the grid square before laser irradiation and Fig. 1c) shows a micrograph collected from the hole marked with a gray arrow. The sample is then illuminated in situ with a 20 µs laser pulse (205 mW), with the laser beam aligned to the central hole, which is marked with a crosshair. As indicated in Fig. 1e), a circular area around the center of the laser focus has melted and revitrified (dashed semicircle). In contrast, the adjacent regions, in which the temperature has remained below the melting point of water, have crystallized , which reveals intact apoferritin particles. We note that in order to ensure a comparable temperature evolution in all melting and revitrification experiments, the laser power  was always adjusted to obtain a revitrified region of similar size to that in Fig. 1e)  compares the density map obtained from a conventional cryo sample  with that obtained after melting and revitrification . We note that the resolution is consistent with that previously obtained on a similar instrument . We obtain near-identical reconstructions of CCMV with resolutions of 4.98 and 5.20 Å for the conventional (left) and revitrified (right) cryo samples, respectively. Secondary structural elements of the viral capsid are well resolved, which is also evident in the details of the density in the vicinity of the quasi-threefold symmetry axis. We note that the viral RNA inside the capsid is disordered and therefore is not resolved. We conclude that the melting and revitrification process leaves the particles intact and that within the spatial resolution of our experiment it does not alter their structure.Single-particle reconstructions confirm that the melting and revitrification process preserves the structure of apoferritin  shows that the average cumulative drift of the apoferritin particles, which is displayed as a function of the electron dose, follows a typical behavior for both the conventional (gray) and revitrified (purple) cryo samples. They initially drift quickly, before slowing to a lower and constant drift rate after a dose of 10–20 electrons Å−2. The revitrified samples exhibit a slightly larger initial drift but a smaller asymptotic drift rate. Their average cumulative drift therefore drops below that of the conventional samples after 15 electrons Å−2. It is 1.5 Å lower at a dose of 60 electrons Å−2, with a total drift of 10 Å. Qualitatively similar, although less pronounced, differences are observed for the beam-induced motion of the CCMV samples . The cumulative drift of the revitrified samples drops below that of the conventional samples after 30 electrons Å−2 and is 1.3 Å lower at a dose of 60 electrons Å−2, with a total drift of 15 Å.While we conclude that the structure of the proteins is not altered, an important related question is whether melting and revitrification changes the properties of the ice. In particular, it is important to establish how it affects the beam-induced motion that occurs during imaging and that limits the obtainable spatial resolution. Such motion arises when mechanical stresses in the vitreous ice, which have built up during plunge-freezing, are released under electron irradiation (Russo & Passmore, 2014es Fig. 3b. The cet al., 2021et al., 2021abet al., 2021et al., 2021et al., 2021We conclude that revitrified samples exhibit comparable amounts of beam-induced motion to conventional samples, an observation that can shed new light on the mechanism that causes a buildup of stress in the ice during vitrification. It has been proposed that this stress results from the large temperature difference between the holey gold film and the grid bars during plunge-freezing. Because of their large heat capacity, the bars cool more slowly and thus exert a compressive force on the vitreous ice film once they contract (Engstrom 4.et al., 2010et al., 2019et al., 2020et al., 2020et al., 2020Our experiments demonstrate that the rapid melting and revitrification of cryo samples leaves the embedded particles intact, providing a central piece of evidence that our approach is suitable to study the dynamics of proteins on the microsecond timescale. We find that proteins do not undergo structural changes within the spatial resolution afforded by our instrument. However, subtle structural differences, such as a side chain adopting a different conformation, may become apparent at higher resolution, a possibility that we have begun to explore. Temperature-resolved cryo-EM experiments have long established that for some proteins different conformational ensembles are obtained when the sample is cooled to a lower temperature before vitrification (Fischer et al., 2021et al., 2021Our experiments also open up new possibilities for studying the vitrification process in real time (Olshin et al., 2021bet al., 2001In the future, we envision using the microsecond melting and revitrification approach to study the dynamics of proteins, as outlined previously (Voss 5.EMD-14767; revitrified apoferritin, EMD-14768; conventional CCMV, EMD-14769; revitrified CCMV, EMD-14772. The corresponding micrographs have been deposited in EMPIAR with the following accession codes: conventional apoferritin, EMPIAR-11016; revitrified apoferritin, EMPIAR-11017; conventional CCMV, EMPIAR-11018; revitrified CCMV, EMPIAR-11019. All other data that support the findings of this study are available from the corresponding author upon reasonable request.The cryo-EM maps presented in this manuscript have been deposited in EMDB with the following accession codes: conventional apoferritin, apoferritin, conventional, EMD-14767EMDB reference: revitrified, EMD-14768EMDB reference: CCMV, conventional, EMD-14769EMDB reference: revitrified, EMD-14772EMDB reference: 10.1107/S205979832200554X/vo5009sup1.pdfSupplementary Figure S1. DOI: