Liquid-activated quantum emission from native hBN defects for nanofluidic sensing

Nanostructures made of two-dimensional (2D) materials have become the flagship of nanofluidic discoveries in recent years. By confining liquids down to a few atomic layers, anomalies in molecular transport and structure have been revealed. Currently, only indirect and ensemble averaged techniques have been able to operate in such extreme confinements, as even the smallest molecular fluorophores are too bulky to penetrate state-of-the-art single-digit nanofluidic systems. This strong limitation calls for the development of novel optical approaches allowing for the direct molecular imaging of liquids confined at the nanoscale. Here, we show that native defects present at the surface of hexagonal boron nitride (hBN) - a widely used 2D material - can serve as probes for molecular sensing in liquid, without compromising the atomic smoothness of their host material. We first demonstrate that native surface defects are readily activated through interactions with organic solvents and confirm their quantum emission properties. Vibrational spectra of the emitters suggest that their activation occurs through the chemisorption of carbon-bearing liquid molecules onto native hBN defects. The correlated activation of neighboring defects reveals single-molecule dynamics at the interface, while defect emission spectra offer a direct readout of the local dielectric properties of the liquid medium. We then harvest these effects in atomically smooth slit-shaped van der Waals channels, revealing molecular dynamics and increasing dielectric order under nanometre-scale confinement. Liquid-activated native defects in pristine hBN bridge the gap between solid-state nanophotonics and nanofluidics and open up new avenues for nanoscale sensing and optofluidics.

attention in nanophotonics as a variety of point defects in its 6 eV band gap were found to be room-temperature quantum emitters [13][14][15] . Although the material engineering toolbox has been widely used to artificially induce emitters in hBN by irradiation 16,17 , strain engineering 18 or carbon doping 19,20 , liquid treatments have been left relatively unexplored. In recent works, we combined liquid and irradiation treatments, to demonstrate that plasma-induced hBN surface defects can be activated by water 21 and binary mixtures of water with organic solvents 22 , while liquid post-treatment of ion beam-exposed hBN can modify its defect emission properties 23 . Additionally, plasma-induced surface defects were found to report on single interfacial charge dynamics such as proton hopping 21,22 . However, even mild plasma treatment induces mechanical and chemical changes, yielding hBN crystals which no longer possess atomically smooth surfaces 24 , and preventing the integration of defects in ultra-flat van der Waals heterostructures which are currently driving progress in angstrom-scale fluidics 4,8 .
Here, we report that common organic solvents can activate visible-range quantum emission from native surface defects in high-quality pristine hBN crystals. This phenomenon relies on the interaction of organic molecules with pre-existing surface defects in high-quality hBN crystals [25][26][27][28] . Using spectral super-resolution microscopy 29 , we reveal defect-mediated molecular random walks as well as couplings between defect dipoles and the liquid medium leading to the dielectric liquid tuning of the emission wavelength. By relying only on intrinsic properties of a widely used two-dimensional material and common solvents, the fluorescence activation mechanism reported here is then used to image nanofluidic structures, with defects acting as nanoscale probes of the order and dynamics of liquid media confined down to the nanoscale.

Liquid-activated fluorescence from hBN native defects
We observed that untreated hBN crystals become fluorescent when brought in contact with most common organic solvents like ethanol. To demonstrate this effect, we exfoliated high-quality hBN crystals 25 using the scotch-tape method onto a glass coverslip, which was then used to seal a liquid-filled chamber placed on an inverted microscope. As sketched in Fig. 1a, the hBN crystal photoluminescence (PL) under 561 nm wide-field laser light illumination (0.35-3.5 kW/cm 2 ) was collected through the coverslip by a high-numerical aperture objective and projected onto a camera chip. As we previously reported, pristine hBN in air or water does not exhibit fluorescence under these illumination conditions 21 . However, we observed that as-exfoliated crystals in contact with ethanol exhibited an intense fluorescence signal (Fig. 1b). The fluorescence intensity gradually decreased under continuous illumination, and stabilized after several seconds, revealing emission from sub-diffraction spots as shown in Fig. 1c and in supplementary movie 1. We attribute this emission to defects present in the as-exfoliated crystal and activated through contact with the liquid. These defects can be super-resolved using single-molecule localization microscopy: each active defect can be localized with an uncertainty σ loc ∼ σ PSF / √ N loc , where σ PSF is the radial extent of the microscope point spread function (≈ 110 nm) and N loc is the number of photons counted in the diffraction-limited spot.
In the imaging conditions used here, we obtain localization uncertainties in the range of 10 − 20 nm. Emitters are randomly distributed on the basal plane of the crystal (Fig. S1), and the strategies for controlling their position by limiting the exposed surface are discussed later in the manuscript. As characterized in Fig. 1d, the number of emitters responsible for the emission decreased under constant illumination and reached a stable value of ∼0.5 emitters per square micron in ethanol. We verified that this process does not deteriorate the crystal (Fig S15-16) and the decrease shown in Fig. 1c is reversible: when left in the dark, the crystal fluorescence recovers within tens of minutes (Fig. S2) without inducing more active defects in the steady state ( Fig. S14).
This fluorescent activation of native defects was found to occur for most common organic solvents, such as n-alkanes (pentane to hexadecane) and primary alcohols (methanol to 1-pentanol). However, we did not observe any emission in pure water, heavy water, and hydrogen peroxide. For quantitative comparison, we imaged freshly cleaved hBN crystals under the same illumination conditions, but exposed to different liquid media such as water, glycerol (Gly), ethylene glycol (EG), benzyl alcohol (BA), dimethylsolfoxide (DMSO), ethanol (EtOH), dichloromethane (DCM), chloroform (CF) and cyclohexane (CH).
The steady state fluorescence under these solvents can be quantified by crystal brightness I crystal defined as the sum of localized emitter intensities per surface unit and time unit (Fig. 1e). Based on these observations, we can define three classes of solvents liquids such as glycerol exhibited only a limited but well measurable defect activation (type II). For water, no fluorescence was observed (type III). We found that the addition of only 10v/v% of water in ethanol could quench most of the emitters, leading to a 5-fold reduction in the fluorescence signal (Fig. S11). The difference between defect activation types could not be explained merely by physical parameters of the solvent (Fig. S3), pointing at chemical specificity. The steady state emitter density was found to depend on the liquid medium ( Fig. S4 & S5), the illumination power (Fig. S5) and macroscopic flow over the crystal (Fig. S6).  Fig. 1b, dense clusters of emission were observed upon turning the laser on (20 ms exposure per frame) in ethanol. After 10 seconds of wide-field illumination, the crystal surface reached a stable number of diffraction-limited isolated emitters. d, Localization microscopy-based counting of the emitters as a function of the illumination time: after 5 seconds, a steady state was reached. The dashed line is a fit to an offset exponential curve. e, Liquid dependency of the crystal fluorescence, showing strongly activating liquids (type I), mildly activating liquids (type II) and no activation in water (type III).
Considering the dynamics of these fluorescent emitters, a striking observation relates to the clear appearance of fluorescent trajectories at the surface of the crystal (see supplementary movie 2). These trajectories point to the correlated activation of neighbouring defects, corresponding to molecular random walks. By linking the super-resolved localization of defects (details in Methods), we can extract the associated trajectories, which are illustrated in Fig. 2b. These trajectories are similar to the ones observed on plasma-exposed hBN in water and binary mixtures of water and alcohols, which were attributed to proton hopping 21,22 . A similar phenomenology (detailed in supplementary section S13) is thus expected here, with (i) defect activation due to reversible charge transfer from and to the solvent and (ii) correlated activation of neighbouring defects involving the motion of charge-bearing solvent molecules which remain physisorbed on the crystal surface while retaining lateral motion (as predicted for water ions 30 ). However, the exact chemical mechanism must be distinct from what was proposed in water due to the aprotic nature of the solvents used here such as neat alkanes. As a comparison, the fluorescent signal in type I liquids is enhanced by over an order of magnitude with respect to plasma-treated hBN in water 21 , and the spectral signature for the

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emission is distinct 21,22 , pointing indeed to a different chemical mechanism, associated with a distinct defect type and reactivity.
As neither hBN nor the liquids used possess visible-range electronic transitions, the activation of emitters at the interface must arise from an electronic structure rearrangement which creates this new optically addressable electronic transition. The chemical selectivity found in Fig. 1e suggests that a necessary and sufficient condition for a liquid molecule to activate native hBN defects is for it to bear a carbon atom in its chemical structure. This result is consistent with the recent finding that carbon plays a crucial role in activating visible-range emitters in hBN 19 . While a direct observation of the exact chemical structure of the emitters is out of reach, the defect-liquid physicochemical interactions and the emitter photophysical properties will guide structural assignment. By imaging a ∼13×13µm 2 crystal area in the steady state for 50k, 6ms-long frames, we counted 700k localizations leading to 100k trajectories. Controlling the steady-state density of trajectories through light and liquid (Fig. S5), we conducted measurements in a regime where single trajectories are found not to split or merge with statistical significance. We therefore assign the trajectories to single molecules binding to single defects. We present in Fig. 2a a subset of these trajectories obtained from 5000 frames, overlaid with the super-resolved image from the same frames. This representation highlights that while the localization spots in some trajectories seem to hop freely, others remain trapped for long times resulting in bright spots in the super-resolved image. In other words, while most emitters only stay active for tens or hundreds of milliseconds, a fraction are found to be stable for several seconds (Fig. 2c) and we observed activations exceeding one minute (Fig. S7).
Under typical illumination conditions (∼ 3 kW/cm 2 ), up to several thousand photons per defect per 20ms frame are counted, which is comparable to state-of-the-art fluorophores typically used for localization microscopy 31 . Therefore, liquid-activated

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solid-state defects act in a similar way as organic fluorophores without suffering from photobleaching, which is one of their main limitations.
To achieve further insight into the observed molecular random walks, we analyzed their statistical properties, with the emitter position described as a random variable of time X(t), and the trajectory residence time on the crystal surface as another random variable T T res . As trajectory residence times comprise complex information on both the chemisorption energy at a single defects and the physisorption energy on pristine hBN in between defects, we also extracted residence times of molecules at single defect sites T D res corresponding to chemisorption only. Both residence times were found to follow a double exponential decay (Fig. 2d), with slow exponential decay components τ D res =35 ms and τ T res =82 ms, respectively. Assuming that the defect residence time constant is given by the Arrhenius equation τ D res = ν −1 e ∆G/kT where ν ≈ 10 12 − 10 13 s −1 is a molecular attempt rate 22 , we obtain a desorption free energy barrier ∆G ≈ 24-27 kT≈ 0.6-0.7 eV which is larger than typical physisorption energies (tens of meV) and smaller than covalent bonding energies (several eV) 32 . This can be rationalized in terms of a lowered energy barrier under illumination 21 , which is consistent with the observed light-induced reduction in number of emitters in Fig.   1d as well as the illumination power dependency of the density of emitters (Fig. S5).
We now turn our attention to the dynamics of the random walks. For Brownian diffusion, the one-dimensional displacement probability distribution function given by PDF( where D is the diffusion coefficient. Here we find that our random walks are best described instead by a two-component Gaussian model (Fig. 2e). The central part of the distribution, with size ∼20 nm, remains of constant size corresponding to the localization uncertainty when a trajectory is trapped. The tails of the distribution however enlarge with increasing lag time, characterizing hopping events. As shown in Fig. 2f, the scaling of the hopping tail size indeed follows Brownian diffusion with D= 9.1×10 −14 m 2 /s, that is 4 to 5 orders of magnitude slower than bulk liquid molecular diffusion coefficients. While this observation makes it impossible to resolve the molecular travel time between defects, it allows for imaging defect-mediated molecular dynamics with standard cameras of 10 ms time resolution, which we propose here as a tool for nanofluidics.

Spectral properties
Having shown robust and intense fluorescence emission from type I liquid-activated hBN surface defects, we further studied the peculiar interactions between solid-state surface defects and the liquid phase by focusing on their spectral properties using spectral single-molecule localization microscopy (sSMLM) 29 . Briefly, the wide-field image signal is divided by a 50:50 beam splitter. Half of the signal is used for spatial SMLM introduced above, and the other half passes through a prism to decompose its wavelength components, allowing for simultaneous localization and spectral characterization, as shown in Fig. 3a. Summing over a large number of single-emitter spectra, ensemble averaged spectra are obtained (Fig. 3b). The single-emitter spectra were found to be homogeneously distributed, with the appearance of a single population of emitters when exposed to the same liquid environment. Spectra were consistently characterized by two peaks, the zero-phonon line (ZPL) and the phonon side band (PSB), which are typical of emitters embedded in a matrix.
Interestingly, these ensemble spectra appeared to depend strongly on the activating liquid, and more precisely on its static dielectric constant ε liq -which remains valid up to the relevant GHz regime for solvents used here 33 . We present in Fig. 3b spectra of emitters obtained in the following liquids of increasing polarity: pentane (P), tert-butyl alcohol (TBA) and DMSO. A significant polarity-induced ZPL redshift of the defect emission spectrum was gradually observed from nonpolar pentane (615 nm) to more polar TBA (626 nm) to highly polar DMSO (641 nm). Beyond the ZPL shift, we observed changes in the PSB, which is less clearly defined for polar solvents. We report in Fig. 3c. the center wavelengths of both peaks as obtained from fitting to a two-Lorentzian model for a number of solvents ordered by increasing ε liq . We tested 1-pentanol (PA), isopropyl alcohol (IPA) and methanol (MeOH) on top of previously introduced liquids to interpolate dielectric constant values and found that both the ZPL and the PSB are redshifted by over 25 nm (∼80 meV) in highly polar liquids compared with nonpolar alkanes.
In the range ε liq < 25, a linear dependence of the ZPL wavelength on the ε liq was observed, with a slope close to 1 nm per dielectric constant unit, indicated by the dashed line (Fig. 3c).

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This positive solvatochromism is expected to occur when the magnitude of the excited state dipole is larger than the ground state one, which is the case for π − π * transitions 34 . The Jablonski diagram presented in Fig. 3d. illustrates the process giving rise to the observed spectra. By absorbing a photon (excitation, green arrow), the defect is excited to a dipolar state which interacts with the solvent and relaxes without radiating (dipole relaxation, curved arrow). Direct evidence of the dipolar nature of our liquid-activated emitters is presented in Fig. 3e where PL changes were monitored while rotating either the linearly polarized light used for excitation (green) or the fluorescent signal from the defects (orange). Details are provided in Fig.   S6. Similar dipolar excitation and emission was reported for several types of hBN defects 35,36 . As sketched in Fig. 3f, the solvatochromic redshift can be described by the presence of liquid molecular dipoles stabilizing the defect's excited state dipole, thus lowering its energy and redshifting the ZPL. After this step, the transition back to the ground state can occur in two ways: direct emission of a photon (ZPL, orange arrow), and phonon-assisted emission (PSB, red arrow), which is redshifted compared to the ZPL as a fraction of the energy leads to lattice vibrations (phonon, zigzag arrow).

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Continuing the analysis of the emission spectra, we evidence further peculiar interactions between the excited defect and solvent molecules. In the case of a mixture of polar ethanol and apolar heptane, the spectrum was very similar to that of the polar liquid, demonstrating a strong affinity between the emitters and polar molecules (Fig. S12). As shown in Fig. 3g, the energy difference between the ZPL and the PSB, which relates to the phonon dispersion of the material, was found to depend strongly on the solvent. The ZPL-PSB energy detuning ranges from 1250 cm −1 for nonpolar pentane to 965 cm This comes with an overall decrease of the phonon-assisted emission from 60% down to 10%, quantified in Fig. 2h as the ratio of the integrated phonon side band over the integrated spectrum (inset). Overall, the vibrational signature of defects in organic solvents points toward covalent bonding between chemisorbed organic molecules and the defect centers. This mechanism is similar to the activation of emitters through the covalent bonding of molecules to carbon nanotubes 46 . However, as discussed in Fig. 2d, the residence time analysis at a defect suggests that this covalent bond is formed only transiently due to a decrease in desorption energy barrier by illumination.

Room-temperature quantum emission in liquid
Liquid-activated native defects reported here host bright dipolar emission, with millions of photons emitted at trapping sites of the trajectories. The dipolar emission patterns shown in Fig. 3e suggest individual emitters, yet clusters of aligned emissive defects could exhibit the same pattern. To demonstrate that the measured fluorescence originates from single emitters, we performed time-correlated single-photon counting. For this, a 0.7 mW continuous-wave 561 nm laser beam was focused to a ∼ 1µm 2 spot onto hBN crystals in liquid, and fluorescence signal was collected by two fiber-coupled single photon detectors (SPDs) in a Hanbury Brown and Twiss interferometer configuration (inset of Fig. 4a). The typical time trace of a stable single emitter in acetonitrile is shown in Fig. 4a. The observed photon count rate, close to a million counts per second, is comparable to previous reports 13 . As shown in Fig. 4b, the analysis of photon arrival times from a 10s-long trace in Fig.   4a exhibited a pronounced anticorrelation between both SPDs at zero delay time, which is the signature of non-classical, sub-Poissonian light. The extent of the photon antibunching dip g (2) (0) = 0.25 ± 0.02 without background correction shows unambiguously single-photon emission (SPE) from hBN defects in liquid. This result implies that the bright spots are single emitters and not clusters, thereby their optical readout truly reports on nanoscale properties of the liquid. The long-lasting emission thus stems from the strong chemisorption interaction between a single activating molecule and a single defect. The onset of quantum emission through the formation of a single covalent bond with a molecule was observed in carbon nanotubes 47 , but the mechanism at play here exhibits the particularity of being fully reversible.
Photon statistical measurements were also performed with pulsed excitation, where the suppression of the correlated pulse peak at zero delay time, shown in Fig. 4c, confirmed single-photon emission. This feature did not depend on the activating 7/16 liquid: it was also present in hexadecane with a measured g (2) (0) = 0.45 ± 0.04. The fluorescent lifetimes, corresponding to the width of the antibunching dip, were found to be 2.73±0.09 ns and 2.20 ± 0.20 ns for acetonitrile and hexadecane, respectively. Taking advantage of the solvatochromic shift reported in Fig. 3, we thus demonstrated liquid-tunable single-photon emission with a ZPL shift of ∼ 20 nm between hexadecane and acetonitrile, shown as an inset in Fig. 4b. The magnitude of the solvatochromic redshift (80 meV ≡ 25 nm) is comparable with the most successful attempts to tune the single-photon emission of hBN defects through strain 48 or electrical control 49 . Such fine control of emitters at interfaces with liquid is appealing from both nanophotonic and nanofluidic perspectives: changes in the liquid environment may be translated into light signal, and conversely light control may be used to tune the properties of nanofluidic devices. Additional insights on spatial features of the quantum emission, including in the high-density regime may be gained by multi-detector quantum imaging with single-photon detector arrays 50 . b, Second order correlation function g (2) (τ) as measured from time correlated single photon counting in a Hanbury Brown and Twiss geometry, in hexadecane and acetonitrile. In both liquids, a pronounced antibunching is observed with g 2 (0) < 0.5 without background correction, proving the non-classical nature of the emitted light. Spectra are shown in the inset, demonstrating liquid-tunable SPE from 620 nm to 640 nm. c, Single-photon statistics under pulsed laser excitation showing a suppression of the central peak due to antibunching.

Integration in single-digit nanofluidic systems
Building upon this extensive spatial, temporal and spectral characterization of liquid-activated defects in pristine hBN, we then took advantage of layered van der Waals heterostructures to probe solid-liquid interfaces in molecular confinement. As sketched in Fig. 5a, we fabricated heterostructures comprising 3 crystals: bottom, spacer and top. The top crystal was chosen to be muscovite mica for its transparency and lack of fluorescent properties, and the bottom crystal was pristine hBN with native defects to be activated by the liquid. The middle layer, composed of few-layer graphene patterned by electron beam lithography, effectively acted as a spacer defining a slit between the hBN and mica sheets, whose height is set by the number of ∼ 3.4 Å-thick graphene layers 4 . Fabrication details are provided in Methods and in Fig. S8.
An optical micrograph of a device with h = 2.4 nm is provided in Fig. 5c. The bottom green region corresponds to the full heterostructure with nanoslits, and the top region corresponds to the open hBN flake masked by graphene spacers but without encapsulation by the mica. We first verified that covering the pristine hBN crystal with a patterned few-layer graphene crystal masks liquid-activated defects, as was observed for other types of hBN defects. 51,52 As shown in Fig. S9, emitters on bare hBN are randomly distributed, but the graphene mask allows for the precise positioning of emitters on the basal plane of hBN in liquid. An overlay of the graphene spacer atomic force microscopy (AFM) topography and the super-resolved image is shown in Fig. 5b, demonstrating the correspondence between the lithographically defined graphene pattern and the optically measured fluorescence from masked hBN defects. We further verified that capping the masked defects with mica does not quench their fluorescence, allowing direct imaging of defects in a confined liquid (Fig. 5d). We show in Fig. 5e that the localization intensity distributions with and without the confining mica top are similar, but the number of emitters in confinement is reduced by two thirds. Residence times inside nanoslits were found to be slightly longer in nanoslits than in masked hBN, as shown in Fig. S17. Therefore, the observed three-fold decrease in emitter number comes not from faster photobleaching of the emitters but rather from confinement-induced slowdown of their activation kinetics. Integrating defects into nanofluidic channels now represents a unique opportunity to probe the effect of confinement on liquid structure and dynamics. We first confirm the observation of trajectories in 1.4 nm confinement in Fig. 5g, where a set of emitters is shown to explore the ∼ 150 nm-wide channel. We then focus on the spectral properties of confined emitters, which can be robustly extracted through sSMLM with relatively low numbers of localizations (<1000). We present sSMLM spectra obtained in the bare, masked and confined geometries for ethanol (Fig. 5h) and acetonitrile (Fig. 5i). For both solvents, bringing the confinement size from 2.4 nm down to 1.4 nm leads to a clear blueshift pinpointed by the dashes indicating the ZPL position. For ethanol, the ZPL blueshifts from 638 nm to ∼ 624 nm, and for acetonitrile, transitions from 636 nm to ∼621 nm under 1.4 nm confinement, bringing the spectral signature of a strongly polar solvent close to that of nonpolar alkanes. This substantial confinement-induced blueshift suggests that emitters experience a reduced dielectric constant of ε conf ∼10 in 9/16 ethanol and ∼7 in acetonitrile when considering Fig. 3c as a calibration curve.
Bringing the top mica wall close to the defects may affect their emission in two ways. First, the wall with lowered dielectric constant (ε wall ≈ 8) 53 may deplete the shell of solvent dipoles interacting with the defect and confine field lines within the channel 54 , thereby destabilizing the excited state. Second, a more indirect way of affecting the emission is by reducing the out-of-plane component of the dielectric constant tensor 8 . As sketched in Fig. 5f the range of interaction between a solvent molecule with dipole µ S and the defect with dipole µ D is dip = (µ S µ D /2πε o k B T ) 1/3 . With the coarse assumption that µ D ∼ a × e where a ≈ 0.25 nm is the in-plane lattice parameter of hBN and e is the unit charge, we obtain µ D ≈ 12 D. Based on the dipole moments of ethanol (1.7 D) and acetonitrile (3.4 D) we obtain dip ≈ 1 nm and 1.3 nm, respectively, which is smaller than the set nanoslit height. Therefore, it seems unlikely that the observed effect under 1.4 nm confinement should arise purely from the top wall depleting the interacting dipole shell around the defect (bottom), and our results may be explained by the confinement-induced reduction of the solvent dielectric constant as was observed for water 8 and predicted for other liquids 55 .
Overall, these results consolidate the picture of dipolar environment-tuned defects, whose properties are affected by changes in the sensing hemisphere with volume ∼ 2/3π 3 dip , which encloses fewer than 100 molecules in the case of acetonitrile. Beyond passive diffusion and dielectric sensing of confined liquids, tracking defect activation dynamics in confinement may be used to directly image nanoscale flow and study its interplay with defects 56 . While a macroscopic control over the ensemble surface charge of nanofluidic devices with light was found to be useful for biomolecule detection 57 and energy harvesting applications 58,59 , the possibility to address single charged defects in confinement suggested by our results may reveal fundamentally new phenomena such as correlated ion transport 54 .

Conclusions
In summary, defects in hBN crystals, already known for a variety of outstanding optical properties, were found to exhibit a peculiar interaction with liquids. Indeed, native point defects on the atomically smooth surface of the as-exfoliated crystal become emissive when in contact with organic solvents. This unique system, where the encounter of a single defect with a single organic molecule yields a single-photon emitter, reconciles solid-state emitters and organic fluorophores to constitute a new tool for the study of solid-liquid interfaces. This tool can readily be applied for a variety of sensing applications as it relies on widespread single-molecule microscopy techniques and commonly available samples. We demonstrated two directions for sensing with liquid-activated hBN defects: activation dynamics report on interfacial charge transfer between defects by single molecules, while the bright emission spectra of emitters offer a readout of their nanoscale dielectric surrounding.
These phenomena were found to hold in single-digit nanometer confinement, allowing for the design of liquid-responsive nanostructures that may find useful applications for the optical imaging of single molecules interacting with solid surface defects in nanofluidic systems operando.

Sample preparation
Pristine hBN flakes from high-quality crystals 25 were exfoliated onto borosilicate glass coverslips (no. 1.5 Micro Coverglass, Electron Microscopy Sciences, 25 mm in diameter, 170 µm thick), using low-adhesion blue tape. The glass coverslips were cleaned either by (i) sonication in acetone followed by rinsing in isopropanol (ii) sonication in 2% Hellmanex III glassware cleaning agent solution, following by sonication in deionized water. No differences were observed between (i) and (ii). The coverslips were rinsed three times in the last solution, after which they were dried with a nitrogen gun covered with a 20 nm-pore filter. Adhesion to the substrate upon exfoliation was promoted by oxygen plasma cleaning of the coverslip (2 min, 100W).
High-quality crystals purchased from HQ Graphene and hBN crystals obtained by crystallization of hBN from molten iron in nitrogen-hydrogen atmosphere were also tested, and exhibited no notable difference with samples grown by the authors. The crystals were immersed immediately following exfoliation, and the PEEK chamber was thoroughly rinsed three times at each The slit-shaped nanochannels are made of a tri-crystal van der Waals (vdW) heterostructure of a spacer layer sandwiched between a top layer and a bottom layer following the same protocol as previously reported 4 . Here, the vdW stack is composed of top mica-graphene spacer-bottom hBN. In brief, thin layered graphene was first patterned via EBL into parallel strips with a width of 1 µm and a separation of 150 nm. A mica crystal (∼ 200 nm thick) was then transferred on top of the graphene spacer, via PMMA based transfer method. Then this mica-graphene spacer stack was lifted and transferred onto a freshly exfoliated hBN layer. This whole stack was then transferred onto a glass coverslip for the imaging. The channel dimensions of the final device is shown in Figure 5a where it has a width of ∼ 150 nm, a length of 20 µm and a height equivalent to the thickness of the graphene spacer. Fabrication flow chart and material requirements are described in detail in supplementary Figure S7.

Chemicals used
All chemicals were purchased with maximum purity grade available, and some were purchased anhydrous. No significant effect of solvent purity or residual water traces on hBN native defect activation was observed.

Optical microscopy
Widefield imaging was performed on a custom wide-field fluorescence microscope, described elsewhere 29 . Briefly, the emitters are excited using 561 nm laser (Monolithic Laser Combiner 400B, Agilent Technologies), which is collimated and focused on the back focal plane of a high-numerical aperture oil-immersion microscope objective (Olympus TIRFM 100X, NA: 1.45). This configuration leads to wide-field illumination of the sample in a circle with ≈ 25µm diameter. Fluorescence emission from the sample is collected by the same objective and spectrally separated from the excitation light using dichroic and emission filters

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(ZT488/561rpc-UF1 and ZET488/561m, Chroma) before being projected on an EMCCD camera (Andor iXon Life 897) with EM gain of 150. An additional spectral path, mounted in parallel to the localization path allows for simultaneous measurements of the emission spectra from individual emitters (see details below). The sample itself is mounted in a sealed fluidic chamber, which is placed on a piezoelectric scanner (Nano-Drive, MadCityLabs) for fine focus. Typical exposure time is 20-50 ms and typical laser power 10-100 mW for the widefield excitation area of 2×10 3 µm 2 , resulting in a power density of 0.35 − 3.5 kW/cm 2 . A typical acquired image stack contained 2-10 thousand frames.
Confocal microscopy measurements were performed in an inverted microscope configuration allowing to image samples in liquid. Here the emission was split between two fiber-coupled APDs (SPCM-AQRH, Excelitas) in an Hanbury Brown and Twiss configuration. Photon correlation measurements were performed using the PicoHarp TCSPC module (PicoQuant).

Spectral SMLM procedure
Acquired image stacks from the wide-field microscope were processed using ThunderSTORM 60 . Spatial SMLM for emitter counting, super-resolution image rendering and trajectory analysis was performed by filtering localization events with 30 < σ PSF < 200 nm. SMLM localization events were rendered as 2D gaussians with a standard deviation of 20 nm unless specified otherwise, reflecting the typical uncertainty on the emitter's position.
Spectral SMLM (sSMLM) was performed following a procedure described in our previous work 29 , which we summarize here. Emitters in the spatial channel are localized using the ImageJ plugin Thunderstorm. Briefly, a wavelet filter is applied to each frame. Peaks are then fitted by 2D-integrated Gaussians. The obtained localizations (x LOC , y LOC ) are matched to a reference spectral point (wavelength of 650 nm) through a matrix transformation of the form (x SPEC , y SPEC ) = A × (x LOC , y LOC ) + B. A is a 2×2 matrix; and B is a vector. The spectrum is then extracted as the vertical profile around (x SPEC , y SPEC ) in a box of 6x40 pixels centered at the emitter. Pixel values are translated as spectra intensities through the linear relationship ∆y SPEC ≈ a × λ , with a ≈0.25px/nm. This value and transformation matrix coefficients were obtained using fiducial markers emitting at known wavelengths. As described in our previous work, this was achieved by using broadband emitters (fluorescent beads) and narrow bandpass filters in the emission path. The signal-to-noise ratio of the single-emitter spectrum is evaluated and all spectra passing a threshold are averaged to yield and ensemble spectrum. We typically used 3000 frames for each solvent and set the threshold to I LOC > 300 photons in all measurements.
Ensemble sSMLM spectra were fit using Python package LMFIT 61 , to a model composed of 2 Lorentzians corresponding to the ZPL and PSB and a constant background. As shown in Fig. 5g-h, only the part of the normalized signal emerging more than 20% over the background was fit, as the background-induced spectra tails could pose fitting issues. For spectra in nanoslits, a spatial filter was applied to avoid counting signal from contamination in between channels (details in Fig. S10).

Trajectory linking
Wide-field fluorescence frames acquired with the EMCCD camera were first localized using ThunderSTORM 60 with parameters described in Methods. Trajectories were obtained by applying the Crocker-Grier linking algorithm 62 to the localization microscopy tables. Briefly, for each localization event at a given frame, the algorithm will link another event if it is found at the next frame within a specified search range. This search range was set to 120 nm in Fig. 2, as the probability of having a 1D displacement exceeding this value is less than 1% according to the analysis in Fig. 1e. The Python package Trackpy was used for the implementation of the linking.

Uncertainty estimations
Typical uncertainty of the ZPL and PSB position were estimated on the order of 3 and 5 nm, respectively (Fig. 3c). The fitting uncertainty is typically smaller than 1 nm, and the assigned uncertainty value reflects measurement-to-measurement variations. For the ZPL-PSB detuning, the error bars were estimated following the same procedure to 80 cm −1 (Fig.3g). For the phonon-assisted emission estimation, the leading uncertainty component corresponds to the evaluation of the height of the peak, which can reach up to 10% (Fig. 3h).