Atomic-scale imaging and charge state manipulation of NV centers by scanning tunneling microscopy

Domain: neuroscience
Published: 2026-01-27
Editorial outcome: minor_revision

Abstract
Nitrogen-vacancy(NV)centersindiamondareamongthemostpromising
solid-statequbitcandidates,owingtotheirexceptionallylongspincoherence
times,efficientspin-photoncoupling,room-temperatureoperation,and
steadilyadvancingfabricationandintegrationtechniques.Despitesignificant
progressinthefield,atomic-scalecharacterizationandcontrolofindividual
NVcentershaveremainedelusive.Inthiswork,weutilizeaconductivegra-
phenecappinglayertoenabledirectimagingandmanipulationofNV⁻defects
viascanningtunnelingmicroscopy(STM).Byinvestigatingover40individual
NV⁻centers,weidentifytheirspectroscopicsignaturesandspatialconfig-
urations.OurdI/dVconductancespectrarevealthegroundstateresonance
approximately300meVbelowtheFermilevelanddensity-of-statesmaps
uncoveratwo-lobedwavefunctionalignedalongthe[111]crystallographic
direction.Remarkably,wedemonstratetheabilitytomanipulatethecharge
stateoftheNVcentersfromNV⁻toNV⁰throughSTMtip-inducedgating.This
workrepresentsasignificantadvanceintheatomic-scaleimaging,spectro-
scopiccharacterization,andcharge-statemanipulationofNVcenters,poten-
tiallypavingthewayforfuturequantumdevicedevelopment.

Introduction
Nitrogen-vacancy(NV)centersindiamond1–8aredefectsconsisting
ofanitrogensubstitutionandanadjacentcarbonvacancyinthe
diamondlattice(Fig.1a).ThenegativelychargedNVcenters(NV-)
haveattractedexponentiallygrowinginterestinrecentyearspri-
marilyascandidatesforquantumsensingandcommunication1,3,9,10.
However,despitethetremendousinterest,amajorgapremainsin
ourabilitytomapoutNV-centersattheatomic-scaleandprobethe
effect of the atomic-scale electrostatic environment around NV-
centers on their qubit properties11–15. Critically, manipulation of
individualNVchargestatesatatomiclengthscaleshascontinuedto
pose a challenge12,16–18. Successful manipulation of local charge
statescouldforexamplebeusedtoimprovequantumsensingwith
simultaneous high sensitivity and spatial resolution by creating
smallnm-scalepocketswithahighdensityofNV-surroundedbya
regionwithoutNV-19,20.
Scanningtunnelingmicroscopy(STM)isoneoftheidealprobes
for atomic-scale imaging, mapping, and manipulation of individual
defects and to map their response to their local environment. The
studyofNV-centersbySTMhasthusfarnotbeenpossibleduetothe
insulatingcharacteristicsofthediamondcrystalhostlattice.Here,we
use a combination of characterization techniques to identify and
manipulate individual NV- centers in diamond. Motivated by recent
STMmeasurementsoninsulators21–24,weintroduceanoveltechnique
in whicha monolayer of graphene is transferred onto the diamond
surface,creatingaconductiveinterfacethatenablesstabletunneling
measurementswhilepreservingtheelectrostaticenvironmentofthe
underlyingatomicdefects.Thegraphenelayeriselectronicallytrans-
parent,allowingustoprobethelowenergyelectronicstatesofNV-;
the
tunnelingprocessandaccessibilityofsubsurfacedefectsarediscussed
furtherinSupplementaryNote1andSupplementaryNote2.Finally,all
STM experiments were carried out in a newly developed laser-STM
systemwhichallowsustousein-situphotoluminescence(PL)asan
independentconfirmationofthepresenceofNV-centers.

Results
Theprimarysamplesusedinthisstudyaretype-1bdiamondcrystals
withthe(100)facetfacingthesurfacenormaldirection.Theywerefine
polishedandstrain-released7beforeirradiationwithanelectronbeam
at 2MeV, with a dose of 2×1016/cm2 to create the high-density
ensemble of NV- defects. As shown in the atomic forcemicroscopy
(AFM)imagesfromFigureS1,thisleadstoverysmoothand
homo-
geneoussurfacesoverlargelength-scales.Wethenuseawettransfer
technique25,26 (see Methods), to cover the diamond sample with
monolayergraphene.Ramanspectratakenafterthisprocedure(Fig-
ureS2a)showtheappearanceofgrapheneG-and2D-peaksinaddition
tothe1332cm-1zone-centerphononpeak27,28ofdiamond.The2D-peak
issignificantlystrongerthantheG-peak,confirmingthegrapheneto
bemonolayer.AsseenintheAFMimagesinFigs.S2b-c,thesurface
remains flat, with the exception of occasional folds in the gra-
phenelayer.
ForSTMstudies,aftertransferringmonolayergraphenewesecure
the graphene/diamond onto our sample holder using a Mo clamp
whichservesasthebiaselectrode.Aschematicofourmeasurement
configurationisshowninFig.1b.STMimages(Fig.1c)showthehon-
eycomblatticeofthesurfacegraphenelayer.Alargerareascanofthis
regionisshowninFigS3.dI/dVspectroscopytakenoveralargeenergy
rangedisplaystheexpected5.5eVbandgapfromdiamondfromthe
valencebandedgeat-3.2eVtotheconductionbandedgeat+2.3eV
(Fig. 1d). A smaller energy-range spectrum reveals the expected V-
shape density of states feature from graphene (Fig. 1d inset). The
abilitytoclearlyseethediamondbandsdemonstratesthatthegra-
phenemonolayerstillallowsaccesstotheelectronicstatesfromthe
bulksubstratebelow.
We firstobtain optical evidenceofthe presence of NV- defects
within our STM scanning field-of-view. To do this, we measure PL
spectraatourtip-samplejunctionusinga532nmexcitationlaserwith
a5μmbeamspotdiameter.AsshowninFig.1e,thecharacteristicPL
signal from NV- defects is found, including the zero-phonon line at
~637nm(insetshowsacameraimageofthebeamfocusedatthetip-
samplejunction).HavingconfirmedthepresenceofNV-centers,we
moveontoatomic-scaleSTMcharacterizationofindividualdefects.
Wenotethatallsubsequentstudiesdescribedinthemaintextwere
performedwithoutlaserillumination.
For clarity ofpresentation,we will hereafter labeldefectsmea-
sured by STM sequentially, as Defect 1, Defect 2, etc. A prominent
defectthatisdiscernedinnegativebiastopographies,labeledDefect1,
is shown in Fig. 2a. dI/dV spectroscopy on Defect 1 shows a char-
acteristicpeakatapproximately-300meV(belowtheFermienergy),
asdisplayedinFig.2c.AsshowninFig.S4,thedefectpeakisrobustat
varioustip-samplejunctionconditionsandalargeenergyrangedI/dV
spectrumshowsnoadditionalpeaks(Fig.S5).Wenotethatthedefect
is not observed in the topography at positive biases (Fig. 2b). This
indicates21,29–34thatthedefectresidesbeneaththesurface—ratherthan
beinganadsorbateonthegraphenelayer—asitdoesnotaffectthe
local topography and is predominantly visible at negative sample
biasesduetothedensityofstatespeakat-300meV.InFig.S6,weshow
examples of defects with different dI/dV peak heights likely arising
fromtheirpositioningindistinctsubsurfacelayersbelowthesurface.
The corresponding topographies also show a sharper contrast
betweenthedefectandthesurroundinglatticeforDefect8,whichhas
astrongerdI/dVpeakcomparedtoDefect9.Asdiscussedfurtherin
SupplementaryNote2,weestimatethatthemaximumdepthofNV
centerswhichwecandetectis~2.5nmbelowthesurface.
Toobtainstatisticalinformationontheseandotherdefectsseen
insamples,wemeasured~45defectsontwodifferenttype-1bdiamond
samples,withmorethan5differentSTMtips;thetwotype-1bsamples
are found to have nearly identical NV- densities based on photo-
luminescencecharacterizationbeforemeasurements.Thehistogram
ofmeasureddefectpeakenergiesshowstwomaxima(Fig.2f).Wefind
thatmostdefectsshowapeakat~-300meV.Comparingtheknown
NV-energylevelsfromthebandedges35,36andusingthepositionofthe
Fermienergyrelativetotheconductionandvalencebandsfromour
spectroscopydata(Fig.1d),weexpecttheNV-groundstatetofallat
approximately -260 meV (|g> in Fig. 2d). The representative peak
energy (-300 meV) of the majority of the defects is therefore con-
sistentwiththeexpectedgroundstateenergylevelofanNV-center.
TheslightlyextendedtailoftheNV-histogramislikelyduetoafew
regionsofthediamondsampleswithshiftedbandedgesasshownin
Fig.S7.Inaddition,afewdefectswithapeakat~+550 eVarealso
found.ThesedefectsarelikelysingleNatomssubstitutingforCatoms
in the diamond lattice (P1 centers). Bias-dependent topographies
confirm that these defects are also subsurface as expected and
typicallyhavedifferenttopographicfeaturescomparedtotheNVswe
image(Fig.S8).Experimental37andtheoretical38workindicatethatP1
centers should show an energy level 1.7eV below conduction band
edgeatwhichwouldbe~+600 eVabovetheFermienergyinourcase
(Fig.2e).Wenotethatouridentificationofthesetwotypesofdefects
asintrinsictothediamondissupportedbyadditionalconsiderations.
Basedonpriorwork39–41,CvacanciesandNadatomsingrapheneare
expectedtoshowpeaksatdifferentenergies.Furthermore,measure-
ments of an electronic grade diamond sample with NV centers
embedded 5nmbelowthe surface,outside the STM probing depth
range,showanabsenceofthesedefectsaspresentedinFig.S9.
HavingidentifiedthemostprominentdefectsinourdataasNV-
centers,wenowproceedtomapoutthedensity-of-statesaroundone
suchdefect,labeledDefect4.Figure3ashowsanNV-defectwithits
correspondingdI/dVspectrum(Fig.3b).AsliceattheDefect4peak
energy(-300meV)isshowninFig.3c.Theelectronicstatesassociated
withDefect4haveadouble-lobestructurewithonelobestrongerthan
theother.Giventhe[111]orientation(seeFig.1a)ofNV-defects3,9,42–45
projectedtothe(100)surfaceofthediamondcrystalsalongwiththe
two-partstructureofNV-centers,thestructureseeninourdI/dVmaps
alignswiththeexpecteddipoleorientationoftheNV-relativetoour
substrateorientation(seearrowsinFig.3candFig.3g).Thesefeatures
areconsistentlyobservedonmultipledefects.ShowninFig.3e-his
anotherNV-defect,labeledDefect5,probedwiththesameSTMtipand
onthesamediamondsampleasDefect4.Defect5showsthesame
asymmetrictwo-lobestructureasDefect4,butisorientedinanother
projected[111]direction,helpingusruleoutanSTMtip-shapeeffectas
anoriginofthetwo-lobedstructure.
Motivated by previous work on STM tip-induced charge-state
manipulationinothermaterials21,46–48,wenextattempttomanipulate
thechargestateofNVdefectsviatip-inducedgating.Figure4ashows
Defect6,anexampledefectwhichistobemanipulated.Weposition
ourtipabovethedefect,retracttheSTMtip0.5-1.0nmoutoftun-
neling,andapplyavoltageof+5Vtothesamplewithrespecttothe
groundedtipfor1min.Aftergoingbackintotunneling,STMimagesof
thesameareanolongershowthebrightfeatureassociatedwithDefect
6,asdisplayedinFig.4b.Additionally,dI/dVspectrarevealatransition
from the characteristic defect peak to a spectrum with only the
backgroundgraphenedensity-of-states,asshowninFig.4c.Thispro-
cedure can be consistently applied to many defects as well as with
differenttypesofSTMtips,asshowninFig.4d-fandFigureS10.To
ensurethatthechangesinthetopographyandspectraarenotdueto
tip-changes, for each manipulated defect, we investigate a nearby
referencedefectwithin20–50nmofthemanipulateddefect,asshown
in Figure S11. No changes are observed for the reference defects,
confirmingthatourtipmanipulationprocedureishighlylocalizedand
tip-changes are not responsible for our observations. Our ability to
controlthechargestateofNVdefectswithoutaffectingneighboring
defectsascloseas20nmawayisanorderofmagnitudeimprovement
onthelocalizationofsuchmanipulationpreviouslyreported,asdis-
cussedfurtherinSupplementaryNote3.
Aplausiblescenarioforthephysicalprocessoccurringduringthe
tip-inducedmanipulationprocedureisacharge-stateconversionfrom
NV-toNV0.Whenalargepositivebiasisappliedtothesamplerelative
tothetip,theelectricfieldcanpushanelectronoutofthenear-surface
NV-defectunderthetip,convertingthechargestatefromNV-toNV0.
ThegroundstateoftheresultingNV0defectisexpectedtobemore
than2eVbelowtheFermienergy35,36,49,50wherethegraphenedensity-
of-statescontributionislargecomparedtothedefectdensity-of-states
(asinFig.S5),whichisconsistentwiththeabsenceofadistinctpeakin
dI/dVspectraafterthetip-inducedmanipulationprocess.Asshownin
Fig. 4, we have found that this manipulation procedure works with
bothstandardWtipsaswellasplasmon-resonantAu-coatedWtips,
opening the door to future studies combining STM and near-field
opticalspectroscopy.Finally,wenotethatwhilereversalofthecharge
statetoitsoriginalconfigurationisinprinciplefeasible,itischallen-
gingtodemonstrateinthiscontext.Theprocessrequirestheappli-
cation of a large negative sample bias, which interacts with the
graphene layer (as shown in Fig. S14f), thereby preventing clear
observation of the reversecharge state transition. We note that we
havealsotriedthisprocedureunderlaserilluminationasdiscussed
furtherinSupplementaryNote4;whilethedefectstatepeakdoesn’t
change under illumination (Fig. S13), the charge state could not be
reversedwithinthestablebiasrange(Fig.S15).
Insummary,wepresentthefirstSTMimagingandcharacteriza-
tion ofindividual NV--centersin diamond, measuring more than40
NVswithourtechnique.Weobtainsupportingevidencethatweare
indeedimagingNV--centersusingourin-situphotoluminescenceset-
up and by comparing samples known to have different NV- depths.
Additionally,usingSTMtip-inducedgating,wedemonstratetheability
to consistently manipulate individual defect charge states. The
manipulationwascarriedoutwithAu-coatedtipswhichareknownto
have a plasmon-resonant response51–55. This is a statistically robust
procedure whichwe have successfully carried out on morethan10
defects.SimultaneouslycorrelatingopticalsignalswithindividualNVs
measured by STM remains a goal for follow-up work. New sample
fabricationtechniquesarebeingdevelopedcurrentlywhichmayallow
forthinNV-enricheddiamondmembranestobeplacedonplasmon-
resonant metallic substrates. As a result, future STM tip-enhanced,
near-fieldPLstudiescouldpotentiallyprovidefurtherconfirmationof
theindividualdefectsandchargestates studied.Giventhepromise
that NV centers hold1–6,9,35,36,43,56, the findings described in this work
markacriticaladvanceintheatomic-scaleunderstandingandengi-
neeringofsolid-statequbits.Futurepossibilitiesinvolveintegrationof
optically-detected magnetic resonance capabilities with STM57–60 to
characterizeandengineerqubitperformanceofindividualdefectsat
theatomic-scaleintechnologicallyoptimaldiamondhostsamples.

Methods
STMmeasurements
Our STM measurements were performed in a Unisoku USM1200LL
instrumentat77KinultrahighvacuumwithaFemtoDLPCA-200pre-
amplifier. dI/dV measurements are taken with a Stanford Research
Systems SR830 lock-in amplifier with a modulation frequency of
911.1Hz. The sample orientation is determined visually, enabled by
optical access to the sample when mounted in the Unisoku
USM1200LL STM. STM tips are electrochemically etched in NaOH
solutions;uncoatedWtipsareannealedinvacuumbyelectron-beam
heating before measurements. To prepare Au-coated tips, we use
electron-beamevaporationtocoat80nmofAuontoetchedWtips;
the coated tips do not have to be annealed in vacuum due to the
chemicalinertnessofAu.FE-SEMimagesinFig.S16showthatthetips
remainsharpaftertheAucoatingprocedure.Alldatashowninthe
manuscript,withtheexceptionofthelabeledplotsinFigs.S12,S13,
andS15,weretakenwithoutlaserillumination.

Samplepreparation
Type-1b diamond crystals are purchased from ElementSix and are
subsequentlyfinepolishedandstrain-released,yieldingaroughnessof
<0.3nm.Thestrain-releasestepistoremovesurfaceresidualstrain
afterfinepolishingandisperformedduetothepotentialforstrainto
affecttheexcited-statestructureofNVdefects61–63.Samplesarethen
irradiatedwithanelectronbeamat2MeV,withadoseof2×1016/cm2.
Followingirradiation,thesamplesaretri-acidcleaned63,64andannealed
at1200°Cfor2h.WeestimateadefectdensityofoneNV-detectable
bySTM(within~2-2.5nmofthesurface)inevery25×25nm2areausing
thesesamplepreparationparameters,withintheapproximaterange
expectedbasedonthepreviouslyreportedresults65–67.Theelectronic-
gradecrystalisnitrogen-implantedwithadoseof5×1014ions=cm2of
15N+ionswith3keVenergyand15°incidenceangleatroomtempera-
ture. The crystals are implanted in a patterned grid yielding ≈16%
coverage and a target depth of 5nm. Our SRIM simulations and
literature64,68–72 yield an estimated density of about one NV- every
20×20nm2 laterally, with the implantation energy used embedding
them~5nmbelowthesurface.Wenotethatthetri-acidcleaningpro-
cess performed on this sample after implantation cleans off
amorphouscarbon64atthesurface,butlikelydoesnotetchawaya
sufficientportionofthediamonditselftobringtheNV-denseregion
enoughtothesurfaceforSTMimaging,basedonourresultspresented
inFig.S9.
Monolayer graphene, grown by chemical vapor deposition on
both sides of a Cu foil, is purchased from ACS Material, LLC. Poly-
methylmethylacrylate(PMMA,CH O )isspin-coatedontoonesideof
5 8 2
graphene/Cu,followingwhichO plasmaetchingisusedtoremove
2
graphenefromthebackside.TheCuisnextetchedawayinammo-
niumpersulfatefor8h;thesolutionisthendilutedandthePMMA/
graphenefilmfloatinginthediluteammoniumpersulfateisscooped
ontodiamondsubstrates.Afterbakingoutthesampleat120°Cfor
1hour,thePMMAisremovedbyimmersionofthesampleinacetone
for6h.Thesampleisthenannealedfor3hat600°Cinvacuumto
remove residues. Following this, the graphene/diamond sample is
loadedintotheSTMprep-chamberthroughair,afterwhichitisagain
annealedat450°Cfor3hinultrahighvacuumbeforetransferringinto
theSTMformeasurements.

PhotoluminescenceandRamanmeasurements
Room-temperature photoluminescence (PL) and Raman spectra are
takenwithaNanophotonRaman11confocalmicroscopeand532nm
excitation.A100× objectivelensisused,yieldingadiffraction-limited
beamspot.PLandRamanspectrafromin-situmeasurementsatthe
STMtip-samplejunctionaretakenat77K,usingaStellarNetHyper-
NOVAspectrometer.Excitationisat532nmwithaThorlabsDJ532-40
DPSSlaser,andthebeamdiameterisapproximately5μm.

Dataavailability
AlldatapresentedinthismanuscriptareavailablethroughtheIllinois
DataBankathttps://doi.org/10.13012/B2IDB-0172361_V1.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors used a new method to conduct STM research on diamond and identified more than 40 defects as NV centers based on dI/dV conductance spectra. The density-of-states mapping of two-lobed wavefunctions aligned along the [111] crystallographic direction supports an anisotropic defect in diamond. However, in view of the following issues, we believe that the identification of NV is not definitive. The following issues need to be properly addressed before publication.\n1. According to the “Sample preparation” part in methods, the NV depth is expected to be ~ 5 nm, while the STM topographies of NV- in fig.2, 3 and 4 all show a size smaller than 0.5 nm. This counterintuitively indicates an atomic spatial resolution with a tip-sample distance at the scale of 5 nm. Besides, the tunneling probability from the tip to the NV also needs an explanation, since it decays exponentially with an increasing distance and should be neglectable under such a distance.\n2. In fig.2 f, the authors show a histogram from 45+ defects, counts from -1 eV to 0 eV are all classified into the ground state of NV-. Could the authors explain why the measured energy levels are so widely dispersed? P.S. the refs. of NV- and P1 energy levels (ref. 24, 25 and 26, 27) seem mislabeled.\n3. In fig.3, the directionality of NV centers should be checked with more reliable methods. For example, polarized excitation laser can be used to identify the NV axis direction (refer to section IV of supplementary information of Nature Photonics 18, 230–235 (2024)).\n4. In fig.4, the authors claim that NV centers’ charge state is manipulated. The straightest way to verify is to measure the photon counts before and after the manipulation (refer to NATURE COMMUNICATIONS 12, 2457 (2021)). It would really help to enhance the reliability of the identification of NV if such data are displayed.", "reviewer_2": "The submitted article by Raghavan et al. presents a compelling study demonstrating atomic-scale imaging and charge-state manipulation of nitrogen-vacancy (NV) centers in diamond using scanning tunneling microscopy (STM) with a conductive graphene capping layer. By imaging the graphene–diamond heterostructure, the authors observe the characteristic honeycomb lattice of graphene and defect signatures attributed to sub-surface NV and P1 centers. These observations are supported by in-situ optical photoluminescence (PL) spectroscopy. Using local differential conductance (dI/dV) spectroscopy, the authors could observe defects that exhibit signatures which can be attributed to NV⁻, as well as P1 centers. The authors also demonstrate the capability to image the two-lobed spatial structure of NV⁻ wavefunctions along the [111] crystallographic direction, and demonstrate tip-controlled charge-state switching from NV⁻ to NV⁰ using a positive sample bias.\nThe article is well written, and the method and results can be of high value to the scientific community. However, before recommending the article for publication, the following points should be addressed:\n1. Tunneling Mechanism\nThe nature of the electronic coupling between the buried defects and the STM tip, mediated through the graphene layer, remains insufficiently explained. Specifically, the manuscript should clarify the nature of the tunnelling current observed in this study. Additionally, any role of band bending, interface dipoles, or charge transfer effects at the graphene–diamond interface should be discussed.\n2. Depth Sensitivity and Lateral Resolution\nA critical aspect of the study is the ability to detect sub-surface defects via STM. The authors should provide an estimate of the depth sensitivity of their method, i.e., the depth of the defects from which they expect a detectable dI/dV signal. Furthermore, the relationship between defect depth and lateral resolution should be addressed. Are there measurable trends in the apparent size or symmetry of the observed NV features (e.g., contrast, sharpness, lobe spacing) that correlate with defect depth? Such analysis should support the sub-surface origin of the features.\n3. Role of Laser Illumination and the Laser-STM Setup\nThe authors mention, that the STM measurements were carried out in a newly developed laser-STM system. However, the importance of this system is not clearly stated. According to the sample preparation the utilized samples should have a homogeneous distribution of NV centers, so the in-situ PL measurements would not give any additional information on the presence of NV centers, compared with PL measurements outside of the STM. Furthermore, it is not stated in the manuscript, if laser-excitation of NV centers during the STM measurements is performed, and/or required for the described measurements. This should be clarified in the text. If laser excitation is used during spectroscopy, any effect of the illumination on the dI/dV spectra (e.g., peak shifts or intensity modulation) should be reported. Control experiments with and without laser illumination could provide important context for interpreting the tunnelling signals.\n4. Defect Density and Statistical Analysis\nThe manuscript reports imaging of approximately 45 defects across two type-Ib diamond substrates, but lacks quantitative analysis of defect densities. The authors should provide the average areal densities for NV and P1 centers detected in STM. This should be compared with expected densities based on the utilized samples. Additionally, it would be informative to discuss the spatial distribution of defects, e.g., whether clustering is observed or whether the defects seem uniformly distributed.\n5. Sample Set and Preparation Parameters\nThe authors state that three diamond samples were used, two nitrogen-rich type-Ib diamonds and one electronic-grade sample, but do not fully explain why two similar samples were required for the main experiments. More detail should be provided regarding the estimated NV and P1 concentrations in each sample, as derived from irradiation and annealing parameters, as well as supporting EPR or optical measurements.\nFurthermore, the statements about the implantation energy, and depth of the electronic-grade crystal(s) are not very detailed. The authors should clarify the sample preparation method of these samples, as well as the motivation for choosing these parameters. Was the NV fluorescence of the electronic-grade sample also verified in-situ in the STM? If NV⁻ centers (even at lower density) were present optically but not detected in STM, this discrepancy deserves a more detailed discussion.\n6. Tip-Induced Charge-State Control\nThe demonstration of bias-induced conversion between NV⁻ and NV⁰ should be better contextualized with respect to a similar previous study (Bian, K., et al., Nature Communications, 12(1), 1–9. https://doi.org/10.1038/s41467-021-22709-9) which demonstrated reversable charge-state control of single NV centers using AFM tip gating. The authors should describe the unique aspects of their approach, and discuss how their observed charge-state dynamics compare to those prior results. Furthermore, they should elaborate on the choice of +5 V sample bias: was this empirically determined? Can a switching threshold voltage be defined? Has laser excitation been attempted to aid reverse the NV charge transition?\n7. Comparison Between NV and P1 Defects\nThe manuscript would benefit from a direct comparison between the STM features attributed to NV centers and those assigned to P1 centers. What spatial topographies do the P1 centers exhibit, and how do they compare to the NVs two-lobed features?\n8. Literature Context and Citations\nThe authors should be aware of a similar graphene-diamond heterostructure system, which has recently been demonstrated to show NV coherence enhancement, and should be referenced in their work (Hao, Y., et al., National Science Review, 12(5). https://doi.org/10.1093/NSR/NWAF076)\nConclusion and Recommendation\nThis manuscript presents important advances in atomic-scale imaging and charge-state control of quantum defects in diamond. However, to fully support its conclusions and improve clarity for a broad readership, the authors must address the above points through additional discussion, clarification, and where possible, supporting data. With these revisions, the study will be a valuable contribution to the fields of quantum sensing and scanning probe microscopy."}, "author_response": "We thank reviewer #1 for thoroughly reviewing our manuscript and providing salient points for consideration. We answer each point in detail below.\n1. According to the “Sample preparation” part in methods, the NV depth is expected to be ~ 5 nm, while the STM topographies of NV- in fig.2, 3 and 4 all show a size smaller than 0.5 nm. This counterintuitively indicates an atomic spatial resolution with a tip-sample distance at the scale of 5 nm. Besides, the tunneling probability from the tip to the NV also needs an explanation, since it decays exponentially with an increasing distance and should be neglectable under such a distance.\nWe would like to distinguish between the two types of samples discussed in our manuscript. One is type-1b and the other is electronic-grade sample. All the defects shown in Fig. 2-4 were measured in type-1b samples which have a relatively large density of NVs distributed uniformly throughout the sample, to the extent that it is reasonable to expect that there is a sizeable density of defects even within 1 nm of the surface in these type-1b samples.\nThe description of NV- depth of ~ 5 nm (mentioned by the reviewer above) is for the case for the other type of sample: electronic-grade. The electronic-grade sample has an extremely low density of natural defects. In this nearly NV-free sample, we applied 15N+ ion irradiation with the conditions which will implement N substitution at around 5 nm depth according to our SRIM simulations and the literature1–3. In contrast to the type-1b samples, we detect no defects in this irradiated electronic-grade sample (Fig. S9b), showing that the probing depth of our STM imaging is less than 5 nm.\nDetails clarifying the implantation parameters for the electronic-grade sample have now been added to the Methods section of the revised manuscript.\nAs for the expected depth at which defects can be probed with STM, literature shows this value to be approximately up to 2-2.5 nm. Here are a few examples aside from ones already discussed in the original manuscript, which we’ve discussed further in the revised manuscript in the newly added Supplementary Note 2:\n• Nguyen, et al. (2018)4 use STM to study Se vacancy defects in the transition metal dichalcogenide PdSe up to 2 nm beneath the surface\n2\n• Kotzott, et al. (2021)5 use STM to study Ag and Ge impurities up to 7 layers, corresponding to 2.5 nm, beneath the surface of copper\n• Wong, et al. (2015)6 use STM to study subsurface hexagonal BN (hBN) defects; data suggests defects up to ~2-2.5 nm below the surface are accessible\n• In another relevant work by Britnell, et al. (2012)7, electron tunneling through insulating layers of BN up to ~1.5 nm in thickness is examined\nRegarding the comment on the lateral size of the NVs in the topographies, it is important to note that the lateral resolution of STM is not depth dependent for defects embedded in insulators. As there is almost no screening electron cloud near defects, how a defect below the surface appears in STM depends on the details of the wave function in the z-direction. Hence, one can often observe atomically resolved small lateral structure of sub-surface defects, as seen from the case of Ni defect at the CuO\n2\nplane below the insulating BiO and SrO planes in Bi Sr CaCu O 8 and Te vacancies in\n2 2 2 2 8\na topological insulator Bi Te at a few-layers depth below the surface9. Also, another\n2 3\nstudy shows that the sub-surface defects in hBN from different layers only show difference in their spectral weight, not in their lateral size6.\nIn summary, the main type of samples used in our measurements were type-1b diamonds which are expected to have homogeneously distributed defects, including in the region from the surface to ~2.5 nm below the surface, which has been shown in the literature to be accessible by STM. Moreover, as is shown in the literature, the lateral resolution for the sub-surface defects in STM is not determined by distance from the surface for the case of insulators.\n2. In fig.2f, the authors show a histogram from 45+ defects, counts from -1 eV to 0 eV are all classified into the ground state of NV-. Could the authors explain why the measured energy levels are so widely dispersed? P.S. the refs. of NV- and P1 energy levels (ref. 24, 25 and 26, 27) seem mislabeled.\nWe agree that further clarification here would be useful. In the new version of the paper we show a comparison between the band edges at different areas of one of our type-1b samples ≈ 150 𝜇𝑚 apart in the Supplementary Information as Fig. S7. We also include it as Fig. R1 below. As can be seen, the band edges with respect to the Fermi energy can shift up to 0.5 eV or so in certain regions of the samples likely due to local potential variation in the sample. This kind of potential variation is often seen in low density of states materials like graphene and topological insulators. We attribute the relatively long tail in the histogram to a few areas of the sample where the band edges may have been particularly shifted. Since the defect energy level is determined from the energy difference to the band edges10,11, this variation of the band edges can also cause changes in the absolute energies of the defect peaks. In principle, it would have been great to have histogram of band edges also, but when taking these large energy range spectra to find the band edges, the tip can interact with the graphene layer leading to instabilities and so we refrained from taking such spectra at each defect individually.\nWe also thank the referee for pointing out that the references in Fig. 2f were mistakenly mislabeled. We have now fixed the labels accordingly.\nFig. R1. dI/dV spectra at two positions approximately 150 𝜇𝑚 away from each other showing variations in the band edges with respect to the Fermi energy\n(V = -4.60 V, I = 10 pA for position 1 and V = -4.50 V, I = 70 pA\nBias Setpoint Bias Setpoint\nfor position 2).\n3. In fig.3, the directionality of NV centers should be checked with more reliable methods. For example, polarized excitation laser can be used to identify the NV axis direction (refer to section IV of supplementary information of Nature Photonics 18, 230–235 (2024)).\nThe paper by Ji, et al. (2024)12 referenced in this comment (and which we’ve now added to the revised manuscript) demonstrates an interesting application of super-resolution STORM microscopy with blinking NV centers. Comparing to our study however, the paper by Ji, et al. (2024)12 uses electronic-grade diamond crystals with a very low number of defects in which single NVs which are ~200 nm apart are created by a laser writing technique and measured in confocal microscopy setups. Since their NV density is compatible with confocal microscopy, the NV polarization can be determined individually by optical techniques.\nIn our work, we use type-1b crystals which have significantly higher NV densities and are not compatible with confocal microscopy for determination of individual defect polarization. We work with this higher defect density environment as it helps us understand the effect of nearby defects and the spatial extent of charge state control in situations similar to those which may be encountered in future technological applications. In this dense NV limit, our method is advantageous in providing information on the directionality of the individual NV with atomic-scale mapping.\nStill, in response to the referee’s suggestions, we made efforts to obtain tip-enhanced photoluminescence (PL) at <10 nm lateral resolution with gold tips, which is described in our response to the next comment, although we eventually found that future development of sample fabrication technology is required before this can be implemented successfully.\n4. In fig.4, the authors claim that NV centers’ charge state is manipulated. The straightest way to verify is to measure the photon counts before and after the manipulation (refer to NATURE COMMUNICATIONS 12, 2457 (2021)). It would really help to enhance the reliability of the identification of NV if such data are displayed.\nThe paper by Bian, et al. (2021)13 as well as a follow-up work by Zheng, et al. (2022)14 led by the same group of Prof. Ying Jiang at Peking University showcase a creative technique to achieve charge-state control of individual NV centers. While we referenced these works in our original manuscript, we’ve now included a further discussion in the revised manuscript as Supplementary Note 3 to distinguish our current work from the previous publications. These previous papers used an electronic-grade diamond substrate with extremely low NV center density such that confocal microscopy can be used and still see a change in photon counts when an individual NV charge state is manipulated.\nOur work, on the other hand, primarily uses type-1b diamond crystals with much higher defect densities. While the NVs in the previous works were ~200+ nm apart, the NVs in our work are less than 20 nm apart, at least an order of magnitude smaller in scale. This high density of NV- defects means that photon counts measurement from confocal microscopy cannot be done directly due to its spatial resolution limitation. This is the reason why we showed the charge state manipulation from atomic scale spectroscopy instead of confocal microscopy.\nNonetheless, for further verification of the charge state manipulation from the photon count, as suggested by the referee, we attempted to obtain PL at nm spatial resolution from tip-enhancement of the local laser electric field. Tip-enhanced PL (TEPL)15–18 is a technique which utilizes a nanocavity between a highly conductive tip and a highly conductive substrate, and is known to potentially allow for spatial resolution of < 10 nm. To this end, we approached a gold tip to the NVs in our type-1b diamond crystals. In new supplementary data added to the revised manuscript as Fig. S12 and displayed in Fig. R2 below, we find that the PL signal doesn’t change noticeably when the gold STM tip is on an NV defect, 50 nm away laterally, and withdrawn 500 nm away in Z from the sample. This is very likely due to the insulating nature of the diamond substrate which doesn’t allow to form an effective nanocavity with the tip for the local enhancement of PL. To make TEPL work, we would need a gold substrate and gold tip to form an effective nanocavity, and a sufficiently thin (≤ 5 nm) diamond membrane with NV centers which can sit in the nanocavity. Such fabrication techniques are not yet available but are the subject of ongoing research19.\nIn summary, despite technical limitations in being able to see a change in photon counts, we have been able to present consistent topographic and spectroscopic evidence for atomic-scale charge state manipulation. In addition, we have presented a platform which can enable measurements of contrast in photon counts from individual NVs with nm-resolution once the sample fabrication techniques evolve further in the coming years.\nFig. R2. Comparison of PL in tunneling on a defect, in tunneling 50 nm laterally away from a defect, and with the tip withdrawn 500 nm away from the sample. Tunneling was with an Au-coated W tip at V = -500 mV and I = 70 pA; laser power is 75\nBias Setpoint\n𝜇𝑊 , wavelength is 532 nm, and beam spot size is ~4 𝜇𝑚 . There is no discernible\ndifference in the PL spectra likely due to the lack of tip-enhanced PL because of the\ninsulating nature of the diamond substrate.\nWe thank reviewer #2 for the thorough comments on our work and providing important points for consideration. We respond to each point in detail below.\n1. Tunneling Mechanism\nGiven that the use of a monolayer graphene layer is an important component of our technique, we agree that further discussion would be useful, and we have now added the discussion and references below to the revised manuscript as Supplementary Note 1. There are several examples in the literature which discuss STM tunneling through graphene, including:\n• Qiu, et al. (2021)20, study the van der Waals magnetic insulator CrI\n3\nthrough a monolayer of graphene\n• Wong, et al. (2015)6 and Velsaco, et al. (2016)21 study defects in hexagonal BN through a monolayer of graphene\n• Gonzalez-Herrero, et al. (2016)22, Tesch, et al. (2016)23, and Leicht, et al. (2014)24 study STM tunneling into metals through monolayer graphene\nIn a few of these papers, density functional theory (DFT) modeling and calculations are performed to understand the nature of tunneling through graphene. For graphene on Cu as an example, Gonzalez-Herrero, et al. (2016)22 perform DFT calculations to show how the graphene 𝜋 bond orbitals from graphene have a significantly less extended wavefunction out-of-plane than the Cu surface states, thus allowing for STM to probe the electronic structure of the Cu underneath. A similar reasoning may apply for tunneling through graphene into diamond defects, however given that ours is primarily an experimental work, we feel it would be more suitable to leave the theoretical modeling of this mechanism to future studies.\nRegarding the effect of charge transfer, we note that Hao, et al. (2025)25, a work which was brought to our attention by reviewer #2 in comment 8 below, do study the effect of charge transfer between diamond and graphene as a function of diamond-graphene distance from 2 Å to 10 Å in their paper using DFT. In the cited work, it was demonstrated that the coherence time of NV centers in diamond could be increased more than twofold by depletion of unpaired surface electron spins by the graphene. Their DFT works shows this depletion becomes effective for graphene-diamond distances less than 3-4 Å.\nWe thank the referee for this question. This discussion has now been added to Supplementary Note 1.\n2. Depth Sensitivity and Lateral Resolution\nWe thank the reviewer for this important question. As for the expected depth at which defects can be probed with STM, literature shows this value to be approximately up to 2-2.5 nm. Here are a few examples aside from ones already discussed in the original manuscript, which we’ve discussed further in the revised manuscript in the newly added Supplementary Note 2:\n• Nguyen, et al. (2018)4 use STM to study Se vacancy defects in the transition metal dichalcogenide PdSe up to 2 nm beneath the surface\n2\n• Kotzott, et al. (2021)5 use STM to study Ag and Ge impurities up to 7 layers, corresponding to 2.5 nm, beneath the surface of copper\n• Wong, et al. (2015)6 use STM to study subsurface hexagonal BN (hBN) defects; data suggests defects up to ~2-2.5 nm below the surface are accessible\n• In another relevant work by Britnell, et al. (2012)7, electron tunneling through insulating layers of BN up to ~1.5 nm in thickness is examined\nRegarding the depth of the defect and lateral size of its topographic feature, we would like to note that the lateral resolution of STM is not depth dependent for the defects embedded in insulators. As there is almost no screening electron cloud near defects, how a defect below the surface appears in STM depends on the details of the wave function in the z-direction. For example, an STM study shows that the sub-surface defects in hBN from different layers only show difference in their spectral weight, not in their lateral size6.\nRegarding measurable trends between the depth of the defect and spectroscopic and topographic features, we have also now added Fig. S6 reproduced as Fig. R3 below where we show a comparison of defects with significantly different peak heights at the NV- defect state energy near -300 meV. The corresponding topographies show that Defect 8, which has a sharper peak in the dI/dV spectrum, also shows a more pronounced topographic contrast compared to Defect 9. This data provides additional evidence that we are accessing defects in distinct subsurface layers. As we tunnel into defects in deeper layers, the dI/dV defect peak height gets weaker and the topographic contrast of the defect gets fainter. This is similar to observations in the literature4,6.\nFig. R3. Depth dependence of defect spectra\nand corresponding topographies. a, dI/dV\nspectra from two defects labeled Defect 8 and\nDefect 9 showing significant differences in the\nheight of the defect peak. This difference likely\noccurs due to the different depths of the defects.\nDefect 8 spectrum was taken from V = -900\nBias\nmV and I = 100 pA; Defect 9 spectrum was\nSetpoint\ntaken from V = -1.00 V and I = 70 pA.\nBias Setpoint\nFor comparison, the spectra are normalized by\ntheir initial point. b-c, Corresponding\ntopographies of Defect 8 and Defect 9. Defect 8\ntopography is at V = -900 mV and I =\nBias Setpoint\n100 pA and Defect 9 topography is at V = -\nBias\n1.00 V and I = 70 pA. It is evident that\nSetpoint\nDefect 8, with the more intense dI/dV peak also\nshows a larger contrast in the topography\ncompared to Defect 9. Both topographies are\nset to the same full-scale height.\n3. Role of Laser Illumination and the Laser-STM Setup\nWe see how the application of the laser in this context could have been unclear and have amended the text accordingly. The instrument was set up such that in-situ PL could be done, especially with a view toward STM tip-enhanced measurements of spatially-resolved PL at the nanoscale to corroborate topographic and spectroscopic data. However, this requires a metallic substrate which allows for plasmonic enhancement of the laser electric field so it couldn’t be used with the insulating diamond crystal platform we are working with in this paper. Since the distribution of defects in these type-1b samples is expected to be homogeneous, we only use the in-situ PL measured as confirmation that there are NV centers in our scan region, although we don’t have reason to expect that the tip will land in an area without NVs given the homogeneous PL seen in measurements outside the STM before inserting our samples into the chamber.\nWhile a change isn’t expected with a continuous wave laser at the power densities we are using, we have still tried measuring dI/dV spectra on a defect with and without 532 nm laser illumination. The spot size is ~4 𝜇𝑚 and power is ~170 𝜇𝑊. As shown in Fig. R4 below, no systematic changes in the dI/dV spectra are found beyond the range of fluctuation. This new figure has also been added to the Supplementary Information of the revised manuscript as Fig. S13. Since an important goal of our work is to allow for atomic-scale flipping of the NV charge state on-demand, we didn’t pursue optical charge state manipulation techniques, which would affect all defects within micron-scale areas. All the STM data presented in the original manuscript were taken without laser illumination; this is now clarified in the Methods section of the revised manuscript.\nFig. R4. dI/dV spectra (V = -1.00 V, I = 70 pA) with and\nBias Setpoint\nwithout laser illumination showing no systematic change beyond\nfluctuations between spectra taken with the laser on and off. Laser\nwavelength is 532 nm, beam spot diameter is ~4 𝜇𝑚, and power at\nthe sample is 170 𝜇𝑊.\n4. Defect Density and Statistical Analysis\nAs our focus is on NV centers, we primarily searched for these defects by scanning at negative biases where the NVs are visible. We show in added additional supplementary data as Fig. S8 and reproduced in Fig. R9 below that P1 centers aren’t visible at these biases. As a result, we aren’t able to provide an accurate estimate for the P1 density from STM. For NVs, we can make a better estimate. However, a primary challenge with these measurements is to find large areas where the graphene is adhered strongly to the diamond such that stable data can be taken while keeping the tip stable for topography, spectroscopy, and charge state manipulation. In addition, despite extensive annealing, portions of the sample still have chunks of polymer residues from the wet transferring process.\nAs shown in the example in Fig. R5 below, we occasionally find 20×20 𝑛𝑚2 areas which don’t have any visible NV centers (Fig. R5a), along with other areas which may have NV centers but are obscured by unstable graphene adhesion (Fig. R5b), and other areas which may have NVs but also have polymer residue on top (Fig. R5c) blocking STM detection of any potential NVs in those spots. Taken together with areas such as those shown in Fig. S9a of the revised manuscript, which is a 40×20 𝑛𝑚2 area with three defects, we can estimate that there may be one NV visible to STM within every 25×25 𝑛𝑚2 area.\nFig. R5. a-c, Derivative images of graphene on diamond showing a clean, scannable area without defects (a), area with spots showing unstable graphene adhesion (b), and an area with a large clump on the top left likely from the wet transfer process (c). All scans shown in this figure are at V = -1.00 V and I = 70 pA.\nBias Setpoint\nWe compare this to literature, including Acosta, et al. (2009)26, Ishii, et al. (2022)27, and Bolshedvorskii, et al. (2023)28 which study the NV- density as a function of various irradiation and annealing parameters. One of the samples in Acosta, et al. (2009) yields a 10 ppm concentration of NV- centers. This corresponds to a density of one NV- within 2.5 nm of the surface in every 15×15 𝑛𝑚2. However, this study used 200 times the irradiation dose, but less than half the N concentration (≤ 100 𝑝𝑝𝑚 instead of 200 𝑝𝑝𝑚), and lower temperature annealing compared to our work (1050 °𝐶 instead of 1200 °𝐶). If we take NV density to scale linearly with irradiation dose and N concentration, and take our annealing procedure to yield double the number of NVs, then we may expect a detectable NV to appear every 100×100 𝑛𝑚2. But, in the same paper, it is found that the NV density doesn’t necessarily scale linearly with dose; for example, comparing two of their samples, a 2.5 times lower dose found only a factor of 1.5 decrease in the NV density.\nThere are also significant differences between results from different groups. For example, Bolshedvorskii, et al. (2013) use a 1.25 times larger dose than Acosta, et al. (2009) but only 800 °𝐶 annealing instead of 1050 °𝐶, and yet find an NV- density 3.7 times larger. Given these variations, we can conclude based on our sample preparation parameters we can expect a detectable NV somewhere between about every 15× 15 𝑛𝑚2 to every 100×100 𝑛𝑚2 and that our observation of an approximate density of a detectable NV every 25×25 𝑛𝑚2 falls within this range. We have now noted our density estimate and relevant literature in the revised manuscript.\n5. Sample Set and Preparation Parameters\nWe used two type-1b samples only to ensure that the data was reproducible; we’ve now clarified this in the revised manuscript. We include here our PL measurements, demonstrating that the NV density is very similar in both type-1b samples as shown in Fig. R6 below.\nWe discuss defect density in the type-1b samples in the response to comment 4 above. For the electronic-grade sample, we used 3 keV implantation of 15N+ ions, a 15° incidence angle, and a dose of 5×1014 𝑖𝑜𝑛𝑠/𝑐𝑚2, with irradiation performed at room temperature. We’ve added these details to the Methods section of the revised manuscript. Based on the available literature2,3,29–31 and our SRIM simulations, we expect NVs to be positioned close to 5 nm below the surface. Combining the results from several previous studies29–32 along with our implantation parameters, we estimate that an NV to would be present in our sample within every 20×20 𝑛𝑚2 area laterally.\nOur motivation in choosing the implantation parameters used here was to understand the depthwise extent to which NVs can be probed with STM. Given that, as shown in Fig. S9 of the revised manuscript, our STM measurements don’t detect any defects in the electronic-grade sample, we can conclude that the probing depth of our technique is less than 5 nm. We note here that while the tri-acid cleaning procedure following implantation and annealing removes amorphous carbon2, it likely doesn’t etch the diamond surface itself sufficiently to bring the NV-dense region close enough to the surface for STM imaging.\nOptically, the NV fluorescence was overall about two orders of magnitude weaker in the electronic-grade sample compared to the type-1b samples, as shown in Fig. S9 of the revised manuscript. Given the low detector and collection efficiency of our STM setup (e.g. transmission of each ZnSe objective and eyepiece lens with respect to visible light is only ~50%), along with limitations on the laser power which can be used inside our STM chamber, it wasn’t possible to get a clear PL spectrum from the electronic-grade sample in-situ. Hence, the comparison shown in Fig. S9d of the revised manuscript is from PL spectra taken in ambient conditions on a Nanophoton Raman11 spectrometer.\nFig. R6. PL measurements comparison on the two different type-1b samples\nused in this work; laser power is 0.65 mW, wavelength is 532 nm.\n6. Tip-Induced Charge-State Control\nThe paper by Bian, et al. (2021)13 as well as a follow-up work by Zheng, et al. (2022)14 led by the same group of Prof. Ying Jiang at Peking University showcase a creative technique to achieve charge-state control of individual NV centers. While we referenced these works in our original manuscript, we’ve now included a further discussion in the revised manuscript as Supplementary Note 3 to distinguish our current work from the previous publications. These previous papers utilize an electronic-grade diamond substrate with extremely low NV center density such that confocal microscopy can be used and still see a change in photon counts when an individual NV charge state is manipulated.\nOur work, which uses type-1b samples with significantly higher defect densities, distinguishes itself in several ways. One is that by using STM we can image the individual nitrogen-vacancy center wavefunction at the atomic scale for the first time. Secondly, while the NVs in the previous works were ~200+ nm apart, we show that it is possible to manipulate individual NVs which are less than 20 nm apart, an order of magnitude smaller in scale which is much below the spatial resolution limit of confocal microscopy identification of individual NVs. The higher defect density environment we work with helps us understand the effect of nearby defects and the spatial extent of charge state control in situations which are akin to those which may be encountered in technological applications.\nRegarding the latter parts of this referee comment (“The choice of +5 V sample bias: was this empirically determined? Can a switching threshold voltage be defined?”), the choice of +5 V sample bias was determined empirically. We’ve found the threshold to be roughly above +2.5 V, however haven’t pursued finding an exact threshold value as it may also depend on the depth of the defect for example. The SPECS Nanonis SPM controller we use has voltage limits of -10 V to +10 V, but as shown in Fig. S14 of the revised manuscript, going close to 10 V can interact with and deform the graphene layer, so we used +5 V since it consistently allowed for charge state manipulation.\nIn addition, addressing the last part of this comment (“Has laser excitation been attempted to aid reverse the NV charge transition?”), we have also tried to use bias manipulation in the -/+ 5 V range with laser illumination to potentially aid in reversing the charge state transition. This configuration with laser illumination has similarities to the work by Bian, et al. (2021)13 and Zheng, et al. (2022)14, although those AFM-based works use much larger voltages above even 100 V. In our case, with the voltage ranges we use which are compatible with subsequent STM measurements, we don’t find an effect of the laser illumination in aiding reverse charge manipulation. We present this additional data in the revised manuscript as Fig. S15 and also display it in Fig. R8 below.\nFig. R8. Comparison of dI/dV spectra (V = -1.00 V, I = 70 pA) before and after\nBias Setpoint\ndifferent manipulation trials with and without laser illumination. The laser illumination is\nwith a 532 nm laser, ~4 𝜇𝑚 spot size, and 1.03 mW power.\n7. Comparison Between NV and P1 Defects\nAs seen from newly added Fig. S8, unlike NV defects, where the double-lobe structure tends to be visible directly in the topography (Fig. S8a), the P1 centers appear to show a single lobe feature (Fig. S8b). In addition, while NV center is only visible at negative bias, the P1 center is only visible at positive bias. These two topographic differences help us to distinguish between NV and P1 centers. This additional figure is also shown below as Fig. R9.\nFig. R9. a, Bias-dependent topographies and dI/dV spectrum from a NV- defect (Defect\n2 in manuscript); topographies are taken with I = 100 pA, and spectrum is from\nSetpoint\nV = +900 mV and I = 100 pA. b, Bias-dependent topographies and dI/dV\nBias Setpoint\nspectrum from a P1 defect, labeled Defect 10; topographies are taken with I = 70\nSetpoint\npA, and spectrum is from V = -1.30 V and I = 280 pA. While the NV- center is\nBias Setpoint\nvisible at negative bias only, the P1 center is only visible at positive bias. Moreover, the\nNV tends to show a double-lobed structure in the topography while the P1 topography\nshows only a single lobe. All scale bars in this figure are 1 nm.\n8. Literature Context and Citations\nWe thank reviewer #2 for bringing to our attention this new work by Hao, et al. (2025)25 published earlier this year. We’ve now added this reference in our revised manuscript with a brief summary of their findings. We note that the factor of 2-2.5 enhancement seen in the T coherence time of NV- defects with a graphene monolayer\n2\ncover would be an additional reason to further pursue studies of these heterostructures aside from the imaging opportunities opened up as shown in our work.\nConclusion and Recommendation\nWe thank reviewer #2 again for important comments which have helped us improve the manuscript. We feel that with these improvements the work is ready for publication in Nature Communications."}, {"round": 2, "reviewer_comments": {"reviewer_1": "This manuscript presents a compelling study on the atomic-scale imaging and charge-state manipulation of nitrogen-vacancy (NV) centers in diamond using STM. The technique and the results, particularly the wavefunction mapping and local charge-state control, are of significant potential interest to the community. While I have one major concern regarding the evidence chain, I believe it can be adequately addressed through clarification in the text.\nThe most significant issue is that the spectroscopic identification of NV centers and their STM characterization are not performed on the same diamond sample.\nElectronic-Grade Sample: As shown in Fig. 1, NV centers were created in this sample via ion implantation (~5 nm depth) and identified optically via their Zero-Phonon Line (ZPL). However, subsequent STM measurements on this sample did not detect any defect signals, which is consistent with the acknowledged STM probing depth limit of ~2.5 nm for defects in insulators.\nType-Ib Sample: The core STM data in Fig. 2-4 are acquired on high-nitrogen Type-Ib samples. The authors argue that the observed defects are NV centers based on their characteristic dI/dV spectra and two-lobed topography aligned with the [111] direction.\nWhile this is not a loop-hole free validation, the authors provide a strong statistical argument. The combination of the STM data from over 40 defects with the ensemble photoluminescence from the Type-Ib samples makes a persuasive, population-level case for the source of the STM signals.\nTo prevent any potential misunderstanding and to accurately represent the nature of the evidence, the authors should explicitly state that the identification of the STM features as NV centers is based on a statistical correlation across a large defect population, rather than on a one-to-one, simultaneous optical and STM confirmation on the same defect. This clarification should be prominently placed in the Abstract and/or the main results summary to ensure readers have the correct context from the outset.\nWith the above clarification added, the manuscript will be significantly strengthened and suitable for publication. The methodological advance and the high-quality data presented are valuable contributions to the field.\nRecommendation: Accept after Minor Revisions", "reviewer_2": "I thank the authors for their responses. Generally, I want to mention that I remain concerned that the method cannot guarantee the detection of subsurface NV centers without simultaneous optical confirmation at the single-defect level (which is beyond this current work). The presented data, while suggestive, does not conclusively rule out the possibility that the observed features originate from surface or other defects than NV centers. Given that the detection scheme lacks a theoretical model to underpin its fundamental mechanism, the evidence for subsurface NV detection remains indirect. The authors should therefore include a statement in the manuscript explicitly acknowledging this limitation.\nFurther comments on the text and figures:\n1.\nIn the abstract the authors state: ‘This work represents a significant advancement in the atomic-scale understanding and engineering of NV centers, paving the way for future quantum device development.‘\nThis sentence seems overstating, as there seem no ‘significant atomic scale understanding’ and also no useful ‘engineering’ in this work which can ‘pave the way for future quantum device development’. I think the main point of this solid work is about the nanoscale imaging of defects, and this sentence should be changed accordingly.\n2.\nThe sentence in the main text: ‘Critically, manipulation of individual NV charge states at atomic length scales has continued to pose a challenge’\nCould be extended to introduce why such manipulation is critical for the ‘development of quantum devices’.\n3.\nThe sentence in the main text: ‘The primary samples used in this study are type-1b diamond crystals with the (100) facet facing the surface normal direction. They were fine polished and strain-released7 before irradiation with an electron beam at 2 MeV, with a dose of 2×1016/cm2 to create the NV- defects.’\nIt should be added that this results in a high-density NV ensemble. Also, as it is mentioned here, what is the purpose/reason for strain releasing, and what is the resulting surface roughness of the diamond? (can also be added to the methods/SM)\n4.\nThe sentence in the main text: ‘Having confirmed the presence of NV- centers, we move on to atomic-scale STM characterization of individual defects.’\nHere would be a good occasion to mention that the further studies are all performed without laser illumination.\n5.\nThe sentence in the main text: ‘In Fig. S6, we show examples of defects with different dI/dV peak heights likely arising from their positioning in distinct subsurface layers below the surface. The corresponding topographies also show a sharper contrast between the defect and the surrounding lattice for Defect 8, which has a stronger dI/dV peak compared to Defect 9.’\nAs mentioned above in my main questions a more detailed discussion about the detection depth limitation should be included, also in the main text.\n6.\nThe sentence in the main text: ‘Using STM tip-induced gating, we are able consistently manipulate individual defect charge states, paving the path to atomic-scale engineering of spin-qubits.’.\nWhilst this statement is partly true, the ‘consistently manipulate ... paving the path to atomic-scale engineering’ is in my opinion slightly overstated, as it would be especially important to manipulate the NV center from its neutral towards its negative charge state.\n7.\nIn the methods part: ‘embedding them 5 nm below the surface’\nShould be better written as ‘∼5 nm’\n8.\nIn the supplement S3:\n‘This higher defect density environment is important in understanding the effect of nearby defects and the extent of charge state control in situations akin to those which could be encountered in technological applications.’\nIt should be stated clearly, why this higher defect density environment is important in understanding the effect of nearby defects\n9.\nIn the supplement S3:\n‘As shown in Fig. S12 however, there is no discernible change in the PL signal while positioning the Au STM tip on top of an NV- defect in tunneling, 50 nm away laterally in tunneling, or withdrawn 500 nm away from the sample. This is very likely due to the insulating nature of the diamond substrate which doesn’t allow for plasmonic enhancement’\nThe effect not being visible is more likely due to the above-mentioned effect of the NV ensemble, and the vanishing effect of the shallow NV centers, which are further quenched by the graphene capping layer.\n10.\nIn supplement S4:\n‘We confirm that under continuous wave illumination with power below 0.2 mW, no systematic changes could be found in the spectra on an NV- defect with and without illumination as shown in Fig. S13.’\nWhy was this low laser power chosen here?\n11.\nIn supplement S4:\n‘However, these previous works, using AFM and no graphene cover layer, use much larger voltages, even beyond 100 V’\nWhilst these works applied higher voltages, the charge state switching already occurred at lower voltages, and should be stated correctly. Also, the NV defects in the cited works were deeper than the ones in this manuscript, this charge state switching for experimentally relevant NV defects is apparently not possible with the STM method.\n12.\nIn Fig. S1:\nIt is unclear which diamond surfaces are shown here, if these are the two type-1b, or the ELSC diamond. Also, the vertical axis should be changed, so that the surface roughness can become better visible. The surface roughness of the diamond substrates should also be mentioned somewhere in the article."}, "author_response": "We thank the referee for a thorough review of our manuscript and are pleased to see the referee’s recommendation for the acceptance of our manuscript following minor revisions. We agree with the referee’s comment that it is important to clarify the nature of this work as providing primarily strong statistical evidence instead of a one-to-one correlation of STM data with optical signals. To address this, in the main results summary we have now added the following part:\n“Additionally, using STM tip-induced gating, we demonstrate the ability to consistently manipulate individual defect charge states. The manipulation was carried out with Au-coated tips which are known to have a plasmon-resonant response51–55. This is a statistically robust procedure which we have successfully carried out on more than 10 defects. Simultaneously correlating optical signals with individual NVs measured by STM remains a goal for follow-up work. New sample fabrication techniques are being developed currently which may allow for thin NV-enriched diamond membranes to be placed on plasmon-resonant metallic substrates. As a result, future STM tip-enhanced, near-field PL studies could potentially provide further confirmation of the individual defects and charge states studied.”\nWe again thank the referee for the valuable comments and suggestions through both rounds of review.\nWe thank the referee for important comments through both rounds of review. To make it clear that while our work provides strong statistical evidence, one-to-one correlation of optical and STM signals from NV centers remains a goal for the future, we have added the following in the main summary of results.\n“Additionally, using STM tip-induced gating, we demonstrate the ability to consistently manipulate individual defect charge states. The manipulation was carried out with Au-coated tips which are known to have a plasmon-resonant response51–55. This is a statistically robust procedure which we have successfully carried out on more than 10 defects. Simultaneously correlating optical signals with individual NVs measured by STM remains a goal for follow-up work. New sample fabrication techniques are being developed currently which may allow for thin NV-enriched diamond membranes to be placed on plasmon-resonant metallic substrates. As a result, future STM tip-enhanced, near-field PL studies could potentially provide further confirmation of the individual defects and charge states studied.”\nFurther comments on the text and figures:\n1. In the abstract the authors state: ‘This work represents a significant advancement in the atomic-scale understanding and engineering of NV centers, paving the way for future quantum device development.’ This sentence seems overstating, as there seem no ‘significant atomic scale understanding’ and also no useful ‘engineering’ in this work which can ‘pave the way for future quantum device development’. I think the main point of this solid work is about the nanoscale imaging of defects, and this sentence should be changed accordingly.\nWe have modified this sentence to state that the work advances “atomic-scale imaging, spectroscopic characterization, and charge-state manipulation of NV centers, potentially paving…” to be more specific about the contributions of our work.\n2. The sentence in the main text: ‘Critically, manipulation of individual NV charge states at atomic length scales has continued to pose a challenge’ Could be extended to introduce why such manipulation is critical for the ‘development of quantum devices’.\nWe’ve added the following sentence with relevant references in the main text to address this motivation for atomic-scale manipulability of NV charge states more directly.\n“Successful manipulation of local charge states could for example be used to improve quantum sensing with simultaneous high sensitivity and spatial resolution by creating small nm-scale pockets with high density of NV-surrounded by a region without NV-1,2”\n3. The sentence in the main text: ‘The primary samples used in this study are type-1b diamond crystals with the (100) facet facing the surface normal direction. They were fine polished and strain-released7 before irradiation with an electron beam at 2 MeV, with a dose of 2×1016/cm2 to create the NV- defects.’ It should be added that this results in a high-density NV ensemble. Also, as it is mentioned here, what is the purpose/reason for strain releasing, and what is the resulting surface roughness of the diamond? (can also be added to the methods/SM).\nWe have added the phrase “high-density ensemble of…” in the main text. The surface roughness of the diamond is <0.3 nm. The strain-release step after fine polishing is performed to remove any surface residual strain that can occur during polishing3,4. This can be important in applications since such strain can potentially affect the excited-state structure of NV defects3,5. We’ve added the following to the Methods section:\n“…subsequently fine polished and strain-released, yielding a roughness of <0.3 nm. The strain-release step is to remove surface residual strain after fine polishing and is performed due to the potential for strain to affect the excited-state structure of NV defects.”\n4. The sentence in the main text: ‘Having confirmed the presence of NV- centers, we move on to atomic-scale STM characterization of individual defects.’ Here would be a good occasion to mention that the further studies are all performed without laser illumination.\nWe’ve included the following sentence here: “We note that all subsequent studies described in the main text were performed without laser illumination.”\n5. The sentence in the main text: ‘In Fig. S6, we show examples of defects with different dI/dV peak heights likely arising from their positioning in distinct subsurface layers below the surface. The corresponding topographies also show a sharper contrast between the defect and the surrounding lattice for Defect 8, which has a stronger dI/dV peak compared to Defect 9.’As mentioned above in my main questions a more detailed discussion about the detection depth limitation should be included, also in the main text.\nWe included the following sentence here to provide a number for the estimated probing depth in the main text (in addition to Supplementary Note 2, where it is discussed in greater detail):\n“As discussed further in Supplementary Note 2, we estimate the maximum depth of NV centers which we can detect to be ~2.5 nm below the surface.”\n6. The sentence in the main text: ‘Using STM tip-induced gating, we are able consistently manipulate individual defect charge states, paving the path to atomic-scale engineering of spin-qubits.’ Whilst this statement is partly true, the ‘consistently manipulate ... paving the path to atomic-scale engineering’ is in my opinion slightly overstated, as it would be especially important to manipulate the NV center from its neutral towards its negative charge state.\nWe modified this sentence to read more concisely as “Additionally, using STM tip-induced gating, we are able consistently manipulate individual defect charge states.”\n7. In the methods part: ‘embedding them 5 nm below the surface’ Should be better written as ‘∼5 nm’.\nWe modified this to say ‘~5 nm’ instead of simply ‘5 nm.’\n8. In the supplement S3:‘This higher defect density environment is important in understanding the effect of nearby defects and the extent of charge state control in situations akin to those which could be encountered in technological applications.’ It should be stated clearly, why this higher defect density environment is important in understanding the effect of nearby defects.\nWe have added the following part in Supplementary Note 3 to provide further details on how the higher defect density environment is important to study:\n“From a practical standpoint, high-density defect environments can be critical in quantum sensing6,7. Also, our atomic-scale manipulation capabilities can be used to probe how the dipolar interaction which is known8 to exist between NV centers at nm-scales precisely varies depending on their distance and the local environment, for example. In terms of broader physical phenomena, dense NV ensembles can be very useful in understanding interacting many-body quantum systems including exotic phases such as time crystals8,9, and the techniques developed in our work can potentially contribute to the further investigation of such fascinating phases of matter.”\n9. In the supplement S3:‘As shown in Fig. S12 however, there is no discernible change in the PL signal while positioning the Au STM tip on top of an NV- defect in tunneling, 50 nm away laterally in tunneling, or withdrawn 500 nm away from the sample. This is very likely due to the insulating nature if the diamond substrate which doesn’t allow for plasmonic enhancement’ The effect not being visible is more likely due to the above-mentioned effect of the NV ensemble, and the vanishing effect of the shallow NV centers, which are further quenched by the graphene capping layer.\nWe meant to convey indeed that the effect is not visible due to the lack of plasmonic enhancement, which is a requirement given the high-density NV ensemble in type-1b diamond. We’ve added the phrase “…required to pinpoint a single NV center within the high-density ensemble in type-1b diamond” to clarify this point. We don’t see the graphene capping layer as a detriment to the NV characteristics, especially given the recent work10 (cited in our revised manuscript) by Hao, Yang, Li, et al., Natl. Sci. Rev. 12, nwaf076 (2025) showing a factor of 2-2.5 enhancement in the T coherence time of NV- defects with a 2 monolayer graphene cover layer.\n10. In supplement S4: ‘We confirm that under continuous wave illumination with power below 0.2 mW, no systematic changes could be found in the spectra on an NV- defect with and without illumination as shown in Fig. S13.’Why was this low laser power chosen here?\nWe apologize for an error here; going back through our data logs, the power used was actually 1.03 mW (not below 0.2 mW as originally stated). The laser conditions were the same as used in Fig. S15 (where the power was stated correctly as 1.03 mW). The values in Supplementary Note 4 as well as in the caption of Fig. S13 have now been corrected.\n11. In supplement S4:‘However, these previous works, using AFM and no graphene cover layer, use much larger voltages, even beyond 100 V’ Whilst these works applied higher voltages, the charge state switching already occurred at lower voltages, and should be stated correctly. Also, the NV defects in the cited works were deeper than the ones in this manuscript, this charge state switching for experimentally relevant NV defects is apparently not possible with the STM method.\nWe agree that the thresholds shown, especially in Bian, Zheng et al., Nat. Commun. 12, 2457 (2021)11 can be lower than the 100 V range, and modified the phrasing here to read “typically beyond 25 V”. Upon further examination, Zheng, Bian, et al., Nat. Phys. 18, 1317-1323 (2022)12 do also work with defects ~2 nm below the surface which we’re likely also able to measure and manipulate with STM (as discussed in the main text and Supplementary Note 2). Given that, we feel that our techniques can be complementary and do not necessarily correspond to defects with different depth ranges compared to those addressed in previous literature.\n12. In Fig. S1: It is unclear which diamond surfaces are shown here, if these are the two type-1b, or the ELSC diamond. Also, the vertical axis should be changed, so that the surface roughness can become better visible. The surface roughness of the diamond substrates should also be mentioned somewhere in the article.\nThe surfaces shown are from two different type-1b samples which were prepared at Argonne National Laboratory. The caption is modified to clarify this. Moreover, the vertical axes on the AFM images are changed so that the surface roughness can be seen more clearly. The roughness is <0.3 nm after the fine-polishing and strain-release procedures, and this value is now stated in the Methods section."}]

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