Cross-sectional electron microscopy, spectroscopy and piezoresponse force measurement reveal the creation of a continuous in-plane charged antiphase boundaries around the implanted depth and a variety of atomic bonding configurations at the antiphase interface, showing the atomically sharp 180° polarization reversal across the boundary. Therefore, this work not only inspires a domain-wall fabrication strategy using He-ion implantation, which is compatible with the wafer-scale patterning, but also provides atomic-scale structural insights for its future utilization in domain-wall nanoelectronics.The deterministic creation and modification of domain walls in ferroelectric films have attracted broad interest due to their unprecedented potential as the active element in non-volatile memory, logic computation and energy-harvesting technologies. However, the correlation between charged and antiphase states, and their hybridization into a single domain wall still remain elusive. Here we demonstrate the facile fabrication of antiphase boundaries in BiFeO 3 remain elusive. Here, the authors report a fabrication of in-plane charged antiphase boundaries in BiFeO3 thin films, revealing the atomic bonding configurations and atomically sharp 180° polarization reversal of such boundaries.The correlation between charged and antiphase states in BiFeO A wealth of physico-chemical properties have been discovered in ferroelectric domain walls, including domain-wall conductivity15, unconventional magnetism16, optical response18, giant electromechanical19 and thermotropic behaviors20.In comparison to the three-dimensional domains in ferroelectric thin films, the two-dimensional domain walls or boundaries, which separate domains of distinct crystallographic and electronic features, typically have the reduced dimensionality of only a few atomic layers. These domain walls possess broken local symmetry and perturbed chemical environments, rendering them with promising aspects for nanoelectronics and nano-optoelectronics21. Specifically, the spontaneous polarization in ferroelectric materials here is defined as the dipole vector drawing from the negative to positive centers of each unit cell (u.c.), and thus in general a domain wall in the head-to-head (or tail-to-tail) state tends to be positively (or negatively) charged, while the defect types accumulating around may affect the charge states. In ferroelectric thin films with out-of-plane polarization, it is normal to observe the neutral domain wall elongating along the polarization direction, while a charged domain wall extending perpendicular to the film polarization direction is barely found in as-grown films , because of the energetically unfavored larger area-to-body ratio and the extra electrostatic energy built up by charging. However, such in-plane charged domain walls are appealing by their non-trivial charged properties and functionalities of more viable controllability, which are unavailable by out-of-plane domain walls, such as the ferroelectric tunnel junction displaying discrete quantum-well energy levels23. Recently, Liu et al. also fabricated the smallest memristor of multiple resistance states facilitated by the layer-by-layer migration of single in-plane charged domain wall in a 4 u.c. BiFeO3 (BFO) film24. Such fascinating quasi-two-dimensional nature of in-plane charged domain walls, in regardless of the film thickness, may enable the design of the electronic circuitry at a sub-nano scale.These domain walls can be categorized as either electrically neutral or charged by the net bound charge accumulated from the normal polarization of neighboring domains26 and twin27 boundaries. In contrast to the twin boundaries involving a reflection of neighboring lattices, the antiphase boundaries feature a planar defect by translating half-unit-cell of atomic registry in reference to the adjacent domain. In addition to the competing effects (either serving as nucleation sites28 or promoting the retention failure29) of antiphase boundaries during the BFO domain switching, they also play critical roles in the anomalous magnetoresistance of Fe3O430 and the antiferromagnetic coupling of NiFe2O431. Since BFO holds robust magnetoelectric coupling above the room temperature, the hybridization of both in-plane charged  and antiphase characteristics into a single-domain wall seems a charming idea and may pave way to the possibility of controlling magnetism with electric field at room temperature33. However, the realization and integration of such in-plane charged antiphase boundary (IP-CAPB) in ferroelectric films has been rather challenging and, to our best knowledge, not reported yet.On the other hand, the domain walls in ferroelectric thin films can be distinguished in respect of their crystallographic geometries, such as antiphase34, the He-ion implantation treatment has been widely applied to a multitude of materials to engineer their microscopic structure and apparent properties to high precision. For example, the negatively charged boron vacancy  and cross-sectional TEM results of the same BFO/LAO film before and after the He-implantation treatment. PFM measurements reveal that the in-plane multi-domain structure is refined into a uniform out-of-plane single-domain polarization near the surface of films. Cross-sectional scanning transmission electron microscopy (STEM) and atomic-resolution energy-dispersive X-ray spectroscopy (EDS) together elucidate various configurations of atomic bonding at the antiphase interface, which interweave to form an IP-CAPB around the implanted depth. Through quantitative analysis of atomic column positions, the atomically sharp 180° polarization reversal across the IP-CAPB is identified. We also discover a single-unit-cell thick antipolar Bi3 (LAO) (001) substrates using the pulsed laser deposition (PLD) technique as detailed in the Method section. The pristine BFO film is dominated by the tetragonal-like (MC) phase as indicated by the X-ray diffraction (XRD) results in Supplementary Fig. C  and weak (002) MA  peaks near the (002) LAO substrate peak41. The topography measurement by atomic force microscopy (AFM) in Supplementary Fig. C matrix when the film thickness exceeds ~40 nm and the substrate constraint relieves39. Because R-like phases come from the distortion of parent matrix to exhibit a smaller c parameter, manifesting lower local height in the topographic AFM map39. As shown in the schematics and the in-plane piezoresponse force microscopy (IP-PFM) phase maps in Fig. 15 ions per cm2. It can be seen that the in-plane domain structures disappear after the He-ion implantation, leaving a uniform IP-PFM contrast in Fig. 42. Due to the insulating nature of LAO substrates, OOP-PFM signal is a bit noisy. However, the homogeneous contrast in both IP- and OOP-PFM results indicates the single-domain state near the surface of BFO films after implantation.We grew BFO films of ~70 nm thickness on LaAlO24. The average and standard deviation values of every u.c. row are plotted as the function of distance away from the CAPB on the right panel of Fig. 24. The cross-sectional microscopic analysis of the BFO/LAO interface can be seen in Supplementary Fig. According to the motivation as discussed previously in the introduction paragraph to hybridize the antiphase and the in-plane charged characteristics into a single-domain wall, we simulated the possible atomic structure by artificially constructing one antiphase interface in the BFO matrix. After geometric and energy refinements, there is spontaneous head-to-head polarization initialized in BFO around the boundary as shown in Fig. M, Fe-K, O-K excitation peaks generates a binary contrast of atomic columns of the respective element, with or without the specific element, in a chemically selective manner. There are three kinds of bonding configurations found in the IP-CAPB: type-I Bi-bilayer, type-II Bi-tetralayer and type-III Fe-bilayer types as listed in Fig. 2O3-BFO, though the normal tetragonal Bi2O3 phase is a metastable non-polar semiconductor43. We also notice that the type-III CAPB involves the direct face-to-face connection of FeO6 octahedra and thus exhibits a high-energy state, making the Fe-bilayer interface rarely observed and spatially discontinuous.Besides the stacking faults (pointed out by red zigzag lines) along the antiphase boundary, CAPB also features a chemical ordering of anti-site occupation in opposite domains as visualized by the atomic-resolution EDS mapping shown in Fig. 21. Our IP-CAPB is also not completely straight as shown in the large-scale image in Fig. εxx and the rotation component εxy) even when the CAPB bends, while the out-of-plane lattice expansion (represented by the tensile out-of-plane strain εyy) can only be tracked in the bending section, which is explained by the configuration switch between type-I Bi-bilayer and type-II Bi-tetralayer in the bending antiphase interface. From the atomic-resolution EDS mapping of this bending region in Fig. It has been reported that a charged domain wall tends to bend its propagation path to reduce the net bound charge accumulated from the normal polarization of neighboring domains2O3 inside the wider domain wall. Such Bi2O3 layer is spontaneously anti-polarized against the surrounding BFO and induces the one-unit-cell thick BFO underneath to switch its polarization direction by 180°, forming an additional tail-to-tail charged boundary that is negatively charged. The creation of antipolar Bi2O3 layer and the resulted head-to-head/tail-to-tail oppositely charged boundaries may compensate the electrostatic potential built up by single charged domain walls (type-I CAPB), corroborating the stabilization mechanism of IP-CAPBs through bending. Combining these atomic observations and the switching experiments performed on the implanted BFO film as shown in Supplementary Fig. The unit-cell-by-unit-cell analysis of polarization in type-I and type-II IP-CAPBs tells us the reason. In Fig. L3,2 onset and the larger L3/L2 ratio in the top region by the atomic-resolution electron energy-loss spectroscopy (EELS) as shown in Fig. 644. Because an antiphase domain is actually a region of anti-site defects in the parent lattice, we thus propose that the switch of A- and B-site ions, leading to anti-site defects, was caused by the high-energy ion bombardment and assisted by the generated oxygen vacancies. The accumulation and migration of oxygen vacancies and anti-site defects finally compose the IP-CAPB at the implanted depth after the implantation process45. The He-implantation-induced IP-CAPB is reproducible as shown in Supplementary Fig. 4. Since the IP-CAPB is charged and possibly electrically conductive, its electronic properties can be probed by cross-sectional conductive AFM22 and exploited in multiferroic tunnel junction devices23.While the direct evidence to explain how the He-ion implantation produces IP-CAPBs remains unclear, it has been observed that the oxygen-vacancy density (the average Fe oxidation state) on the top side of IP-CAPBs is higher (lower) than that on the bottom side. This difference has been demonstrated by the lower chemical shift of Fe-2O3 resides in the bending region of CAPB, relieving the built-up electrostatic energy. Therefore, this work not only proposes a domain-wall fabrication strategy by the He-ion implantation, but also provides direct atomic-scale observation on such IP-CAPBs for their future utilization in domain-wall nanoelectronics.To sum up, we demonstrate the facile fabrication of continuous IP-CAPBs in BFO thin films using the He-ion implantation process. This technique is highly scalable, making it suitable for wafer-size lateral patterning. PFM data confirm the disappearance of stripe domain wall structures near the surface of films. Cross-sectional STEM and EDS spectroscopy then reveal a variety of atomic bonding configurations at the implanted depth, where they interweave to create the antiphase interface. Besides the atomically sharp 180° polarization reversal across the IP-CAPB, a single-unit-cell thick antipolar Bi2 pressure of 100 mTorr and cooled under 1 atm oxygen atmosphere. After deposition, the He-ion implantation was carried out at 8 keV with a fluence of 2.5 × 1015 ions per cm2 at the Ion Beam Center (IBC) at the Helmholtz–Zentrum Dresden–Rossendorf.A series of epitaxial ~70-nm-thick BFO films on LAO (001) substrates were grown by the pulsed laser deposition (PLD) technique at 680 °C under an OThe topographical measurement was conducted by the tapping mode of our Scanning Probe Microscope (model: Asylum Research Cypher) using Al-coated Si probes.The PFM measurement was conducted by the vector PFM mode of our Scanning Probe Microscope (model: Asylum Research Cypher) using Pt-coated conductive probes. The cantilever of probes is coated with Al to enhance the reflectivity. During scanning, the tips were applied with 800 mV AC voltage for reading the piezoresponse of BFO films.The XRD and RSM data, including 2theta-omega line scan and 3D-RSM, were obtained from the six-circle diffractometer equipped with both 0D and 2D detectors in the 1W1A beamline station of the Beijing Synchrotron Radiation Facility using the X-ray wavelength of ~1.548 Å. The 2D RSM images were extracted from the 3D-RSM dataset using MATLAB.Focused ion beam (FIB) techniques by FEI Helios G4 UX were applied to prepare the cross-sectional membranes of BFO/ALO films. Before the ion milling processes, the surface was coated with thin carbon paint and deposited 500-nm-thick Pt protecting straps using a low-energy (2 kV) electron beam. Then thicker Pt protecting straps (1.5 μm thick) were deposited using the ion beam. After lifting out a 1-μm-thick plate of the chosen crystal orientation to a copper finger, step-by-step thinning processes using 30, 5, 2, 1, and 0.5 kV ion beams well minimized the beam damage. STEM imaging and spectroscopy were performed using JEOL JEM ARM 200CF equipped with a cold field emission gun and an ASCOR fifth-order probe corrector. HAADF imaging, STEM-EDS and STEM-EELS experiments were carried out under 200 kV accelerating voltage with a 26 mrad convergence semi-angle and 24-120 pA probe currents for optimal information transfer and minimal electron irradiation damage. The collection semi-angles for ADF signal were set to 68–200 mrad. In STEM-EELS measurements, energy dispersion of 0.25 eV per channel and 91 mrad collection semi-angle of Gatan Enfinium ER spectrometer was set up. Zero-loss peak and core-loss edges were simultaneously recorded in dual EELS mode. The simultaneous ADF signal was acquired with a 93 mrad inner collection semi-angle.Further information on research design is available in the Supplementary InformationPeer review fileLasing Reporting Summary


Escherichia coli, the DNA polymerase IV, or DinB, plays this crucial role in coping with these type of mutations via the so-called translesion DNA synthesis. Despite the availability of several high-resolution crystal structures, important aspects of the functional repertoire of DinB remain elusive. In this study, we use advanced solution NMR spectroscopy methods in combination with biophysical characterization to elucidate the crucial role of the Thumb domain within DinB’s functional cycle. We find that the inherent dynamics of this domain guide the recognition of double-stranded (ds) DNA buried within the interior of the DinB domain arrangement and trigger allosteric signals through the DinB protein. Subsequently, we characterized the RNA polymerase interaction with DinB, revealing an extended outside surface of DinB and thus not mutually excluding the DNA interaction. Altogether the obtained results lead to a refined model of the functional repertoire of DinB within the translesion DNA synthesis pathway.In order to cope with the risk of stress-induced mutagenesis, cells in all kingdoms of life employ Y-family DNA polymerases to resolve resulting DNA lesions and thus maintaining the integrity of the genome. In   Escherichia coli, Dpo4 and Dbh in yeast, and DNA polymerase η, ι, κ, as well as Rev1 in eukaryotes , and the bacterial translesion DNA polymerase IV, encoded as dinB is one of those genes (2-furfurly-deoxyguanosine (N2-fdG) as well as ultraviolet (UV) radiation-induced thymine-thymine cyclobutane pyrimidine dimer (CPD) kind of lesions with high catalytic efficiency  substrates  to study the different interactions permitted us to elucidate the key role of the Thumb region in facilitating these as well as confirming the auxiliary role of the PAD. Together our dynamic and interaction data enabled us to refine the functional picture of DinB, which likely has implications for the vast majority of its related proteins spanning all kingdoms of life.Crystallographic studies of cleotide  reveal tcleotide . Experimcleotide ,26. Desp1–351 with an amino-terminal His6-tag. The individual domain constructs (DinB-NTD (DinB1–165), DinB-Palm , DinB-Fingers (DinB11–73), DinB-Thumb (DinB166–241) and DinB-PAD (DinB228–351)) were cloned into a pET28_SUMO vector using a pET28_SUMO_Hsc70 plasmid (a kind gift of B. Bukau (Heidelberg)) as a template, yielding amino-terminally SUMO-tagged DinB constructs using standard methods. DinBΔPAD (DinB1–230) was cloned by introducing a stop codon at amino acid 231 using standard single-point mutagenesis. Plasmids and primers used in this study can be found in The used DinB constructs were subcloned from a pET28b(+)-DinB construct (purchased from Genscript), yielding DinBE. coli BL21 (λDE3) cells and subsequently grown in 1 l of LB (Luria Bertani) medium supplemented with 50 μg/ml kanamycin at 37°C. Upon reaching an OD600 of 0.6–0.8 the cells were induced with 1 mM IPTG  and expression was continued overnight (12–16 h) at 25°C. The cells were harvested by centrifugation at 5000 × g for 10 min at 4°C, resuspended for lysis in 40 ml buffer A , supplemented with 5 mM MgCl2 , cOmplete EDTA-free protease inhibitors cocktail (Roche Diagnostics GmbH), and HL-SAN DNase I (ArcticZymes), and incubated for 30 min at 4°C. Thereafter, the cells were lysed by three passes through an Emulsiflex C3 (Avestin) homogenizer at 4°C. Cell debris was removed via centrifugation at 19000 × g for 50 min at 4°C. All different DinB variants were purified using a 5 ml Ni2+-Histrap HP column  column equilibrated with buffer A and subsequently eluted with buffer B (buffer A supplemented with 1 M imidazole). Eluted fractions of DinB and DinBΔPAD were dialyzed overnight against Buffer C , while His6-SUMO-variants were dialyzed against buffer D , respectively. The His6-SUMO tag was subsequently cleaved with human SenP1 (Addgene #16356), equilibrated in their respective dialysis buffer, and eluted by an NaCl gradient containing 1–1.5 M NaCl. Eluted fractions from the Heparin step were pooled and concentrated using appropriate Vivaspin centrifugal concentrators . DinB and DinBΔPAD, respectively, were additionally purified on a size exclusion chromatography column ) equilibrated with buffer E . All the purified proteins were concentrated to a range of 0.3–1 mM and stored –80°C until further usage.Each of the DinB constructs were chemically transformed into e #16356) and sepa2O supplemented with (15NH4)Cl for [U-15N]-labeled protein or with both (15NH4)Cl and d-(13C)-glucose for uniform double labeling , or D2O supplemented with (15NH4)Cl yielding -labeled proteins or D--glucose for uniform triple labeling ). For specific methyl group labeling, Met, Ala, Leu, Val and Ile (MALVIproS)-labeled DinB sample was produced in Bioexpress rich (2%) D2O-based 2× M9 media supplemented with (15NH4)Cl, d--glucose as well as 50 mg/l 2-Ketobutyric acid-4–13C,3,3-d2 sodium salt hydrate (Isoleucine), 4 vials/l DLAM-LVproS-kit (2-(13C)-methyl-4-(D3)-acetolactate , 50 mg/l  methionine, and 50 mg/l 2-[2H], 3-[13C] L-alanine  precursors added 1 h prior to induction  oligonucleotides (18 mer: 5’-TCTAGGGTCCTAGGACCC-3’ and 5’-GGGTCCTAGGACCCTAGA-3’) were purchased from Eurofins in lyophilized form. The oligonucleotides were resuspended in annealing buffer  to a concentration of 340 μM. To obtain double-stranded DNA (dsDNA), equimolar concentrations of the ssDNA were mixed together and heated for 5 min in a 95°C heating block. Thereafter, the heating block was turned off to allow the ssDNA oligonucleotides anneal together while gradually cooling down to room temperature. The sample was exchanged into appropriate NMR buffer for protein–dsDNA NMR titration using Zeba™ Spin Desalting Columns . For the dynamics measurements of holo-DinB, the annealed dsDNA was buffer exchanged into H® ion-exchange purification step was exchanged with a size exclusion chromatography step using a Superose6 10/300 GL column  pre-equilibrated with PBS buffer (RNA polymerase core enzyme was expressed from plasmid pIA900 (Addgene #104401)  and puriS buffer  supplemeNMR measurements were performed on Bruker Avance III HD 700, 800 or 900 MHz spectrometers, running Topspin 3.5/3.6 and each equipped with a cryogenically cooled triple-resonance probe. All experiments were performed at 298 K in NMR buffer as listed in 15N,1H]-TROSY-HSQC  version, as well as 3D (H)CC(CO)NH, H(CC)(CO)NH and HCCH-TOCSY-experiments –i–(i + 1) was applied to the raw data : Alaβ (31/31), Ileδ1 (22/22), Leuδ2 (33/38), Metϵ (9/10) and Valγ2 (21/24).For the methyl group assignment we used a -DinB and measured -NMR spectra  and the 15N longitudinal (R1) relaxation rates we probed the pico- to nanosecond motions of the H–N bonds. In these type of experiments, high hetNOE values and small R1 rates indicate rigid and stably folded regions whereas the inverse, low hetNOE values as well as high R1 rates, points to flexible and unfolded segments. Consistent with our structural characterization, the hetNOE data indicated that the folded parts of the Fingers, Palm and PAD are stably folded as evident by high hetNOE values, whereas linker and loop regions are more flexible as indicated by low hetNOE values  of 0.86, indicating the presence of global fast motions within the whole domain in solution. These motions are most pronounced within the Thumb domain as this region shows the lowest average values, which might also stem from inherent dynamics as this domain also showed quite distinct chemical shift changes between separated form and within the full-length protein as discussed above.To address the conformational dynamics of DinB urements ,55. By ms Figure , B. In sR1 rates for the folded segments of DinB could be determined to be 0.36 s−1, in line with the magnitude of the obtained hetNOE values. We observe only a marginal increase for the Thumb domain to 0.38 s−1 and a more notable one for the carboxy-terminal residues 342–351 with 0.76 s−1, indicating that this segment is intrinsically unstructured.The obtained average 15N transverse relaxation rates. We measured the R2-rates derived from the R1ρ rates (R2(R1ρ)), which report on the motions on the lower micro-second timescale, because under the used spin-lock radio frequency (RF) field of 2000 Hz all exchange contributions (Rex) much slower than 80 μs would be leveled out. In line with the previous analysis of the fast timescale motions, we observed a largely planar profile for the folded segments with an average value of 29 s−1 compared to almost identical 28 s−1 for the Thumb domain  rates we next determined the rotational correlation time τc of the DinB with ∼22 ns for the structured region  rates. To assess if this modulation of the relaxation rates can be attributed to different global dynamics of the DinB-PAD, we analyzed next the R1/R2(R1ρ) quotient as the distributions provide direct insight into if the analyzed domains tumble as (partly) independent units  values .To obtain a more detailed picture of the underlying dynamics within DinB and especially within its Thumb domain, we exploited the increased sensitivity of methyl groups to get access to the side-chain dynamics of the DNA polymerase IV. We chose specific labelling of isoleucine, alanine, and methionine, as well as stereo-specific labeling of valine and leucine methyl groups as these are well dispersed among the whole DinB, providing specific probes Figure . The excproS-DinB , which reports on the extent of the amplitude of motions on the fast NMR timescale .,50.2axis13C,1H]-NMR spectrum of MALVIproS-DinB had already shown some indications of specific line-broadening, which can possibly be attributed to conformational exchange processes. Therefore, we used a multiple quantum (MQ) Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion experiment  on the micro- to millisecond timescale by measuring the difference in the relaxation rates at two different CPMG fields (25 and 750 Hz). The obtained values indicated that the vast majority of the methyl groups are not involved in chemical exchange processes as the values were close to zero in agreement with a stable protein fold , which was used previously in crystallographic studies of a DinB construct lacking the ten carboxy-terminal residues . Upon adkon-rate constants ranging from 10 × 105 to 7 × 105 M−1 s−1 for DinB and DinBΔPAD, respectively  in comparison to DinBΔPAD (2.0 ± 0.08 nM) Figure –C, suggey Figure . In addiy Figure .R1 rates within Thumb domain also seem to be quenched within the holo-state. These effects also resulted in a reduction of the Rex values within the DNA binding pocket, in line with our initial hypothesis that these inherent dynamics could play a role in priming the DNA interaction. Interestingly on the outside surface of DinB an extended surface spanning the Fingers, Palm, and Thumb sub-domains still experience enhanced dynamics pointing to an additional interface possibly used for protein:protein interactions  to -DinB  between DinB and the RNAP core-enzyme was determined to be 6.4 ± 0.07 nM  via an extended flexible linker . This finding of the importance of an unstructured linker for the correct fold of the associated domain here matches a previous finding for the CTD of bacterial UvrD, also named helicase II . Based oFocusing subsequently on the backbone relaxation properties of full-length DinB showed in general a stable protein fold with a value 0.73 for the full-length protein, which in general indicates a stable fold, but is lower than the expected maximum values under the chosen experimental conditions of 0.86 ,70, indiOn the lower micro-second timescale, we observed a largely planar profile for the folded segment of the full-length protein, which is almost identical to that of the Thumb domain, and in agreement with the previous analysis of the fast timescale motions. Analyzing the fast and slow motions together revealed that the PAD is able to tumble to a certain extend independently of the catalytic core domain , which iS2axis•τC), which reports on the extend of the amplitude of motions on the fast NMR timescale gave a maximum value of ∼23 ns, which is in good agreement with the τc for the protein backbone.Expanding the analysis of the inherent dynamics towards the methyl bearing amino-acid side chains we initially determined the product of the side-chain order parameters and the correlation time of the overall molecular tumbling (R2eff values indicate that the vast majority of the methyl groups are not involved in chemical exchange processes. Nevertheless, within the core of the Thumb domain in particular several residues experience large exchange rates or were even exchange broadened, which is further proof of the existence of structural flexibility within this domain and can possibly be attributed to conformational sampling to facilitate substrate interactions.The obtained CPMG Δvia helices α8 and α11 of the Thumb domain and then the dsDNA is bound on the inner cavity of the catalytic domain encompassing Palm and Fingers and in a last step locked in by the PAD-domain, that reaches around the dsDNA and enables optimal positioning ensuring efficient catalytic activity. The obtained binding affinities by BLI clearly confirm this as the DinBΔPAD construct shows almost the same affinity as the full-length DinB despite the NMR data showing a less extended binding interface (Figure Rex exchange contributions (Figure In line with this hypothesis, NMR titrations with dsDNA show a distinct subset of the DinB backbone amide resonances exhibiting signal intensity attenuations and thus binding to the DNA. Most of the residues undergoing intensity changes, are located at the supposed entry point of the DNA the helices α8 and α11 of the Thumb domain, parts of the Palm and Finger domains pointing to the inner cavity of the full-length DinB protein, and the central β-sheet within the PAD comprised of strands β9–β12. Thus, our data indicates that the initial recognition possibly occurs e Figure . As expes Figure . NeverthU-2H,15N]-DinB show the backbone amide resonances of DinB exhibited severe line-broadening for a large number of resonances, indicating a specific interaction and the formation of a large DinB–RNAP complex with more than ∼330 kDa in size, and thus in line with previous observations for other enzymes involved in DNA repair (In a last step, we also examined if DinB can make direct contact with RNAP at a DNA lesion site, NMR titrations of unlabeled RNAP core-enzyme with [A repair  and 7. FA repair , as wellvia allosteric signaling through the catalytic core as well as likely through the linker towards the PAD, thus ensuring a stable DNA encounter complex (Figure In summary, we show that the Thumb domain of DinB plays a central role in the binding of the DNA substrate and is modulated by inherent dynamics to exhibit conformational flexibility required for a stable initial interaction. The inherent dynamics are then transferred x Figure . Anotherx Figure . Neverthgkad490_Supplemental_FileClick here for additional data file.


An automated pathway to statistically disentangle and detect domain structure transformations using deep autoencoders, providing a pathway towards real‐time analysis is also established. These results show a clear difference in the temperature at which phase transition occurs and the domain behavior between various environments, with a peculiar domain reconfiguration at low temperatures, from a‐c to a‐a at ≈60 °C. The vacuum environment exhibits a rich domain structure, while under the oxidizing environment, the domain structure is largely suppressed. The direct visualization provided by in situ gas and heating STEM allows to investigate the influence of external variables such as gas, pressure, and temperature, on oxide surfaces in a dynamic manner, providing invaluable insights into the intricate surface‐screening mechanisms in ferroelectrics.Ferroelectrics, due to their polar nature and reversible switching, can be used to dynamically control surface chemistry for catalysis, chemical switching, and other applications such as water splitting. However, this is a complex phenomenon where ferroelectric domain orientation and switching are intimately linked to surface charges. In this work, the temperature‐induced domain behavior of ferroelectric‐ferroelastic domains in free‐standing BaTiO Ferroelectric surfaces can enable dynamic surface chemistry control due to their polar nature and reversible switching. This study investigates temperature‐induced domain behavior in free‐standing ferroelectric films under various gas environments using in situ scanning transmission electron microscopy and deep autoencoders. Real‐time analysis of domain structure under different environments provides invaluable insights into intricate surface‐screening mechanisms, responsible for surface chemistry control. For decades, the polar surfaces of ferroelectrics have been considered extremely interesting entities, where the possible controlled exchanges between the chemical species in a chemical environment and polarization can be exploited to tailor surface reactivity for electrochemical, catalytical, and other energy harvesting applications. Importantly, the relationship between the chemical environment (atmospheric adsorbates) and ferroelectric polarization is two sided: i) the (polar) domains can significantly influence the local surface potential and surface free energy, thus, influencing the chemistry at the ferroelectric's surface, and ii) the chemical environment can provide charged species which can be used to control the polarization orientation and domain switching. It is crucial to understand both aspects of this relationship to effectively control the properties and behavior of ferroelectric materials for their development towards novel applications aimed at electrochemistry and controllable surface chemical reactions, which have shown promising for applications such as water splitting, and piezo‐catalysis. This is a complex phenomenon and the current understanding of this remains an open theoretical and experimental challenge. Another important aspect to consider when investigating surface effects on oxides is temperature. Morozovska et al. demonstrated theoretically that temperature plays a non‐trivial effect on the polarization and nature of the surface charge. Thus far, experimental efforts attempting to disentangle the role of surface chemistry, polarization direction, and surface structure have been dominated via X‐ray scattering, X‐ray photoelectron spectroscopy (XPS), and low energy electron diffraction (LEED), most of them on bulk. To date, a limited number of publications have reported on the simultaneous imaging of the domain structure under different chemical environments, such as Kim et al., who reported chemical switching in BiFeO3 thin films using piezoresponse force microscopy (PFM).Ferroelectrics are polar materials characterized by their spontaneous polarization that can be reversibly switched by applying an external electric field. The resulting domain structure, and switching dynamics, are determined by electrostatic boundary conditions. In particular, the compensation of polarization‐induced surface charges is a key factor in stabilizing ferroelectricity. In typical ferroelectric devices, charge compensation is achieved through the screening provided by the metallic electrodes, where domains will be formed to minimize the depolarization field energy. However, any deviation from this ideal scenario, including the presence of surface charges such as charged ionic species adsorbed from the atmosphere or vacancies,1, 23 (BTO) films under distinct atmospheres via in situ scanning transmission electron microscopy (STEM) techniques. The term 'free‐standing' refers to the fact that, unlike traditional epitaxial heterostructures, the ferroelectric material is not attached to a substrate. By using free‐standing films, we eliminate substrate‐related effects such as rigid clamping, chemical gradients, etc. Additionally, free‐standing ferroelectrics have recently gained great interest as they exhibit interesting effects that differ from the bulk.Here, we investigate the temperature‐dependent behavior of ferroelectric‐ferroelastic domains in free‐standing BaTiOthe bulk.29 and other phenomena. However, to our knowledge, this is the first instance of in situ gas STEM being used to study ferroelectric domains and phase transformations under controlled environments. We complemented the STEM observations with in situ heating PFM under ultrahigh vacuum (UHV) conditions. Importantly, we also establish an automated pathway to statistically disentangle and detect changes in the domain structure using deep autoencoders, providing a pathway toward real‐time analysis. In summary, we offer valuable insight via direct domain imaging, into the delicate link between key ferroelectric characteristics and the chemical environment, reporting clear variations on the phase transition temperature (TC), domain size, and periodicity presented. This direct visualization demonstrates in situ gas STEM as a novel technique to explore the effect of external variables  on oxide surfaces in a dynamic manner, particularly, to investigate surface‐screening mechanisms in ferroelectrics.In situ TEM gas cells have been successfully used to investigate catalysis17 and2To thoroughly discuss the results presented here, it is essential to first introduce i) the sample configuration, along with the in situ TEM system, and ii) the image segmentation method used to calculate the domain area fraction.2.1Figure 3 (BTO) and LaAlO3 (LAO) single crystals by focused ion beam (FIB) and transferred to a DENSsolutions Climate MEMs bottom chip with the lamellae positioned as shown in Figure −1. High‐angle annular dark‐field (HAADF) and bright‐field STEM (BF‐STEM) images were acquired every 5 °C. First, an open cell configuration , Figure −1, respectively, and, both parameters were stabilized prior to in situ heating.In situ heating and controlled gas flow were performed on a DENSsolutions Climate system, as shown schematically in 2.2Figure  Finally, a logarithm is applied, and the large values are reduced. The preprocessed data is a stack of  windows of length T•n•n, where T is the number of temperatures sampled and n is the number of windows sampled along each side of the image. Applying FFT to windowed images provides localized information about periodicity and reduces bias caused by sample warping. Next, the FFT dataset runs through a model inspired by the joint rotationally‐invariant variational autoencoder, where input is compressed into a latent space with an additional discrete rotation representation, then reconstructed to compare to the original image. The model used in this article disentangles the input datasets scaling, rotational, and translational elements and appends them to the latent representation. The encoder receives a batch of  images, which are down sampled and flattened to a feature vector with 8 points, as shown in Figure n,n) images (see Supporting Information). These identify domains, sample warping, and contamination, embedding channels  in vacuum, Channel 3 in all environments, and Channel 4 in oxygen, respectively. Then the relative areas shown in Figure Bright‐field STEM images were segmented and analyzed using a three‐part pipeline, where deep learning disentangles the bias caused by sample warping. During preprocessing, the images were cropped, and Gaussian background subtraction was applied. Then, the Pycroscopy package was used to sample  sized sliding windows and apply a Hanning window and Fast Fourier Transform (FFT), shown in Figure 31 Fin2.3Heating under UHV is the most common condition for in situ heating TEM experiments, under which oxygen vacancies are expected to form given that for oxides in general, the vacuum presents a reducing environment during annealing. Thus, as a first instance, UHV provides a reference environment and point of comparison of the domain behavior with other environments here investigated.−1). Four domain wall orientations or variants are identified, referred to as pairs of a‐a and a‐c domains, marked by the color scheme shown in the inset of Figure pc, a‐c domains, spanning across the whole lamellae. As the sample is heated, the domain wall mobility increases rapidly, with domains moving (retracting) at temperatures as low as 30–40 °C. Notably, upon heating, the a‐c domains decrease until they disappear at ≈60 °C  than that of bulk BTO, at ≈190 versus 120 °C. To verify the accuracy of the temperature measurement, a LaAlO3 (LAO) lamella of similar dimensions was heated under UHV conditions, with identical conditions as the BTO lamella. In this case, the lack of domain contrast was observed near the bulk's TC value . Annealing under UHV conditions, even under low temperatures, is known to promote the formation and redistribution of 𝑉o, as well as partially removing atmospheric adsorbates from the sample surface. The concentration and distribution of 𝑉o are known to modify the structure via changes in the oxidation states and their coupling with lattice strain. 𝑉o are associated with domain pinning, accumulation close to 90° domain walls, and ferroelectric degradation. Importantly, Vo are positively charged defects and can significantly affect the electrostatic boundary conditions, hence influencing the screening mechanism. It has been reported that under reducing conditions oxygen deficiency favors BaO evaporation, creating a Ti‐rich surface. In fact, thermal annealing between UHV and atmospheric conditions in PZT thin films has been shown to induce a reversible effect, between insulating and conducting behavior, demonstrating that surface adsorbates alter the boundary conditions . Whilst the exact origin for the higher and diffuse TC is not clearly known to the authors, we suggest that it can be rationalized by the creation of a defective surface with a highly nonstoichiometric nature, e.g., the creation and redistribution of Vo, or, forming a surface layer with different chemistry to that inside the “bulk” of the lamellae. Next, we compare the behavior observed under UHV to that under an oxygen‐rich environment, where a lower concentration of 𝑉o would be expected.Diffuseness on the phase transition resembles what is widely reported for strained ferroelectric thin films,53 elu2.42, an inert gas that possesses no charge and was not expected to influence the screening mechanism. Hence, considered a control test before exposing the sample to another environment (reducing or oxidizing). However, the domain behavior observed under N2 was unexpectedly different from that under UHV and very similar to that under 20% O2/Ar. For this manuscript's brevity, the full data for the N2 environment is discussed in Figure 2, the sample was directly exposed to an oxygen‐rich environment: 20% O2/Ar. In both cases, the NR pressure was 900 mbar, with a flow of 0.3 mL min−1, and these conditions were stabilized before annealing. The annealing conditions were identical to that of the UHV cycle: RT – 250 °C – RT at 1°C s−1.After the UHV cycle, the sample was removed from the TEM, and the full NR was assembled. The first environment tested was No induced by the UHV cycle. The latter is particularly interesting because a fully reversible system would be extremely attractive for chemical switching applications.The motivation for exposing the ferroelectric surface to an oxygen‐rich environment is twofold: i) to compare the domain behavior to that under UHV, which, whilst commonly used for most in situ TEM experiments, such conditions are not representative of working environments for a ferroelectric, and ii) to lower the population of Vications.133 has been characterized sometimes as a Ti‐O and BaO rich depending on the atmosphere. Spasojevic et. al. identified BaO2 formation on bulk BTO in an XPS study during heating in an O2 atmosphere. However, additional investigations are required to fully identify the surface chemistry.Analogous to the UHV cycle, the domain behavior in the oxygen‐rich environment exhibited repeatability, in a hysteretic fashion for the full annealing cycle Figure . Howeveras a Ti‐O71 andC, domain size, and periodicity.Figure 2, and 20% O2/Ar) at 90 °C, as this is the temperature at which more domains are observed under the N2 and 20% O2/Ar environments. The domain size exhibits no evident trend, slightly increasing for the oxygen‐rich environment. On the other hand, the domain periodicity continuously decreases as the sample is exposed to different environments, with the lowest value measured under the oxygen environment. It should be noted that the domain size is measured from wall to wall of the same domain, and domain periodicity is measured from the first wall of one domain to the first wall of the neighboring domain; both measured directly from intensity profiles across domains on HAADF images. The TC in all three environments is diffused and much higher than that of the bulk TC (≈120 °C), and that reported in previous BTO lamellae work. The lowest TC is observed for UHV (≈195 °C), followed by oxygen (≈215 °C) and nitrogen (≈225 °C).Additionally, it was observed that both the oxygen and nitrogen environments affect the TC observed in this work significantly differs from the TC reported in previous BTO lamellae work. However, there are some important differences to be noted.Firstly, the lamella in this work has only a thin top electrode (Pt), unlike the work reported by Chang et al. It is well known that electrodes alter the boundary conditions and thus induce different screening phenomena. Additionally, our samples were annealed and observed in situ, under subsequent environments with no exposure to ambient conditions between imaging. The authors attribute the changes observed in the N2 and O2 environments, particularly the apparent domain suppression, to two likely phenomena: a decrease of Vo and a change in surface species. The surface species could form a thin layer that differs from the depth of the sample, similar to a dead layer on the surface, which would alter the screening mechanism and prevent the reduction of oxygen vacancies. We propose that the domain suppression observed in the oxygen environment is indicative of the material trying to reach a monodomain state, similar to previous studies on PbTiO3 thin films. There are several potential reasons for this monodomain state not to be realized. Although there is strong evidence that significant surface compensation is achieved by ionic adsorption for relatively thick films, the lamella thickness might offer different screening mechanisms to that of an epitaxial thin film. The lamella contains two free surfaces, whereas a thin film without a top electrode only contains one. Thus, negatively charged O2‐ ions can accumulate at both free surfaces, where the top surface could be more exposed to the gas flow and would create surface layers with uneven stoichiometry that could result in a full preferential polarization orientation not being achieved. It could also be possible that a complete monodomain state is not viable in this configuration. In addition to polarization, BTO requires the spontaneous strain in the system to be compensated when cooling through TC, via the formation of 90° ferroelastic domains. Previous work has modeled the effect that the FIB‐induced surface damage has on the domain configuration, where it was found that the driving stress for the formation of periodic 90° domains is provided by an encapsulating surface layer. Overall, it should not be ruled out that some of the behaviors here observed are intrinsic to free‐standing ferroelectrics with boundary conditions that diverge from the typical two metal electrodes or thin‐film design, and that the surface ionic and electronic screening would become comparable to the bulk‐free energy of the ferroelectric.As previously mentioned, the T2, and O2 environments, without being exposed to air , the domain behavior could be replicated. However, this reproducibility is lost once the sample is removed from the TEM and exposed to ambient conditions. In this case, high‐temperature annealing can be used to induce the initially observed domain behavior, strongly indicating the effect of adsorbates from the environment.One important aspect to consider is the effect of the sample's history on modifying the screening mechanisms. Interestingly, it was observed that if the sample is directly cycled between UHV, Nbehavior,40 str3 lamella), with no polarization component. It was found that the resulting domain structure at RT does not change between the UHV and the nitrogen/oxygen‐rich environments, and the domain structure is not suppressed in any way  and temperature constant. It is worth mentioning that some degree of bulging can be expected from the silicon nitride window as the temperature increases. This deflection, in the Z‐direction, is more significant at higher temperatures (>300 °C) and typically observed by a drastic change in focus, which was not observed here. Therefore, this effect is expected to be minimal for the temperature here chosen (80 °C).The experimental design of the DENSsolutions Climate holder allows for a small pressure to be achieved (max ≈ 1 bar). Additionally, there is a natural compromise to achieve between flow, temperature, and pressure to avoid the windows of the nanoreactor (NR) from breaking. Nevertheless, it is possible to vary the pressure while keeping the gas flow rate . For example, the polarization orientation can be inverted in ultra‐thin PbTiO3 by changing between low and high pO2. Furthermore, the temperature dependence of the polarization is a strong function of the external pO2. At intermediate values, the TC in the film is strongly suppressed. Therefore, it could be that the increased pressure causes more adsorbates to bind to the surfaces of the lamella, changing the screening conditions and successfully compensating the depolarizing fields.Under 20% O2/Ar, it was found that reducing the NR pressure by 200 mbar at a constant flow rate and temperature favors the nucleation of new domain walls, as shown in r Figure  than at r Figure . An impohigh pO2.62 Fur2 atmosphere was also found to change the domain structure significantly. Thus, it could be the flow or pressure of N2 changing the current adsorbate structure  on the surface from the start, which would change the screening conditions and hence the preferred polarization orientation for subsequent environments. In support of this argument, there is the work of Segura et al.which revealed a gradual incorporation of positive charges onto the surface of a PZT thin film during low humidity (N2) experiments, and considered it unlikely that such charges came from new adsorbates in the environment, but this was not fully rationalized.However, the introduction of the inert Neriments,82 andVo, or the pressure and flow in the NR, that causes the significant changes to the domain microstructure between UHV and nitrogen/oxygen enviroments. Potentially, a limiting factor is the FIB processing, which creates an amorphous gallium‐implanted surface layer that encapsulates the material of interest. This would limit the ability of the charge to be compensated by adsorbates, which is purely a surface phenomenon. This is an important aspect to address in future experiments, because the full potential to study the ferroelectric surface by this technique may be limited by the sample preparation.In this work, it is difficult to ascertain whether it is the presence of adsorbates, the creation, and diffusion of Finally, for completeness, the temporal effect was also investigated and in general, it was observed that the domain structure did not change as a function of time, see Supporting Information.3TC, we examined the behavior of the domains under three different environments: ultrahigh vacuum (UHV), nitrogen (N2), and oxygen‐rich (O2/Ar) conditions. Our results revealed distinct domain behavior under each environment. Specifically, the UHV environment promoted a dense metastable configuration of competing a‐c and a‐a domain variants, which was corroborated by in situ heating PFM studies. In contrast, both N2 and oxygen/argon (O2/Ar) environments largely suppressed the domain structure immediately after gas flow, and the oxygen environment created a mottled feature indicative of a surface reaction. Additionally, we established an automated pathway to statistically disentangle and detect domain structure transformations using deep autoencoders, providing a pathway toward real‐time analysis. Our results emphasize the importance of considering the chemical environment when performing in situ experiments on ferroelectric materials, particularly under UHV conditions. Additionally, we suggest that the pressure effect should be further investigated as a route to understand and control the interaction between the ferroelectric surfaces and the environments’ chemical species. To the authors' knowledge, this is the first time that such experimental techniques (in situ gas and heating STEM) have been applied to study environmental effects on domain configurations in free‐standing ferroelectric films, which have recently emerged as an exciting platform as they exhibit interesting effects that differ from the bulk. Hence, we expect that this study would motivate further experimental and theoretical in situ gas STEM studies on ferroelectrics.In this study, we employed in situ STEM gas and heating to investigate the impact of different chemical environments on ferroelectric‐ferroelastic domains in free‐standing films of barium titanate (BTO). By cycling the sample from RT to 250 °C, past 43 (BTO) and LaAlO3 (LAO) single crystals using a dual‐beam FIB TESCAN LYRA3 and transferred to DENSsolutions climate MEMs chip via ex situ lift‐out technique, with the lamellae positioned as shown in Figure  A sister sample was prepared for piezoresponse force microscopy (PFM) with a similar procedure as for the STEM samples, but with a thickness of ≈300 nm on a platinized substrate; and acid washed with HCl to remove the gallium oxide layer expected to form at the surface from the FIB sample preparation.Lamellae of ≈150 nm thickness were prepared from BaTiOechnique,83 wit STEM was performed on an FEI TALOS F200 G2 at 200 kV. Low‐current imaging (≈50 pA) was used to prevent electron beam‐mediated domain nucleation. The temperature was gradually changed using a ramp of 1°C s−1, and an image was acquired every 5 °C with a dwell time of 20 µs. In situ heating PFM under ultra‐high vacuum (UHV) conditions was performed on a Scienta Omicron VT‐AFM system, between ≈27 °C and ≈187 °C with vertical and lateral PFM scans taken at 7‐predetermined temperatures, guided by the STEM in situ heating data. Temperature stills from this heat cycle are shown in Supporting Information. The temperature of the stage was controlled via a resistively heated cold finger and was monitored with a gilded copper thermalization block located in contact with the sample holder. Under these conditions, it should be noted that the temperature at the sample surface is estimated to be ≈5–10 °C lower than is recorded on the temperature sensor. During in situ experiments, the TEM column operated at a vacuum level of 10−8 mbar and the base vacuum of the UHV PFM system was ≈10−11 mbar.Scanning transmission electron microscopy (STEM) images were acquired simultaneously on high‐angle annular dark‐field STEM imaging (HAADF) and bright‐field STEM (BF‐STEM) detectors, to image 90° ferroelectric‐ferroelastic domains, as the contrast can arise from differences in the Bragg condition ; 180° domains are observable to a lesser extent due to the contrast arising due to the possible failure of Friedel's law.86 STEThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supplemental Video 1Click here for additional data file.Supplemental Video 2Click here for additional data file.Supplemental Video 3Click here for additional data file.Supplemental Video 4Click here for additional data file.Supplemental Video 5Click here for additional data file.


In this review, we attempt to summarize the DMPK characteristics of antisense oligonucleotide (ASO) therapeutics and discuss some of the issues regarding how to optimize the evaluation and prediction of the DMPK and DDI of ASOs.Oligonucleotide therapeutics are attracting attention as a new treatment modality for a range of diseases that have been difficult to target using conventional approaches. Technical advances in chemical modification and drug delivery systems have led to the generation of compounds with excellent profiles as pharmaceuticals, and 16 oligonucleotide therapeutics have been marketed to date. There is a growing need to develop optimal and efficient approaches to evaluate drug metabolism and pharmacokinetics (DMPK) and drug–drug interactions (DDIs) of oligonucleotide therapeutics. The DMPK/DDI profiles of small molecule drugs are highly diverse depending on their structural and physicochemical characteristics, whereas oligonucleotide therapeutics share similar DMPK profiles within each chemistry type. Most importantly, the mechanisms and molecules involved in the distribution and metabolism of oligonucleotides differ from those of small molecules. In addition, there are considerations regarding experimental approaches in the evaluation of oligonucleotides, such as bioanalytical challenges, the use of radiolabeled tracers, materials for  Oligonucleotide therapeutics are gaining attention as a new drug modality to reach therapeutic targets that cannot be treated using existing drug modalities, such as genetic diseases ,2. Earlyin vitro metabolism/DDI studies, and methods to study biodistribution. This review outlines the DMPK characteristics of antisense oligonucleotide (ASO) therapeutics and discusses the issues and perspectives regarding how to optimize the evaluation and prediction of the DMPK/DDI of oligonucleotides.There is a growing need to develop optimal and efficient approaches to evaluate the drug metabolism and pharmacokinetics (DMPK) and drug–drug interactions (DDIs) of oligonucleotide therapeutics. Currently the DMPK/DDI profiles of oligonucleotide therapeutics are generally evaluated according to the concepts and methodologies applied to small molecule drugs, but it has become clear that the factors that characterize the DMPK profile of oligonucleotides, especially the mechanisms and molecules involved in their distribution and metabolism, differ from those for small molecules. In addition, there are considerations regarding the experimental approaches used in the evaluation of oligonucleotides, such as bioanalytical challenges, the use of radiolabeled tracers, materials for Oligonucleotide therapeutics are generally considered to be “chemically synthesized drugs that consist of an oligonucleotide containing about one dozen to several dozen unmodified or modified nucleic acids and directly exert pharmacological actions without being translated into proteins.” DNA- and messenger RNA-based gene therapy products are also modalities composed of nucleic acids but differ from oligonucleotide therapeutics in that they are translated into proteins and are manufactured biologically or enzymatically. The main categories of oligonucleotide therapeutics are presented in The ASOs currently used clinically exhibit one of two types of mechanisms of action  Fig. 1aFig. 1a. The successful development of oligonucleotide therapeutics requires the oligonucleotide to be stable in the body and to bind potently to the target RNA; a wide variety of nucleotide modification techniques has been developed and used to meet these ends  11–13]..11–13]. The ASOs currently under clinical development can be broadly classified as either PS-oligonucleotides, whose nucleic acids are all PS modified, or PMOs, whose nucleic acids are all morpholino nucleic acids. As discussed later, the DMPK profiles of oligonucleotide therapeutics are highly dependent on their chemical modifications.N-acetylgalactosamine  conjugation and lipid nanoparticles are highly affected by the nature of the drug delivery systems; however, the ADME profiles of ASOs have been well characterized and can be summarized by chemistry type .,28.24,28PS-ASOs are distinguished from PMOs by their protein-binding characteristics. The negatively charged backbone of PS-ASOs interacts with the hydrophilic regions of plasma proteins , conferring overall high plasma protein binding ,29,30. SThe molecular mechanisms underlying the cellular uptake and intracellular transport of oligonucleotides are complex and insufficiently characterized, which poses a challenge to better understanding the distribution of oligonucleotide therapeutics. Because oligonucleotides are taken up into nonparenchymal cells  or accumulated in the lumen of lysosomes of parenchymal cells, overall tissue concentrations may not be indicative of their effective concentrations in those tissues.The cellular uptake process can be broadly divided into two steps, namely the adsorption of ASOs to cell surface proteins and internalization . Variouset al. identified a set of intracellular proteins with which PS-ASOs interact [Internalized ASOs need to be released from endosomes to interact with target RNAs in the cytosol or nucleus, and it is considered that these intracellular ASO trafficking processes are regulated by interactions with multiple proteins in the cytoplasmic and nuclear compartments. Using an affinity selection approach with biotinylated PS-ASOs, Liang interact . A receninteract . Compare14C is often used to label small molecules, for ASOs with a larger molecular weight, the resulting molar specific activity after 14C labeling may be insufficient for sensitive detection and quantification. The random labeling of ASOs with tritium, which has a higher molar specific activity than 14C, may be an alternative, but there are some disadvantages to using tritium. Specifically, tritium can be re-exchanged with hydrogen, and quantitative interpretation is difficult when metabolized. Efforts have also been made to develop tag-based labeling methods, but it is necessary to confirm that the tag does not alter pharmacological activity and PK properties [Distribution studies for oligonucleotide therapeutics will therefore likely benefit from microscopic approaches, such as fluorescence imaging , mass imoperties .in vitro in hepatocytes, liver microsomes, and other liver preparations. After the study drug is incubated with an animal or human liver preparation, a sample of the resulting mixture is analyzed using LC/MS/MS or another analytical technique to evaluate metabolite stability, the kinetics of metabolic reactions, the structure of major metabolites, metabolic pathways, and interspecies differences in metabolism. Metabolism is generally evaluated in vivo by analyzing samples  from animals dosed with a radiolabeled drug using LC/MS/MS connected to a radioactivity detector to elucidate the structure and abundance of metabolites. The presence of a metabolite specific to humans requires further investigation of the relevance of that metabolite to efficacy, toxicity, and DDIs.Metabolic studies investigate the process of structural conversion of a drug and identify metabolites with potential efficacy or toxicity, including cross-species differences. In the case of small molecule drugs, metabolism primarily takes place in the liver and is thus often evaluated In terms of metabolism of oligonucleotide therapeutics, PS-ASOs are hydrolyzed by endonucleases and/or exonucleases present in the plasma and tissues throughout the body. The stability of oligonucleotides against these nucleases and which site in the sequence is cleaved depends on the chemistry type of oligonucleotides (gapmer or SSO) and sugar modifications . Gapmersin vitro metabolic studies of oligonucleotide therapeutics have not been well established. Although the metabolic profiles of oligonucleotide therapeutics can be evaluated with a radiolabeled compound, as for small molecule drugs, the design of the labeling site and data analysis should take into account the possibility that the label can be removed as the compound is degraded into shortmers. It should also be noted that shortened metabolites from the ends may have pharmacological activity.Because the metabolic processes of oligonucleotide therapeutics mediated by nucleases are completely different from those associated with small molecule drugs, such as hepatic cytochrome P450 (CYP) oxidation and Phase 2 conjugation reaction, the materials and methodologies suitable for According to the guideline for preclinical safety assessment of oligonucleotide therapeutics issued by the Ministry of Health, Labour and Welfare in Japan, safety is not considered to be a particular concern for naturally occurring nucleic acid components degraded by nucleases; however, metabolites and degradation products containing chemically modified moieties need to be evaluated for nonclinical safety in accordance with ICH M3(R2), similar to conventional chemical products ,43. The et al. reported comprehensive biotransformation of volanesorsen, a PS-ASO gapmer, in preclinical animal species and humans [In the case of PS-ASOs, similar metabolic pathways for mice, monkeys, rats, and humans have been observed for several compounds , demonstd humans . The plad humans . Based oExcretion is evaluated to quantitatively determine how the parent drug and its metabolites are eliminated from the body. In general, for small molecule drugs, radioactivity in the urine, feces, bile, and expired air of animals dosed with a radiolabeled drug is measured over time with a liquid scintillation counter to determine the main routes of excretion and the amount excreted over time. The rates of excretion of the parent drug and major metabolites can also be measured using LC/MS/MS analysis of samples collected after an unlabeled drug is administered, although this requires chemically synthesized reference standards for quantification.PS-ASOs are primarily excreted in the urine as the intact form and nuclease-shortened metabolites, whereas PMOs are excreted intact ,28. The in vitro studies.DDI studies, in general for small molecule drugs, evaluate both the effects of the study drug on the pharmacokinetics of concomitant drugs  and the effects of concomitant drugs on the pharmacokinetics of the study drug  . Key molLike small molecule drugs, the DDIs associated with oligonucleotide therapeutics are evaluated both as a perpetrator and a victim of DDI based on the experimental approaches described above. It has been reported that oligonucleotide therapeutics do not (or only weakly) inhibit or induce drug-metabolizing enzymes and drug transporters and are not substrates of these enzymes and transporters . Althougin vitro DDI assessment of ASOs demonstrated that 2′-MOE-PS-ASOs did not significantly inhibit CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, or CYP3A4 in cryopreserved human hepatocytes at concentrations up to 100 μM ASO  [A comprehensive study on  100 μM) . In addi 100 μM) . That st 100 μM) .et al. compared human liver microsomes (HLMs) to cryopreserved human hepatocytes for the in vitro assessment of DDI, including inhibition of CYP enzymes and UGT by PS-ASOs [50: 0.8–4.2 μM), CYP2C8 (IC50: 1.1–12 μM), and UGT1A1 (IC50: 4.5–5.4 μM). In contrast, in hepatocytes there was little to no direct inhibition of CYP by PS-ASOs, demonstrating that the results are dependent on the test system used [In another study, Kazmi  PS-ASOs . When HLtem used . As descin vitro experiments, how to set clinically relevant concentrations, and cutoff criteria in the decision tree.Because the processes and molecules involved in the metabolism and distribution of oligonucleotide therapeutics are different from those of small molecule drugs, it is necessary to discuss whether the rationale and approaches for evaluating DDIs associated with small molecule drugs are directly applicable to oligonucleotide therapeutics, particularly in terms of the choice of materials for Concentrations of oligonucleotide therapeutics are determined not only with LC/MS/MS, which is generally used for small molecule drugs, but also with hybridization assays.For LC/MS/MS analysis of oligonucleotide therapeutics, ion-pair chromatography using hexafluoro-2-propanol and triethylamine is primarily used for sample separation. Although the LC/MS/MS-based approach has the advantage of relatively fast method setup, it has the disadvantages of degrading the mobile phase and analytical columns, making long-term, robust analysis difficult .In a typical procedure for hybridization assays, a template oligonucleotide that includes a sequence complementary to the analyte is immobilized on a solid surface and incubated with the analytical samples for hybridization, followed by ligation of a tagged probe sequence and then detection using enzyme-labeled antibodies . The detThe hybridization assays enable highly sensitive analysis, but a method for quantification of parent compounds may also detect shortened metabolites such as (n–1) mer, so attention should be paid to the selectivity of the parent and metabolites. To overcome the drawbacks in specificity and sensitivity of the conventional hybridization assays and LC/MS/MS methods, an alternative bioanalytical method has recently been developed by combining hybridization-based sample pretreatment and LC-MS/MS-based detection . This hyBecause each bioanalytical technique has its own pros and cons, it is important to choose a platform according to the purpose, nature of the samples, and stages of drug discovery and development.Various DDS approaches have been developed and used for oligonucleotide therapeutics. The major functions of DDS include: (i) targeted delivery to tissues; (ii) protection from degradation; and (iii) prolongation of exposure and pharmacological effects. There are two major types of approaches for DDS: chemical conjugation-based methods and carrier-based methods.in vivo [The conjugation of ASOs to a receptor ligand can facilitate the entry of ASOs into target cells and tissues. The most well-established of these systems is the asialoglycoprotein receptor (ASGPR)-mediated uptake of oligonucleotides conjugated to GalNAc, a natural ASGPR ligand, into liver hepatocytes. This technology has already been used in marketed siRNA as givosiran, lumasiran, inclisiran, and vutrisiran , and itsin vivo .Other examples of specific receptor targeting include the conjugation of glucagon-like peptide-1 receptor (GLP1R) agonist ligands as carrier peptides for targeted delivery of ASOs to pancreatic β cells ; a neuroLipid nanoparticles (LNPs) are a representative example of carrier-based approaches. LNPs have been applied to siRNA and mRNA and put to practical use as patisiran (an RNAi therapeutic) and in vaccines for SARS-Cov-2 . The appAs described above, there has been significant progress in ASO delivery technology, particularly in targeting the liver, with extrahepatic delivery attracting considerable attention as a future challenge.in vitro metabolism/DDI studies, and methods to study biodistribution:ASOs are taken up into nonparenchymal cells or are accumulated in lysosomes, and thus, overall tissue concentrations may not be indicative of efficacy.In addition to conventional distribution studies  used for small molecule drugs, microscopic approaches, such as fluorescence imaging, mass imaging, and immunohistochemistry, are beneficial in analyzing the cellular and subcellular distribution of oligonucleotides.Radiolabeling of ASOs is generally more difficult compared with small molecules, and attention should be paid to the position of the label because terminal labels can be metabolically removed.in vitro metabolic studies of ASOs are still not well-established; it is important to profile shortmers from the ends because they may have pharmacological activity.The materials and methodologies suitable for Highly sensitive bioanalytical methods need to be developed to determine low plasma concentrations of ASOs in the elimination phase as a surrogate of tissue concentrations.In addition to LC/MS/MS analysis, hybridization assays enable highly sensitive analysis, but a method for quantification of parent compounds may also detect short-chain metabolites such as (n–1) mer, so attention should be paid to the selectivity of the parent and metabolites.Because the excretion of PS-ASOs is slow, it is difficult to evaluate elimination profiles of PS-ASOs until completion of excretion; a longer period for the collection of excreta and measurement of residual radioactivity in the carcass at the final observation point should be considered.in vitro experiments, how to set clinically relevant concentrations, and cutoff criteria in the decision tree) may not be directly applicable to ASOs.Because the molecules involved in the ADME of ASOs are different from those of small molecules, DDI assessment approaches for small molecule drugs  has recently published a recommendation article regarding protein binding and DDI of siRNA . In addi


As one of the common techniques for measuring coseismic deformations, optical image correlation techniques are capable of overcoming the drawbacks of inadequate coherence and phase blurring which can occur in radar interferometry, as well as the problem of low spatial resolution in radar pixel offset tracking. However, the scales of the correlation window in optical image correlation techniques typically influence the results; the conventional SAR POT method faces a fundamental trade-off between the accuracy of matching and the preservation of details in the correlation window size. This study regards coseismic deformation as a two-dimensional vector, and develops a new post-processing workflow called VACI-OIC to reduce the dependence of shift estimation on the size of the correlation window. This paper takes the coseismic deformations in both the east–west and north–south directions into account at the same time, treating them as vectors, while also considering the similarity of displacement between adjacent points on the surface. Herein, the angular continuity index of the coseismic deformation vector was proposed as a more reasonable constraint condition to fuse the deformation field results obtained by optical image correlation across different correlation window. Taking the earthquake of 2021 in Maduo, China, as the study area, the deformation with the highest spatial resolution in the violent surface rupture area was determined (which could not be provided by SAR data). Compared to the results of single-scale optical correlation, the presented results were more uniform . At the same time, the proposed index also detected the strip fracture zone of the earthquake with impressive clarity. As one of the severest forms of natural disaster, earthquakes seriously threaten the safety of human life and property. As such, coseismic deformation is an essential source of data for assisting the parameter estimation of surface deformation models during devastating earthquakes . SatelliCo-registration of Optically Sensed Images and Correlation (COSI-Corr) allows for more accurate and robust measurement of the relative displacement between two images . COSI-CoEven so, COSI-Corr still has some problems which need to be solved. Firstly, general optical images with lower or medium spatial resolution are occasionally insufficient to measure fine tomographic displacements . This caOne particularly notable case was the 2021 Maduo earthquake, to which four different correlation scale sizes were applied, where the variation among the maximum values approached a dozen meters. Even based on these four different outcomes, it was impossible to determine the most reliable technique. A simple average was also attempted for post-processing to seek results agreement, but the effect was not ideal. Furthermore, traditional post-processing divided the east–west and north–south directions without establishing a typical two-dimensional relationship. Therefore, considering the similarity of adjacent surface deformations, high-resolution optical remote sensing data were used to fuse different OIC deformation results for this earthquake, from which consistent results for multi-scale correlation shape changes could be obtained. The method’s reliability was demonstrated by comparing its results with those of Sentinel-1-based D-InSAR, POT, and other published studies.On 22 May 2021, a magnitude 7.4 earthquake occurred in Maduo County, Guoluo Tibetan Autonomous Prefecture, Qinghai Province, China. The earthquake took place about 70 km south of the East Kunlun Fault Zone on the northern boundary of the Bayankala block and was caused by an internal fault within the block. The Bayankara massif is extensively exposed to Triassic flysch construction. The strata are controlled by the regional tectonic movement, and the strata strike in the same direction as the regional tectonic line. The earthquake’s surface deformation was extensive, creating a complex coseismic deformation zone. The overall deformation showed a left-lateral strike-slip motion with a minor component of reverse faulting. The rupture mainly occurred along the Jiangcuo fault located in the southern part of the East Kunlun Fault Zone, with an overall trend of N105° E and a length of approximately 151 km . The res2.The optical remote sensing images used in this article are high spatial resolution data from the Planet satellite, which is a new type of remote sensing satellite with high-resolution, high-frequency, and global coverage capabilities . This arIn the method validation section of this article, radar data were used to obtain coseismic deformation in the study area, serving as a reference and comparison for this article’s method. The radar data were collected on 20 May and 26 May, ascending- and descending Sentinel-1 satellite C-band data. The study area and data range are shown in We used the GAMMA2020 software to process the collected Sentinel-1 acquisitions with the D-InSAR and POT techniques. Initially, the single and multi-look complex images (SLC) from the upward and downward Sentinel-1 Synthetic Aperture Radar (SAR) data were extracted for corresponding bursts in respective areas. Using the satellite orbital parameters, the Shuttle Radar Topography Mission (SRTM) digital elevation model (DEM) with a resolution of 90 m was converted to the radar coordinate system. The coarse and fine registrations of the interferometric pairs of ascending and descending SLC data were performed to improve azimuthal registration accuracy. By eliminating the terrain phase and the flat earth phase by DEM and SAR orbit parameters, respectively, the raw differential interferogram was obtained. To suppress phase noise and improve the signal-to-noise ratio (SNR), multi-looking processing was performed with azimuthal and range views 10 and 2, respectively. An adaptive filter was applied to the obtained interferograms to eliminate phase noise and reduce the difficulty of phase unwrapping. To improve the accuracy of phase unwrapping, the low-coherence areas were masked before performing two-dimensional phase unwrapping and converting the deformation phase to displacement values in the LOS direction. Finally, the obtained interferograms were transformed to the geographic coordinate system through geocoding to produce the coseismic interferogram and deformation field for ascending and descending SAR images.Severe decoherence and sizable missing areas are noticeable in the InSAR results. The POT generates more noise, and the POT results from downgraded data show small missing areas in the epicenter region. This phenomenon occurs due to the extreme horizontal displacement changes in these regions.According to data from the China Continental Tectonic Environment Monitoring Network, a permanent east–west displacement of up to 25 cm has been recorded at the Maduo station, located over 30 km away from the epicenter. Meanwhile, the InSAR also detected significant deformation, with the maximum relative Line of Sight (LOS) displacement of about 1.87 m and 2.32 m in ascending and descending directions, respectively . Our meaWhile COSI-Corr has been designed to extract sub-pixel displacements between optical images, it is important to acknowledge that factors such as uncorrected topography, noise, and limited resolution can impact measurement results despite maximizing image utilization . When caThe frequency correlation process involves two main steps. The first step requires an initial estimation of the pixelwise displacement between two patches. If the image is noisy or there is an expected large displacement, a large initial window size should be selected. In theory, the window size should be twice the expected displacement. However, it is recommended to use a larger ratio, such as a 128 or 256 initial window size with an expected displacement of 15 pixels. Once the initial displacement is estimated, a second and final correlation step is performed to retrieve the subpixel displacement. For the final window size, it is customary to choose the smallest size that provides an acceptable level of noise. The researchers suggest that a window size of 32 × 32 pixels is the minimum necessary, while a window size of either 32 × 32 or 64 × 64 is usually sufficient to yield good results with high-quality images . This isSo in this paper, in order to explore the effect of different correlation windows on the results, with the same settings for the remaining parameters , correlation was conducted by COSI-Corr at scales of 64 × 64, 128 × 128, 256 × 256, and 512 × 512 pixels. The east–west and south–north displacements are obtained. Where north and east are positive, south and west are negative. The displacement results are shown in A vertical section line is drawn in the middle of the image, and the change of coseismic deformation with the distance of the section line under each window is analyzed, as shown in To select the appropriate fusion scale, profile analysis was carried out, which determined that the four windows detected faults in the same column. The 64 × 64 pixels window was similar to its 128 × 128 counterpart, wherein both exhibited more wavelet peaks. Analogously, the 256 × 256 and 512 × 512 pixels windows were also similar. Overall, across all window sizes, the performance was relatively flat. Nevertheless, determining which method was correct or incorrect was difficult, hence the attempt to fuse the results from a range of correlation scales to achieve greater uniformity. According to the above analysis, the subsequent steps selected the 128 × 128 and 256 × 256 pixels windows for interpolation fusion.To address the issue of the correlation technology of optical images being affected by the size of the correlation window, this study employs a vector analysis method to fuse deformation results of varying scales during the correlation process. The study considers the spatial continuity of the deformation vector and applies weighted fusion to the results of differently sized correlation windows for optical images.With the exception of fault rupture areas, the surface displacements of adjacent points are usually correlated in geophysical processes such as earthquakes, and ignoring this correlation limits the accuracy of determining displacement . Taking Displacement fields resulting from coseismic or inter-seismic events can be represented by a three-dimensional vector for any given ground point. In the local reference system, this vector may be expressed as its north, east, and vertical components. When generating a 2D displacement image, each pixel represents a single ground point displacement measurement. This is achieved by taking the scalar product of the real displacement and a vector  that represents the projection of the measurement onto the local reference system . The defIf each pixel center is taken as the origin, with the east-west direction as the x-axis and the north–south direction as the y-axis, with the positive direction being eastward and northward, then the actual horizontal displacement of each pixel can be represented by a two-dimensional vector, denoted as To study the variations of the deformation angle between adjacent pixels, it is necessary to calculate the angle between two adjacent vectors. Two calculation methods are proposed: one is to calculate the angle between the two vectors directly using their dot product, i.e.,The other is to first calculate the angle between each vector and a reference positive direction  separately, denoted as:Then take their difference:After standardizing all deformation vectors, the formulas for calculating the angles using the two methods are:I ranges from 1 to 9. Due to the lower time complexity, the first method of calculation is chosen.The Vector Angular Continuity Index (VACI) is defined as the average absolute difference in angle between the displacement vectors of a pixel and the eight surrounding pixels. This index describes the variation in displacement direction between a pixel and its neighboring pixels. The range of values for VACI should be between 0 and 1, and the unit is in radians. A higher VACI value indicates a larger difference in displacement direction between a pixel and its neighboring pixels, reflecting a more discontinuous and fragmented surface deformation in the neighborhood. A smaller VACI value indicates a smaller difference in displacement direction, reflecting a more continuous and consistent deformation direction in the neighborhood. Below are the formulas for calculating VACI using the two aforementioned methods:To test the reliability of VACI in detecting the angular properties of deformation vectors in the rupture and non-rupture zones, its distribution with respect to the deformation results obtained using POT from Sentinel-1 was calculated . The VACTo comprehensively describe the deformation characteristics at different deformation-related scales, the deformation vectors obtained at different scales are merged. The common merging method is to simply average the east–west and north–south components separately. However, from the perspective of vector analysis, this merging process is actually an interpolation process for two vectors of different sizes and directions. If a simple linear interpolation is used, the endpoint of the computed vector will always fall on the path between the difference of the two vectors, and the size of the resulting vector will always be smaller than the original vector, which is obviously not consistent with the actual situation of coseismic deformation vectors. Therefore, the Spherical Linear Interpolation (SLERP) method commonly used in computer graphics to describe vector movement is considered, since it has long been used in computer animation to interpolate movements between two 3D orientations . The weiVarious errors exist in the horizontal deformation field obtained from data processing, including decorrelation noise, orbital errors, and terrain shadow errors. For decorrelation noise, a signal-to-noise ratio (SNR) threshold of 0.9 is set in the deformation field, and a mask is used to remove regions with an SNR below the threshold. Since there are no large mountains or terrain undulations in the study area, terrain shadow errors are not considered. As optical image correlation methods theoretically cannot detect deformations greater than half the size of the correlation window, extremely large and small deformation values greater than half the window size are replaced with null values, and the missing values are then filled using inverse distance weighting, thereby removing decorrelation noise.Orbital errors appear in the deformation results as a global intrinsic quantity and are often removed using a polynomial surface fitting model. Reference points are established in the non-deformation areas, and the desired parameters are solved using the least-squares principle. The simulated orbit error component of the whole image is subtracted from the original deformation field to obtain the true surface deformation field .The final result obtained by using the VACI method introduced in Part Three after error processing is displayed in In order to verify the accuracy of optical data fusion results, the VACI OIC method results were compared to those obtained via the traditional D-InSAR and SAR POT techniques for the southern part of Eling Lake and the western end of the rupture zone as shown in Severe decoherence and sizable missing areas were noticeable in the InSAR results. Meanwhile, the POT generated more noise, and its results from downgraded data showed small areas missing from the region around the epicenter. This phenomenon was observed due to the extreme horizontal displacement changes which occurred in these regions. The modulus of the deformation vector was calculated to facilitate comparison. Deformation data from areas with poor coherence could be extracted using optical remote sensing images, and the resolution which resulted from this was much higher than that of POT. In remote sensing technology for obtaining coseismic deformation, optical image correlation has advantages such as high precision and immunity to the influence of decorrelation in the epicentral area. Moreover, POT was only able to obtain more intuitive results in the distance direction, while the Sentinel-1 orbit descent POT method results were very blurred and almost no deformation could be seen in them.Among the methods surveyed, SAR POT is the one that best matches the results of this study and provides superior visual effects. Its spatial resolution, however, is limited, and it struggles to capture detailed features . In contrast, the use of high-resolution optical imagery, illustrated in In order to verify that the method proposed in this paper is much more effective than simply averaging the results of randomly selecting two sizes of correlation windows, we conducted pairwise averaging of the east–west and north–south results obtained by four sizes of correlation windows, taking SAR POT results as reference. Their mean absolute error (MAE) is calculated, and their standard deviation (STD) is plotted in the form of an error bar to compare with our method, as shown in This method significantly reduces MAE. It is worth noting that compared with the fusion results of the same correlation scale, the trends of north–south deformation and east–west deformation are not the same, and the mean MAE of north-south results is higher. For the result using a 64 × 64 pixels window, adding a larger window improves the accuracy of its east–west results, but reduces the accuracy of its north–south results. For the result using a 512 × 512 pixels window, adding smaller window results improves the accuracy of its east–west and north–south results (except for the north–south shift fused with a 64 × 64 pixels window). This observation shows that the correlation scale and deformation direction are not uniform, which confirms the influence of the choice of correlation scale on the results. Therefore, in order to improve the performance of point target analysis in non-uniform moving regions, it is crucial to accurately evaluate the similarity of two candidate pixels, especially when using small-scale correlation windows.The accuracy of the proposed method is better than that of any combination of Windows. The constraint based on displacement vector angular continuity avoids the above problems, and the adaptive weight can better adjust the balance of larger or smaller correlation windows. Additionally, the proposed method also significantly improved the peak signal to noise ratio (PSNR) and reduced the root mean square error (RMSE) as shown in When using the optical correlation offset tracking technique to detect coseismic deformation, the spatial sampling and measurement accuracy depend greatly on the local image correlation. The variability of the phase shift on the scale of the sliding window provides an estimation of the measurement’s consistency . AlthougIn this work, the conventional correlation of surface displacements between adjacent points in the non-fault rupture region was considered. This offered an innovative approach, taking the resonance deformation vector as the research object and including the angular continuity index of the deformation vector (VACI). This index was implemented as a constraint to fuse the deformation results of different correlation scales obtained by OIC to obtain a vector field better than any relevant individual scale. Taking the 2021 Maduo earthquake as the subject of our proposal, high-resolution optical images allowed the generation of two-dimensional deformation fields and vector maps of the study area, from which the rupture zone was successfully detected via VACI.The effectiveness of the method we proposed here was demonstrated by comparison with the traditional single correlation scale and the simple average of two-scale combinations, which showed that the accuracy of the east–west deformation results was clearly improved. Additionally, in contrast to POT technology, our processing process was able to provide a two-dimensional deformation field with higher spatial resolution and local detail information. When compared to D-InSAR, our method demonstrated superior effectiveness with respect to information from areas with large deformations and severe decoherence near the rupture zone. However, the deformation field obtained in this study only accounted for a small portion of the area affected by the Maduo earthquake deformation field, due to the limited range of available Planet images. Consequently, the VACI was defined within the context of only eight neighborhoods. Therefore, where this parameter can be improved is to use more neighborhood pixels or to adaptively determine the number of surrounding pixels used based on the distance from the epicenter, such that more surrounding pixels are considered, which may be more appropriate for large correlation scales.