Imaging solvated oxygen atoms with a femtosecond laser

Domain: physics
Published: 2025-11-24
Editorial outcome: major_revision

Abstract
As a powerful oxidant, atomic oxygen (O) holds considerable promise for a variety of biomedical and industrial applications. However, the inability to quantify solvated oxygen atoms has prevented the determination of the fundamental parameters governing its behaviour in relevant aqueous environments. Here, we directly image ground-state oxygen atoms in water using femtosecond two-photon absorption laser-induced fluorescence. Measurements show that oxygen atoms persist for tens of microseconds in water, penetrating hundreds of micrometres into the liquid. This observed longevity has significant implications, suggesting a need to re-evaluate existing models of solvated atomic oxygen reactivity and transport. Beyond atomic oxygen, this technique is broadly applicable to other solvated atomic species of interest, including nitrogen (N) and hydrogen (H). This work establishes that radical atomic species can be quantified in liquid with ultrafast laser spectroscopy, providing the basis for the determination of key properties including reaction rates, chemical lifetimes, and Henry’s law constants.

Introduction
Several recent studies have demonstrated the potential of atomic oxygen (O) for a variety of uses in biomedicine and chemistry1–4, primarily due to its oxidative capacity5. These nascent applications are often contingent on O atoms reacting in liquid6–8. However, the behaviour of O in aqueous environments (O ) remains enigmatic, as little is known about its reaction rates9, diffusion coefficients, or transport from the gas phase10,11. This lack of understanding persists due to the inability to accurately measure concentrations of O in liquid, an issue compounded by the absence of the aforementioned chemical reference data12.

Attempts to characterise O with chemical probes have proven problematic, as O often degrades the probes or reacts indistinguishably from other reactive oxygen species (ROS)13–15. Understanding the dynamics of solvated O requires a selective and quantifiable diagnostic capable of spatial and temporal resolution. The prerequisites for such a method include the ability to probe the atomic ground state, where the vast majority of O atoms reside at room temperature, as well as a mandate to leave the ambient liquid environment unperturbed.

Optical techniques applied to the liquid phase are natural candidates for the direct detection of solvated O. Of these, two-photon absorption laser-induced fluorescence (TALIF) offers the highest degree of spatiotemporal resolution and can measure atoms in the ground state. TALIF is commonly used to quantify several reactive atomic species in the gas phase, including O16,17, N18, and H19,20. In O, it relies on the simultaneous absorption of two photons tuned to the O(2p43P)→O(3p3P) resonance. In the absence of other de-excitation mechanisms, O(3p3P) fluoresces at 844.6 nm with a radiative lifetime of ~35 ns21. The principal challenge for applying TALIF in liquid is obtaining a detectable signal, as the liquid phase strongly limits the efficiency of both the excitation and the fluorescence of the atomic species. Excitation is inherently limited by the pulse energy that can be applied without appreciably heating the liquid, while the highly collisional liquid environment rapidly quenches the laser-excited state. However, with a sufficiently fast excitation and detection scheme, it is possible to overcome these constraints.

Here, we demonstrate that solvated O atoms can be imaged using a femtosecond (fs) laser for TALIF in liquid. With fs-TALIF, the limitations of existing methods for measuring O can be effectively addressed. Direct measurements highlight the stability of O atoms in water, suggesting a chemical lifetime of at least tens of microseconds. With an induced flow, this allows O to reach depths of several hundred micrometres. Ab initio molecular dynamics (AIMD) simulations are also used to approximate the collisional quenching rate of laser-excited O . This enables estimates of solvated O densities, along with a Henry’s law constant. These results establish fs-TALIF as a viable technique for directly quantifying solvated atomic species and provide a framework for determining the fundamental parameters that govern their behaviour.

Results
Detection of atomic species using TALIF is fundamentally about two processes: the efficiency of excitation and the effective branching ratio, which describes the fraction of laser-excited atoms that subsequently emit a photon in the wavelength region of interest21. The primary constraints for applying TALIF in liquid concern these processes, as limitations on pulse energy restrict excitation efficiency, while the highly collisional environment results in a very low branching ratio. However, both of these can be addressed using an ultrafast (fs) laser17.

For excitation of ground-state O to occur, two photons with the appropriate combined energy must concurrently reside within the absorption cross section of the atom. Assuming an identical beam geometry and pulse energy, a laser pulse that is n-times faster will produce an n-fold higher density of photons. As TALIF is a two-photon process, this will increase instantaneous two-photon absorption by a factor of n2 and the total number of excitation events by n over the duration of the laser pulse. As a result, a faster laser proportionally increases the excitation efficiency. This is of particular importance for aqueous environments where the laser should not considerably heat (or boil) the target liquid. The use of a femtosecond laser allows for a lower pulse energy while still achieving sufficient excitation for detection.

The second major limitation in applying TALIF in a liquid environment is the high collisional frequency compared to the gas phase. This has consequences for both the two-photon excitation and the effective branching ratio. Considering the vibrational modes of H2O, nearly all collisions between O and H2O likely result in the quenching of laser-excited O. If a laser pulse is not appreciably faster than the rate of collisional de-excitation, two-photon pumping will not sufficiently populate the upper state to allow for detection of the fluorescence signal. Moreover, the highly collisional environment raises the detection threshold considerably. For solvated O atoms, collisional quenching occurs several orders of magnitude more frequently than radiative de-excitation21, so the resultant effective branching ratio is very small. This makes the additional excitation efficiency provided by the ultrafast laser critical for O detection.

To provide an efficient source of O atoms to a target liquid, O delivery was mediated by the COST Reference Source22, a microscale atmospheric pressure plasma jet (μ-APPJ). A well-studied and reproducible reference source23,24, the COST jet generates O in the active plasma through dissociative collisions between high-energy electrons and molecular oxygen (O2) present in the feed gas25. The O atoms produced in the active plasma are subsequently expelled via the gas flow into the plasma-free effluent (also free of electrons22, metastables9,26, and electric fields22). In this study, peak O densities in the effluent were measured to be around 1.5×1015 cm−3, representing a dissociation fraction above 2% at a gas temperature of 350 K. As a continuous-wave source, the plasma delivered a consistent flux of O atoms to a volume of deionised water held in a quartz cuvette 5 mm from the nozzle of the jet. Previous modelling studies of the COST jet10,27, when applied to the operating conditions used here, suggest a total O flux as high as 3×1016 s−1 at the water interface. The highest density of solvated O is then expected directly below the deformation zone created by the incident gas flow on the water surface, as shown in Fig. 1.

Given the constraints for a laser diagnostic to image solvated O atoms, a series of measurements was required for the confirmation of a TALIF signal from O (see Methods). The first measurement recorded the fluorescence with the plasma on and the laser tuned to the two-photon resonance of O (225.7 nm). Next, a laser background was taken at the same excitation wavelength with the plasma off. For the laser background, the gas flow was maintained to ensure a consistent air–water interface between the two measurements. Another measurement was then recorded with the plasma on, but with the ICCD gated after the laser pulse. With the delayed gate, if the fluorescence originates from O, the signal below the water surface should be quickly quenched, while the gas-phase fluorescence would persist due to its longer lifetime. Finally, the first two measurements were repeated with the laser tuned off resonance. For the off-resonance measurements, any observed signal cannot be attributed to O TALIF.

The results of this series of measurements are shown in Fig. 2a–d and conclusively demonstrate the presence of an on-resonance O fluorescence signal, below the water surface, with a laser-excited lifetime significantly shorter than gas-phase O. Fig. 2a shows the on-resonance, plasma-on measurement after subtraction of the 225.7 nm laser background. The background-subtracted signal in the water peaks at ~30 counts s−1 near the interface. It decreases away from the surface but is evident several hundred micrometres into the water. Fig. 2b shows the signal recorded with the ICCD gated 3 ns after Fig. 2a. As expected, it indicates that the fluorescence in water is quickly quenched while the gas-phase signal remains. For the delayed gate, the intensity of the gas-phase fluorescence is approximately halved. This confirms that the signal observed below the water surface in Fig. 2a does not originate from O2, either directly or as a reflection. In Fig. 2c, the ratio of the on-resonance signal and the laser background is shown for pixels below the water surface with a raw intensity of at least 12 counts s−1. The signal-to-background ratio is at least five for the majority of pixels along the laser path and has a clear maximum near the water surface, where O densities should be highest. Coupled with the results of the delayed-gate measurement, the magnitude and spatial extent of the signal-to-background ratio are further evidence that solvated O atoms are responsible for the observed fluorescence below the water surface. Finally, the laser background-subtracted off-resonance measurement is displayed in Fig. 2d. The absence of an off-resonance signal, along with the ultra-narrowband interference filter used on the ICCD, reaffirm that O TALIF is shown in Fig. 2a, b and lends additional confidence that O is being imaged below the water surface.

To further investigate the quenching environment in the gas and liquid phases, a complementary study was performed to record effective lifetimes of laser-excited O with a 0.5 ns ICCD gate. To accomplish this, a technique was used that differentiates the fluorescence of species affected by distinct quenching environments28, where the lifetime and relative signal strength at each pixel are recovered from a series of time-delayed images. The resulting lifetime data are shown in Fig. 2e. Only data with a signal-to-noise ratio of at least 1 are displayed. In the gas phase, effective lifetimes range from 1 to 5 ns, decreasing in proximity to the water surface. This is likely due to the higher concentration of H2O in the gas phase near the liquid, which efficiently quenches laser-excited O20,29. Below the water surface, effective lifetimes are less than 0.25 ns—the detection limit of this technique. This reflects very efficient quenching of the laser-excited species, as would be expected for solvated O atoms. This clear distinction between quenching environments further confirms that the fluorescence observed below the water surface originates from O.

The spatial extent of the signal below the water surface must also be reasonable to claim that it originates from O. Fig. 2a shows that fluorescence is observed at a depth of several hundred micrometres. To reach this depth, O must have limited reaction pathways in water. It was previously assumed that solvated O would react directly with water molecules to produce hydroxyl radicals. However, molecular dynamics simulations have identified an energy barrier that prevents this reaction30,31, a finding supported by several empirical studies10,13,32. Alternatively, O can react with solvated O2 from the gas phase to form O39,12. In the absence of other reaction partners in DI water, this is the dominant extinction pathway of O9. Assuming that the extended duration of He/O2 gas flow on the small water surface effectively outgases the DI water in the cuvette, the concentration of solvated O2 can be estimated using Henry’s law11. At an effluent temperature of 350 K, the extrapolated concentration of solvated O2 is 2.0×1015 cm−3. With a rate constant of k=5×10−12 cm3 s−1 for the reaction between O and O2 in the liquid phase27,33, a chemical lifetime of 100 μs for O can be estimated. As a result, a flow velocity of ~1 m/s in the half mm to the surface would be sufficient to explain the observed spatial extent of O. Fluid modelling with similar APPJs has indicated that this is realistic near the water surface considering the angled orientation of the plasma jet, the small water volume, and 2 slm flow rate used here27,34,35.

The direct detection of solvated O demonstrates that femtosecond TALIF can be used to image atomic species in liquid. However, an accurate density calibration is essential for the successful application of this technique. For TALIF measurements in the gas phase, this is regularly completed with noble gases of known density20. Xenon (Xe), for instance, is often used to calibrate TALIF measurements of O, given its nearly identical two-photon excitation and fluorescence scheme21,36–38. An essential feature of this calibration procedure is the ability to establish the effective branching ratio of the two species. In water, the nearly instantaneous collisional quenching of laser-excited O and Xe precludes an accurate in situ determination of their respective branching ratios. Given the additional uncertainties introduced by changes in the experimental setup required for Xe TALIF, an alternative approach to density calibration was considered.

Here, O densities are calibrated by comparing the time-normalised intensities of the TALIF signals in the gas and liquid phases37. This is advantageous as it only requires an estimate of the effective branching ratio for O in liquid, as opposed to a technique incorporating Xe TALIF in liquid. The equation for the density calibration of O is given in equation (1). The subscripts aq and g denote the aqueous and gas phase, respectively, for the effective branching ratios (a), normalised signal intensities (S), and O densities (n):

n_Oaq = a_g / a_aq * S_aq / S_g * n_Og (1)

Atomic O densities in the gas phase (n_Og) were calibrated using Xe TALIF measurements in accordance with the procedure outlined previously21,37,38. The gas-phase calibration included in situ effective lifetime measurements of laser-excited O and Xe atoms at atmospheric pressure. For the liquid phase, the effective branching ratio of O was estimated with AIMD simulations of solvated O atoms39–42. By accounting for the diffusion of O in room temperature water and assuming that all collisions result in quenching of the laser-excited state, AIMD simulations estimated the collisional quenching rate of O(3p3P) to be 1.45×1011 s−1. The corresponding effective branching ratio (a_aq) is then 1.99×10−4, almost three orders of magnitude less than the value of a_g recorded in situ for the operating conditions used here. With an estimate for a_aq, solvated O densities were calculated using equation (1). The result of the calibration applied to the laser background-subtracted measurement (Fig. 2a), is shown in Fig. 2f. Verbatim, it depicts solvated O densities on the order of 1016 cm−3 near the water surface. However, uncertainty in the calibrated densities of O is substantial, based on the assumption that all collisions between O and water molecules result in de-excitation. If collisions occur without quenching, the branching ratio would increase accordingly, reducing the estimated values of n_Oaq. Therefore, the calibrated O densities presented here should be interpreted as upper-bound approximations, with accuracy limited by uncertainty in the collisional quenching coefficient of laser-excited O by water.

To further examine O transport at the air–water interface, the laser was scanned horizontally across the region where the gas flow impinges on the surface. The resulting measurements are compiled in Fig. 3a. The density calibration for O was applied for Fig. 3b, c, where n_Oaq is shown as a function of penetration depth.

As expected, the highest concentration of O was found on the left side of the deformation zone, where the gas flow is nearly perpendicular to the surface. This corresponds to the region where x<0.5 mm in Fig. 3a. Here, maximum densities of solvated O are as high as 1016 cm−3 near the surface of the water and decrease with depth. No O is observed beyond a depth of 0.4 mm. On the right side of the deformation zone, where the gas flow is more parallel to the surface, only noise is evident below the air–water interface. In this region, the penetration depth of solvated O is mediated by diffusion rather than the induced flow. Applying the diffusion coefficient estimated by AIMD simulations for O, a 0.5 μm penetration depth is predicted within one chemical lifetime, well below the resolution of this experimental setup. The observed spatial distribution of O is therefore in good agreement with expectations based on the distinct transport regimes for solvated O. These spatially resolved measurements highlight the importance of the induced flow for both the direct detection of O, as demonstrated here, and applications that require penetration depths >1 μm.

In addition to probing liquid-phase dynamics, fs-TALIF enables the study of interfacial transport of atomic species. While this experiment was not explicitly designed to determine a Henry’s law constant, one can still be estimated if local equilibrium is reached between the gas and liquid phases near the water surface11. This assumption is likely valid in the present setup, given the steady-state nature of the O flux and induced flow, along with the relatively long chemical lifetime of O in DI water. A dimensionless Henry’s law constant, defined as H_CC = n_Oaq / n_Og, can then be established from measurements of O on either side of the air–water interface. In this study, O densities appear to reside around 1015 cm−3 above the water, with higher than usual uncertainty due to imaging the fluorescence through the deformed surface. This value for n would give a dimensionless Henry’s law constant of H_CC ≈ 10. Considering the caveats associated with the absolute calibration of n_Oaq, this should be regarded as an upper-bound estimate. Nevertheless, the Henry’s law constants for other atomic reactive nonmetals suggest that this value is reasonable. Those with available data have dimensionless Henry’s law constants that fall between hydrogen (H_CC = 6.4×10−3) and chlorine (H_CC = 57), including fluorine (H_CC = 0.50), iodine (H_CC = 2.0), and bromine (H_CC = 30)11. In addition, a recent study modelled the effect H_CC would have on phenol consumption in plasma-treated solution27. It was determined that a wide range of prospective Henry’s law constants could give the observed trends in phenol consumption, with model and experiment diverging as H_CC approached 20. This further supports the estimate of H_CC = 10 derived from direct imaging of O, if appropriately interpreted as an upper-bound value.

Additional studies employing fs-TALIF should make a concerted effort to refine the collisional quenching coefficients of solvated laser-excited atomic species, as the uncertainty in the branching ratio propagates to all other quantities derived from the density calibration. An accurate assessment of the collisional quenching coefficient would allow many currently unknown parameters to be quickly established, including Henry’s law and reaction rate constants, chemical lifetimes, and diffusion coefficients for atomic species of interest. This would aid in the implementation and optimisation of a host of novel applications predicated on solvated reactive atomic species.

In summary, this work demonstrates the direct imaging of ground-state O in liquid using femtosecond TALIF. This technique provides spatiotemporal resolution of solvated O, offering an alternative to existing indirect detection methods. We also introduce an approach for density calibration that utilises AIMDs simulations to estimate the effective branching ratio of laser-excited O. This allows in situ quantification of solvated O without the need to employ inherently problematic chemical probes. Our findings definitively indicate that O atoms are stable in water and persist for tens of μs based on the presence of O several hundred micrometres below the water surface. In addition to atomic O, this non-intrusive approach is applicable to other solvated atomic species of interest, including N and H. This diagnostic has significant implications for determining the fundamental parameters of solvated atomic radicals, including reaction rates, diffusion coefficients, and Henry’s law constants. The determination of these properties will enhance understanding of the behaviour of these species in the aqueous phase and facilitate their use for a variety of biomedical and chemical applications.

Methods
COST reference microplasma source
The atomic oxygen delivered to the water in these experiments was produced by the COST jet, a reference atmospheric pressure plasma jet22. The COST jet is a 13.56 MHz RF plasma source with a 1 mm × 1 mm × 30 mm plasma channel bound by two stainless steel electrodes, along with two quartz panes that allow optical access to the active plasma. Unlike other plasma jets, the COST jet is uniquely suited for selective ROS delivery as the active plasma is not in contact with the ambient air. The perpendicular orientation of the gas flow and electric field ensures that most electrons22,23 and metastables9,26 are restricted to the active plasma. This, along with the expansive literature available for the COST jet as a reference source, makes it an ideal tool to study O atoms9,23.

To give the best opportunity for O detection, the COST jet was configured to maximise O flux at the water surface. To do so, a more aggressive 2 slm flow was employed along with a 0.3% O2 gas admixture in the He feed gas. Although maximum O densities are found at higher O2 admixtures25,29, ozone formation, via reactions between atomic and molecular oxygen, scales with O2 25. The lower O2 admixture was used to increase the chemical lifetime of O in the gas and liquid phases. In addition, a relatively high voltage of 300 V RMS was applied to the powered electrode to ignite the plasma and optimise O production. Under these operating conditions, the total flux of O atoms at the water surface may exceed 3×1016 s−1, depending on the surface loss probability. This value is based on the densities of O measured in the gas phase, in addition to the transport efficiency of O to the liquid target. To achieve the stated flux, 70% of the O produced by the jet would have to reach the water 5 mm from the nozzle. Two modelling studies of O transport in the effluent of a He/O2 COST jet suggest that this is more than reasonable when considering differences in flow rate and O2 admixture10,27.

Xe TALIF measurements for the absolute density calibration of O in the gas phase were performed by flowing a helium/air/xenon admixture through the jet without plasma ignition, similar to the technique used in Myers et al.29. The total concentration of Xe in the feed gas was 0.03%.

Optical measurements
The laser source used for the fs-TALIF experiments is an amplified femtosecond laser (Solstice ACE, Spectra-Physics), equipped with an optical parametric amplifier (TOPAS Prime Plus, Light Conversion), capable of generating 100 fs pulses with >2 μJ energy/pulse, tunable ~225 nm at a repetition rate of 1 kHz. A fused silica prism was used to separate UV light from all other frequencies, and a 20 cm focal length fused silica lens focused the laser on the surface of the water. The imaging system consisted of a low noise CCD camera (Pixis 512, Princeton Instruments) equipped with a gated double-stage intensifier (Quantum Leap, Stanford Computer Optics) capable of varying gating times as low as 0.3 ns. A variable-focus 50 mm lens (Nikkor) with a spacer provided imaging from a distance of 5–10 cm. Collectively, the imaging system allowed for a minimum ICCD gate of 2.2 ns, utilised for all images presented here. The time delay between the laser excitation and ICCD gating was controlled by a digital delay generator (DG645, Stanford Research Systems).

For TALIF measurements, the laser system was tuned to the appropriate wavelength for two-photon excitation (225.7 nm for O and 224.3 nm for Xe36), and the fluorescence collected by the imaging system was passed through interference filters centred at 844.6 nm for O (bandwidth of 0.8 nm) and 830 nm for Xe (bandwidth of 10 nm). The 0.8 nm ultra-narrowband filter for O was of particular importance for O detection, isolating the fluorescence signal and excluding inelastically scattered laser light from the bulk liquid.

The experimental setup employed to image solvated O atoms is depicted in Fig. 1. It consists of a source of O atoms (the COST jet) incident on a water target, a femtosecond laser with the requisite optics, and an ICCD to collect the fluorescence signal. The effluent of the plasma jet was directed onto a volume of deionised (DI) water in a quartz cuvette. DI water was used to maximise the chemical lifetime of O atoms in the aqueous phase, allowing the best chance for detection. The fs laser was focused on the surface of the water vertically along the z axis and the plasma jet was positioned towards the interface at an angle 22.5 degrees from the normal. The ICCD was then placed along the y axis at a distance of 5 cm from the imaged region. To limit the effect of evaporation on the water level during the course of the measurement, the cuvette was linked to a large external reservoir of DI water (~10 litres). Despite this, slow evaporation does occur, lowering the water level ~35 μm per hour. This is derived from a shift of 22 μm in the water level between two measurements 37 minutes apart. This shift is almost imperceptible over the timescale of a single measurement (~15 minutes), is considerably less than the uncertainty in the location of the interface, and is accounted for by recording the location of the surface periodically over composite measurements. The gas flow from the plasma jet impinging on the air–water interface creates a deformation of the water surface on the order of 1–2 mm. To determine the exact position of the water surface, the ICCD recorded images with microsecond gating times, and the resulting images were processed using edge detection filters. Matlab image processing was able to identify the location of the interface with a half width at half maximum (HWHM) precision of approximately 100 μm. The mean full width at half maximum (FWHM) uncertainty for each measurement is indicated by error bars in Figs. 1, 2e, f, and 3a.

To identify fluorescence from solvated O, a series of measurements was required: First, the TALIF signal was recorded with the laser tuned on-resonance (225.7 nm), plasma source on, and the ICCD gated to include the laser pulse (t=0 ns). This contained the fluorescence signal from solvated O atoms, as well as contributions from unwanted sources. Next, the laser background was taken with the plasma off (no O atoms) and the same gate delay. To identify the O fluorescence, the laser background was then subtracted from the first plasma-on measurement. The resulting image is displayed in Fig. 2a, with the location of the water surface annotated by the dashed red line.

On-resonance measurements were also performed with a delayed ICCD gate. By gating the ICCD after the laser pulse, it was possible to differentiate the very short-lived TALIF signal originating from solvated O (on the order of picoseconds) and fluorescence from gas-phase O, which has an effective lifetime of several nanoseconds. Fig. 2b shows the background-subtracted image at t=3 ns. A sharp cutoff in fluorescence is apparent at the water surface, demonstrating the stark difference in lifetimes between the gas- and liquid-phase signals. To account for any contribution of scattered gas-phase O fluorescence to the signal observed below the water surface, the delayed-gate image was subtracted from Fig. 2a before the density calibration was applied. This results in slight differences in the spatial distributions of O in Fig. 2a, f, and 10–20% lower O densities than would be found otherwise.

A complementary study was completed to investigate the temporal dynamics of the O fluorescence in greater detail. For this test, a 4 μJ per pulse UV pump laser was used to generate the fs-TALIF signal from O in the gas and liquid phases. The fluorescence was collected with a PI Max 1024i and an intensifier gate of 0.5 ns, allowing for superior temporal resolution to the 2.2 ns gate used initially. The results, shown in Fig. 2e, confirm the aggressive quenching environment for solvated O atoms with effective radiative lifetimes of <0.25 ns.

To further validate that solvated O was the source of the recorded signal below the water surface, measurements were also performed with the laser tuned off-resonance (224.7 nm). With the ICCD gated with the laser pulse, no signal was visible after background subtraction (Fig. 2d). The absence of signal off-resonance, coupled with the much faster decay of the on-resonance fluorescence below the water surface, is clear evidence that solvated O is being imaged directly.

To obtain a 2D image of the O distribution both in water and above the surface, the laser beam was scanned horizontally. Measurements were performed at 13 locations, ~125 μm apart. The resulting images were compiled after accounting for the laser background, and only pixels with more than eight counts s−1 of signal were included. The resulting spatial map is shown in Fig. 3a. During this process, care was taken to account for the water evaporation. For Fig. 3b, a spatial map of the densities below the water surface was compiled using the calibrated signals along vectors normal to the interface. As the orientation of the interface is nearly constant in the region of interest, distortion in the x-direction of the 2D image should be minimal. Only points below the HWHM uncertainty in the location of the surface are included in Fig. 3b, c. As a result, the zero position in the y-direction for both figures is defined as the location of the water surface plus the HWHM uncertainty.

For measurements in air, the COST jet was oriented vertically, and the laser beam was focused horizontally to pass through the effluent region. The 2.2 ns ICCD gate was timed concurrently with the laser excitation, and the fluorescence of the atomic species (O or Xe) was imaged. The jet effluent was vertically scanned to give a 2D spatial map of O and Xe, while the time delay between the laser and the ICCD gate was varied to measure the fluorescence lifetimes of O and Xe. The recorded effective lifetimes were then used to calculate the branching ratio for the absolute density calibration21,29,38. The uncertainty in the gas-phase O density calibration with in situ lifetime measurements was previously found to be ~37%29.

Applying the calibration, O densities in the effluent were found to peak at 1.5×1015 cm−3 near the nozzle of the jet. In the absence of a liquid target, O densities 5 mm from the nozzle were 76% of their initial value. This supports the assertion that the total O flux at the water surface was as high as 3×1016 s−1, which would require 70% of O atoms to reach the liquid. The persistence of O in the effluent is a function of the relatively high flow rate (2 slm) and small O2 admixture (0.3%). For comparison, a 1 slm He, 0.6% O2 gas flow produced a 30% higher initial O density, but only 43% of O atoms remained 5 mm from the nozzle.

Laser-water interaction
As the TALIF signal depends quadratically on laser intensity, it is important to know the influence of the water on the propagation of the femtosecond laser. The linear absorption of the 224–226 nm laser beam in the water was found to be negligible. Using a fused silica cell with water, the transmission of the UV laser beam vertically through varying water thickness was found to be unaffected by the amount of water over several cm. This finding agreed with the reported values for the water UV absorption coefficient43, which is <0.1 m−1. This indicates that absorption is not a factor within the few mm probed in these experiments.

The laser intensity may also be influenced by the dispersion of the water. In particular, the group velocity dispersion (GVD) leads to an increased pulse width and a reduction in the peak laser pulse intensity. Using the procedure to estimate the pulse increase due to the GVD reported for 100 fs laser pulses in water at 800 nm44,45, the pulse width τ after propagating through a water layer of thickness L is given by:

τ = τ_0 * sqrt(1 + (4 ln(2) * G * L / τ_0^2)^2) (2)

where τ_0 is the initial pulse width and the GVD coefficient G is given by the second-order dispersion of the refractive index n as a function of the wavelength λ:

G = (λ^3 / (2 * π * c^2)) * (d^2 n / d λ^2) (3)

Using the Sellmeier equation for the dispersion of the index of refraction of water46:

n^2 = 1.7616034 - 0.0119397 * λ^2 + 0.00644940 / (λ^2 - 0.0148669) (4)

The GVD from equation (3) for water at 225 nm is 330 fs2/mm, and, using equation (2), the pulse width acquires an insignificant increase from 100 fs to 100.1 fs after passing through 0.5 mm of water.

The conclusion from the linear absorption and pulse stretching studies is that the intensity of the pump laser beam is not significantly affected after propagation through the first few hundred micrometres of water. As a result, the fs-TALIF signal accurately maps the spatial distribution of solvated O atoms in the water, as shown in Figs. 2 and 3.

Molecular dynamics simulations
To determine the collisional quenching rate and resulting branching ratio of laser-excited O in water, AIMD simulations were performed to estimate the collision frequency of O with the ambient water molecules. The collision frequency is the average number of ballistic collisions per unit time for a given particle within a given medium:

τ_c = V_cyl * ρ_med (5)

where τ_c is the collisional frequency, ρ_med is the atomic density of the medium of interest, and t is the time of interest. V_cyl is the volume of the collisional cylinder, which is defined as:

V_cyl = v_p * σ_p * t (6)

where v_p is the velocity of the particle and σ_p is the collisional cross section. The collisional cross section of atomic oxygen is assumed to be 1.9×10−15 cm2, which is half that of diatomic oxygen47. The velocity of O in water at room temperature is unknown. The most accurate way to determine how atomic O diffuses in water is by utilising AIMD, which can account for the electronic, short-range van der Waals (vdW), and long-range vdW interactions, while evolving in time through Newton’s equations of motion.

AIMD simulations of liquid water and liquid water with atomic O were performed using the strongly constrained and appropriately normed (SCAN) meta-GGA functional48. SCAN is inherently non-empirical, developed by satisfying all 17 known exact constraints on semilocal exchange-correlation functionals. Thus, the results obtained from SCAN are purely predictive and do not rely on training data. SCAN has been shown to correctly predict liquid water that is denser than ice at ambient conditions. SCAN accurately describes covalent and H bonds due to an improved description of electronic structure and captures intermediate-ranged vdW interactions that further improve the structure and thermodynamics of liquid water49.

Systems were investigated using the Vienna ab initio Simulation Package (VASP)39–42. The projector augmented wave (PAW) method50,51 was utilised within the density functional theory framework52,53. PAW pseudo-potentials within VASP with the h designation were utilised for all elements. The Gaussian smearing method was used with a width of 0.05 eV to determine the partial occupancies for each wave function. A gamma-centred Monkhorst-Pack54 1×1×1 k-point mesh was employed for Brillouin zone sampling. The energy cutoff was increased to 1000 eV to improve the accuracy and stability of the simulations. The electronic self-consistent loop exit criterion was set to 10−4. Simulations are spin-polarised to account for unpaired electrons.

A 90-atom supercell, which includes 30 water molecules, was constructed using the PACKMOL package55 to ensure the initial structure. Dynamics were carried out in the canonical (NVT) ensemble with a Langevin thermostat to control temperature. The Langevin friction coefficient for the atomic degrees-of-freedom was set to 100 ps−1. The time step was set to 0.5 fs, and the initial structures were equilibrated at room temperature for 4 ps. The trajectories of the total supercell energy and pressure were analysed as a function of time to ensure that an equilibrated state had been reached, and each of these quantities was oscillating around a given value. While the temperature of interest is room temperature, it has been shown that increasing the equilibration temperature in water by 30 K mimics nuclear quantum effects (NQEs)56. Thus, all calculations were performed at 328 K. The density of water was determined by varying the volume of the supercell, calculating the pressure at each individual volume, fitting a second-order polynomial to the pressure versus volume curve, and identifying the volume at which the pressure is zero. Three simulations were performed at each specified volume, and the system was evolved for 2000 time steps with a time step of 1 fs. This method yielded a density of 1.07 g/cm3, which is within 2% of the previously reported value from the SCAN XC potential48.

Given this density prediction, a single O atom was inserted into the supercell and equilibrated for 5 ps using the above simulation conditions. Subsequently, diffusion calculations were performed in which the system was evolved for 20,000 steps with a time step of 1 fs, for a total system time of 20 ps. Ten unique simulations were conducted to gain statistical significance in the results. The mean-squared displacement (msd) and the displacement were determined for the total system, H atoms, O atoms, and the free O atom. This allowed for the determination of both the velocity and the diffusion coefficient. The diffusion coefficient was fit to the msd as a function of time per Einstein’s equation (D=r2/2dt), where d is the number of dimensions, which in this case is three. The velocity is taken as the linear fit to the displacement as a function of time. The first 1 ps was neglected in the determination of the slope of displacements versus time.

Although ten unique simulations were included for the mean-squared displacement as a function of time for an O atom in water at 328 K, there is uncertainty in the data that increases with time. However, a linear trend is observed (R2 = 0.96), indicating reasonably converged diffusive behaviour.

The velocity derived from this set of trajectories was determined to be 2399.5 cm/s, and the diffusion coefficient was calculated as 1.5 × 10−9 m2/s. There are no prior experimental or computational data available for comparison. Using this velocity, the experimental density of water, and the assumed collisional cross section of O, the collisional frequency can be estimated. This method predicts a collisional frequency of 1.45×1011 collisions per second. If it is assumed that all collisions result in quenching of the laser-excited state, the effective radiative lifetime of O is 6.9 ps. This is approximately three orders of magnitude lower than the effective lifetime of laser-excited O atoms in the plasma effluent. Uncertainties associated with the prediction of the collisional frequency include the collisional cross section for monatomic O in water and the velocity of O in water determined by AIMD.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript provides noteworthy results suggesting the measurement of atomic oxygen in the liquid phase. I fully agree with the authors that this would be a unique measurement providing new insights in experimentally unexplored liquid phase chemistry.\nWhile the work is done carefully, the reviewer has doubts about the liquid phase measurement that can be addressed by the following analysis and experiments:\n(1) Figure 1 shows a rather blurry deformed gas-liquid interface at which the O radicals have been measured which does not give much confidence that the resolution of the interface is much better than the reported penetration depth of O radicals. As O radicals have been measured at this interface, the accuracy and reproducibility of the determination of this interface needs to be reported.\n(2) It should be firmly established whether the gas-liquid interface is static over time and does not oscillate or shift during the measurement. This is to be expected as a gas flow is impinging on the liquid and evaporation will occur over time with a dry gas flow. These effects could hugely impact the result in figure 3 c.\n(3) To validate the measurements beyond doubt, one should add a known scavenger of O in the liquid and confirm that the reduction of O density by the scavenger significantly reduces the liquid TaLIF signal. See for example the work of prof. Go`s group (Nature Communications 6, 7248 (2015)) who confirmed a novel measurement of solvated electrons for similar conditions.\n(4) The quenching constant which is critical to obtain the O density is estimated by the collisional frequency. In addition, it seems authors took half of the gas-kinetic cross section which is doubtful to be valid in the liquid. A detailed discussion of hydration effects, if important, and estimates of possible errors by comparison with known/measured collision frequencies of atoms in water should at least by provided. It seems based on the provided information that the accuracy of the reported O density might not be better than an order of magnitude (although no detailed uncertainties have been provided). In this case the data is not quantitative and will not be as useful as claimed by the authors.\nWhile the above comments and suggestions are critical to provide confidence in the reported result, the comment below is concerned with the impact of the developed measurement.\n(5) The authors state their measurements provide 'the basis for establishing a litany of currently unknown parameters for aqueous oxygen atoms including reaction rates, chemical lifetimes and Henry`s Law constant'. One would expect that the manuscript would at least report one of these parameters to show the impact of the measurement but no other data or analysis than Figure 3 is reported. The reporting of such parameters requires a detailed uncertainty analysis of the measurements and how this impact the determination of the parameters.\nAfter addressing the above comments in detail, I would highly recommend the work for publication in Nature Communications.", "reviewer_2": "The authors report on imaging of O atoms by means of femtosecond laser and two-photon absorption laser-induced fluorescence (fs-TALIF) in the process, where effluent of a microscale atmospheric pressure plasma jet (μ-APPJ) is in the contact with water surface and the TALIF signal originates both from the gas phase and from the liquid water. The authors provide an evidence that the O fluorescence signal from the water is not a reflection or result of laser light interaction with water and suggest a calibration method for O atom density under the liquid surface based on the comparison of the gas phase O density and quenching rates above and under the liquid surface. The collisional rate of O atoms with water molecules and the O diffusion velocity in water are estimated with the help of ab initio molecular dynamics (AIMD) simulations.\nThe fs-TALIF is the diagnostics of choice for quantitative laser-based O atom detection in plasmas (e.g. much better performance compared to ns-TALIF) and it has a potential to detect and quantify O atoms in the liquid phase as well, bringing significant progress into to understanding of O behavior in aqueous solutions. The measurements the authors performed and the data they are trying to obtain are of high importance and can bring the field significantly further. However, there are too many critical issues regarding the presented data, their analysis, and AIMD simulations, which have to be clarified before any publication. I am, therefore, not recommending this manuscript for publication.\n1) The main concern I have is the unrealistic density distribution of O atoms above and below gas-water interface.\n1a) O atoms are generated inside the plasma jet and transported mainly through convection with the He gas flow towards the liquid. The O density decreases with increasing distance from the jet (increasing residence time in the gas phase outside of the jet) due to three body recombination reaction with O2 molecules or due to losses to the liquid once the gas flow reaches the water surface. The TALIF signal above the water surface as shown in figure 3a) should represent this O atom spatial distribution above the water, with highest density “coming from above” from the jet nozzle 5 mm far away and decreasing monotonously as O atoms are transported along the gas flow streamlines in the direction of the water surface and then along this surface. Instead, an egg-shaped region of the fluorescence signal is observed with very sharp boundary between regions with 0 counts/s and 30 counts/s. This is not the expected O atom distribution and it looks more like a “saturated” detector signal in the region with the highest signal intensity. Or was the representation of the measured signal limited only to the 30 counts/s? Still, I would expect that the O atoms will follow the streamlines and their density should change more smoothly above the liquid. The authors say that O density determined in the gas phase agrees with the literature values, but they are not showing any figure with calibrated gas phase densities. I would find it useful to show these results as well and to compare them to existing spatial distributions available in the literature.\n1b) The O density distribution under the water surface has unexpected spatial distribution as well. Why is the O signal measurable just in the very narrow region between -0.2 and 0.3 mm on the horizontal axis in the figure 3? Why is it not detectable everywhere there, where the high measured gas phase O density is facing the liquid? The asymmetric signal distribution makes a bit the impression that there is a turbulent flow above the surface, which is also influencing the stability of the gas-water interface. The presented signal in fig. 3a and 3b can still be fluorescence of O dissolved in water, but not in the depth of 0.4 mm, but just at the surface of some small temporarily formed bubbles. The signal integration over many laser pulses results then in a “diffuse-like” signal distribution. The asymmetry is then caused by the tilted position of the jet. This impression is corroborated by estimation of the O diffusion length based on its diffusion constant and life time. Using the mentioned life time of 64 μs and the O diffusion constant of ~ 2.8 10-9 m2/s (OH diffusion constant in water) one obtains a diffusion length of L ~ √Dτ ~ 0.4 μm, three orders of magnitude smaller than measured. This also indicates that it is not straightforward to extrapolate the diffusion transport of O atoms in the water bulk based on the AIMD simulation with just 40 water molecules. The authors have mentioned that they have determined the diffusion coefficient of O atoms from the simulation as well, but the value is not given. Is this value similar to the known OH diffusion constant?\n2) The AIMD result are also used for determination of the O density in water and the resulting absolute O density just below water surface is given as ~3.3x1016 cm-3 (see fig. 3c). This is ten times larger than the O density above the water. O atom as a nonpolar species is expected to have low Henry’s law constant and usually the value known for O2 is used for it (dimensionless O2 Henry’s law constant is around 3x10-2). This would in this case result in the O density in water of just 9x1013 cm-3. Are there any arguments for much larger Henry’s low constant of O atoms? This discrepancy indicates that the estimated effective branching ratio is not correct or not every O collision with water results in non-radiative quenching.\n3) Regarding the concerns given in points above the authors have to provide much more information to make sure that their measurements, simulations, and data analysis are correct. First, they do not comment on the stability of the gas-water interface. Its ICCD image in figure 1 shows some complex “shadow” structure above the surface. Is this interface stable in time and is the gas flow laminar? The used jet can be operated at smaller gas flows, which would result in less strong deformation of the interface and maintain laminar flow with higher certainty. Schlieren imaging could provide some additional information about the gas flow. Second, the authors have to document all data processing steps by showing all the 2D images, which are used in the equation (1) before their subtraction. It is not clear to me, why there is still the large red area above the water surface in the figure 2b). The signal above the water should disappear or at least get much weaker after the subtraction of the signal measured at 3 ns after the laser pulse.\nThere are some other issues, which needs to be checked and addressed in any future manuscript:\n4) The shape of the gas-water interface is inconsistent in figures 2b) and 3a), see the figure attached separately to my comments, where both 2D images are set roughly to the same scale. The parallel red dashed lines show that the steep increase of the water surface has a different slope.\n5)The figure 1 should provide the photograph of the much larger surface area as well, including the jet nozzle. It should also make clear whether the 5 mm distance are measured to the undisturbed water level, or to the bottom of the deformed water surface. Additionally, I expect that the 22.5 degrees are measured to the normal, but it is not 100% sure from the text.\n6) How is the depth in figure 3b determined? Is it just a vertical distance to the line representing the surface or it is really a depth measured in the perpendicular direction to this line?\n7) Water parameters (pro analysis?, purity?) are not mentioned in the manuscript.", "reviewer_3": "The manuscript “Imaging solvated oxygen atoms with a femtosecond laser” by B. Myers et al. demonstrates nuances of application of femtosecond two-photon absorption laser-induced fluorescence (TALIF) for direct imaging of ground-state oxygen atoms in water. It looks like the authors are the first examining this technique for this particular purpose. The current manuscript builds on their previous work that showed fluency of using femtosecond version of TALIF technique for imaging oxygen atoms in plumes of atmospheric pressure plasma jets (APPJ). Nanosecond version of the method has been applied about 15 years ago to quantify absolute atomic oxygen densities in analogous type of APPJs. It was not clear whether oxygen atoms can penetrate and survive in aqueous phase long enough to get involved in any relevant chemical reactions without reacting with water first. In recent years, however, it has been unequivocally demonstrated by multiple studies of product generated in aqueous solutions exposed to oxygen APPJs that hydrated oxygen atoms survive long enough to freely diffuse and react with the aqueous phase solutes. The authors have cleverly recognized that, with the expansion of TALIF technique into femtosecond domain they can succeed in probing oxygen atoms, which cross plasma/water interface into aqueous medium and become hydrated. It can only occur if population of the upper excited state is produced faster than its heavy collisional quenching in condensed phase. Authors demonstrate that indeed they can clearly observe atomic oxygen TALIF signals under the surface of water at the area exposed into plasma jet containing small percentage of oxygen atoms. Based on the spatial distribution of intensities of the hydrated atomic oxygen TALIF signals researches are able to estimate that O atoms can diffuse up to 400 microns deep under the liquid surface, yet with the distance the signal intensity substantially drops. Varying the delay between plasma exposure and TALIF signal detection, using gated intensified CCD, the authors estimated that lifetime of the observed fluorescence signal originating in liquid phase is no longer than few nanosecond which is more than one order of magnitude shorter than in the plasma phase. Based on their ab-initio molecular dynamics simulations authors projected that hydrated oxygen atoms can migrate up to 1.5 mm if not scavenged by any solutes or involved in mutual recombination.\nI believe that the manuscript deserves publication in Nature Communications as is after the authors consider answering optional comments indicated below.\nComment1:\nThe authors estimated chemical lifetime of hydrated O at about 64 microseconds assuming that the main path of Oaq decay is pseudo first order reaction of Oaq + O2aq giving ozone. The concentration of oxygen in bulk water authors estimated based on Henry’s constant for molecular oxygen in the plasma gas phase. Do authors have evidence that indeed molecular oxygen concentration in their cell was at the level of single micromoles? Was the water outgassed before the exposure to APPJ? Chemical physics intuition would suggest that if authors used cuvette filled with air saturated water the concentration of molecular oxygen in bulk water would be in the order of 260 micromoles which would drop the lifetime of Oaq in water to submicrosecond range due to very same reaction O + O2 =O3.\nComment2:\nWas the water ultrapure and free of any impurities that could react with Oaq and decrease its apparent life time?\nComment3:\nIf the response to above 2 comments indicate that indeed water was ultrapure and free of oxygen from air than single nanosecond decay of aqueous phase TALIF signals would indicate Oaq involvement in second order reactions with other intermediate species like solvated electrons or OH radicals etc. Is that a plausible explanation of the apparent observation of the very short lifetimes of Oaq or is the concentration of these transient species too low at the near surface depths to become relevant?\nComment4:\nIs it possible to introduce Oaq scavenging solutes (phenol, chloride ions etc.) to APPJ exposed aqueous solutions and measure TALIF signals dependence on their concentration and penetration depth to unequivocally prove these signals indeed originate from Oaq?"}, "author_response": "Dear Editors and Reviewers,\nThank you very much for the time and effort you put into our manuscript. Your much appreciated and valuable comments helped us to refine and improve the manuscript. Please see the point-by-point responses to your comments in blue:\nReviewer #1 (Remarks to the Author):\nThe manuscript provides noteworthy results suggesting the measurement of atomic oxygen in the liquid phase. I fully agree with the authors that this would be a unique measurement providing new insights in experimentally unexplored liquid phase chemistry.\nWhile the work is done carefully, the reviewer has doubts about the liquid phase measurement that can be addressed by the following analysis and experiments:\n(1) Figure 1 shows a rather blurry deformed gas-liquid interface at which the O radicals have been measured which does not give much confidence that the resolution of the interface is much better than the reported penetration depth of O radicals. As O radicals have been measured at this interface, the accuracy and reproducibility of the determination of this interface needs to be reported.\nResponse: This is an important point worth clarifying and (along with comment 2) key to our reported result. The location of this interface is determined using Matlab. The FWHM of the interface has also been established and is now included in Figs. 1, 2d, and 3a as error bars plotted along with the interface. The HWHM of the interface, corresponding to the uncertainty extending into the liquid, is generally around 100 microns. Additional text has been included in the manuscript to this effect. The HWHM with respect to x-position for the surface depicted in Figures 1, 2, and 3 is shown below. Keep in mind the region for the deepest recorded penetration depth of O is centered around x=0 mm.\nOld text (pg. 14, paragraph 2): To determine the exact position of the water surface, the ICCD recorded images with microsecond gating times and the resulting images were processed using edge detection filters. The image processing allowed identification of the interface (shown in Figs. 1, 2b, and 3a) with a precision of 25 µm.\nNew text (pg. 16, paragraph a): To determine the exact position of the water surface, the ICCD recorded images with microsecond gating times and the resulting images were processed using edge detection filters. The image processing in Matlab allowed identification of the interface with a HWHM precision of approximately 100 µm. The FWHM uncertainty is denoted by error bars in Figs 1, 2d, and 3a.\nOld text (pg. 4, Figure 1 caption): Figure 1: A schematic representation of the experimental setup with the laser incident perpendicular to the water surface. An ICCD image of the water surface during plasma operation is shown at right. With a 2 slm gas flow, the atmospheric pressure plasma jet provided an O flux upwards of 1016 s-1.\nNew text (pg. 4, Figure 1 caption): Figure 1: A schematic representation of the experimental setup with the laser incident perpendicular to the water surface. An ICCD image of the water surface during plasma operation is shown at right with error bars denoting the FWHM uncertainty in the location of the interface. With a 2 slm gas flow, the atmospheric pressure plasma jet provided an O flux upwards of 1016 s-1.\n(2) It should be firmly established whether the gas-liquid interface is static over time and does not oscillate or shift during the measurement. This is to be expected as a gas flow is impinging on the liquid and evaporation will occur over time with a dry gas flow. These effects could hugely impact the result in figure 3 c.\nResponse: The interface does shift very slowly over time and the rate of this shift has been included in the Methods section. As noted, the rate of evaporation is very slow due to the 10 L external reservoir and is almost imperceptible over the measurement time of ~15 minutes (includes an initial measurement, laser background, and time-delayed). Over multiple measurements, the water surface was imaged periodically to update the location of the interface, relevant for Figure 3(a). The shift in the interface, recorded before and after two complete measurements of O , is shown below. The location of the surface was recorded 37 minutes apart and a 22 micron drop in the water level was observed (green line t=0 min, red line t=37 min). This also demonstrates the consistency in the shape of the interface over long periods of time.\nOld text (pg. 14, paragraph 2): For the measurements of O in liquid, as shown in Fig. 1, the laser was focused on the surface of the water vertically along the Z-axis and the plasma jet was directed to the interface at 22.5 degrees in the X-Z plane.\nNew text (pg. 16, paragraph 1): For the measurements of O in liquid, as presented in Fig. 1, the effluent of the COST jet was directed onto a volume of deionized (DI) water in a quartz cuvette. The cuvette was linked to a large external reservoir of DI water (~10 litres) to limit the effect of evaporation on the water level over the course of the measurement. Even so, slow evaporation does occur, lowering the water level about 35 micrometres an hour. This is derived from a shift of 22 micrometres in the water level between two measurements 37 minutes apart. This shift is almost imperceptible over the time scales of a single measurement (~15 minutes), is considerably less than the uncertainty in the location of the interface (discussed at the end of this paragraph), and is accounted for by recording the location of the surface periodically over composite measurements, such as Fig. 3a.\n(3) To validate the measurements beyond doubt, one should add a known scavenger of O in the liquid and confirm that the reduction of O density by the scavenger significantly reduces the liquid TALIF signal. See for example the work of prof. Go`s group (Nature Communications 6, 7248 (2015)) who confirmed a novel measurement of solvated electrons for similar conditions.\nResponse: While the authors agree that this would reaffirm the presence of solvated O, we believe that the off-resonance measurement, in conjunction with the temporal evolution of the signal in liquid vs. air, conclusively demonstrates that O is being measured. To further convince readers of this, additional figures (2b and 2c) have been added, along with some corresponding text.\nOld text (pg. 6, paragraph 3): The on-resonance signal is displayed in Fig. 2a after the contribution from the laser background is subtracted. The signal below the liquid interface peaks at around 30 counts s-1 and is discernible several hundred micrometres from the surface (depicted in Fig. 2b).\nNew text (pg. 6, paragraph 3): The on-resonance measurement is displayed in Fig. 2a after the contribution from the laser background is subtracted, while Figs. 2b and 2c confirm that the signal originating in the water is not observed after a short time delay (3 ns) or off-resonance (224.7 nm). In Fig. 2a, the signal below the liquid interface peaks at around 30 counts s-1 and is discernible several hundred micrometres from the surface.\nOld text (pg. 7, Figure 2 caption): Figure 1: (a) The on-resonance, laser background-subtracted O fluorescence signal, recorded with the ICCD gated to capture the laser pulse and TALIF signal in water. (b) Absolute densities of atomic O with the location of the water surface annotated. Note that the density calibration shown here is only accurate below the water surface where the appropriate branching ratio applies. In (b), a delayed gate measurement 3 ns after the laser pulse has been subtracted from the initial measurement (see Methods), as indicated by the distorted shape of the TALIF signal in air. Due to the aggressive quenching of laser-excited O in the liquid environment, no O fluorescence is visible after this time delay. This then accounts for the contribution of isotropic scattering from the TALIF signal originating in the gas phase, highlighting the fluorescence from solvated O atoms.\nNew text (pg. 8, Figure 2 caption): Figure 2: (a) The on-resonance, laser background-subtracted O fluorescence signal, recorded with the ICCD gated to capture the laser pulse and TALIF signal in water. The water surface is annotated by the red line. (b) The time-delayed, on-resonance O fluorescence signal recorded 3 ns after (a). Due to the aggressive quenching of laser-excited O in the liquid environment, no O fluorescence is visible after this time delay. (c) The off-resonance (wavelength centred at 224.7 nm), laser background-subtracted signal, gated to include the laser pulse. (d) Absolute densities of solvated atomic O. Note that the density calibration applied here is only accurate below the water surface where the appropriate branching ratio applies. Here, (b) has been subtracted from (a) to account for scattering from the intense gas-phase fluorescence, resulting in the distorted shape of the signal above the water surface.\n(4) The quenching constant which is critical to obtain the O density is estimated by the collisional frequency. In addition, it seems authors took half of the gas-kinetic cross section which is doubtful to be valid in the liquid. A detailed discussion of hydration effects, if important, and estimates of possible errors by comparison with known/measured collision frequencies of atoms in water should at least be provided. It seems based on the provided information that the accuracy of the reported O density might not be better than an order of magnitude (although no detailed uncertainties have been provided). In this case the data is not quantitative and will not be as useful as claimed by the authors.\nResponse: The reviewer is absolutely correct that there are significant uncertainties in the estimate of the collisional quenching coefficient of O in water. The collisional cross section of monatomic oxygen is unknown, but since this quantity largely correlates to the size of the species of interest, assuming a cross section that is half of molecular oxygen is reasonable. Additionally, the atomic cross section of Ne is approximately the same as that used in this work for monatomic O. If error is present in this assumption, it is quite minimal and should not affect the conclusions. We would also note that the gas-kinetic scattering cross section is used in Ref. 43 to explore diffusional phenomena in water. To clarify these points and discuss how this uncertainty propagates through the density calibration and other parameters contingent on its use, additional text has been added to the manuscript in two locations. We believe this will help readers take our results in context and motivate the next iteration of experiments.\nOld text (pg. 9, paragraph 1): The corresponding effective branching ratio (a ) is then aq 1.99×10-4, almost 700 times less than a under identical plasma conditions22. g\nNew text (pg. 10, paragraph 1): The corresponding effective branching ratio (a ) is then aq 1.99×10-4, almost three orders of magnitude less than a recorded in situ for the operating conditions used here. The uncertainty in the calibrated densities of O is substantial, based on the following caveat: If a smaller fraction of O collisions with water molecules result in de-excitation, the branching fraction would increase accordingly. Therefore, the calibrated densities for O presented here should be viewed as an upper bound approximation limited by knowledge of the collisional quenching coefficient of O in water.\nOld text (pg. 11, paragraph 1): Further study of O densities in solution will likely be key to determining reaction rate constants, assuming the chemical lifetime permits O detection. By employing this imaging technique, the chemical lifetime can be quickly established, an important facet in understanding O behaviour.\nNew text (pg. 12, paragraph 2): Further studies employing fs-TALIF should make a concerted effort to refine the collisional quenching coefficients of solvated laser-excited atomic species, as the uncertainty in the branching ratio propagates to all other quantities derived from the density calibration. An accurate assessment of the collisional quenching coefficient would allow many currently unknown parameters to be quickly established including Henry’s law and reaction rate constants, chemical lifetimes, and diffusion coefficients for atomic species of interest. This would aid in the implementation and optimization of a host of novel applications predicated on solvated reactive atomic species.\nWhile the above comments and suggestions are critical to provide confidence in the reported result, the comment below is concerned with the impact of the developed measurement.\n(5) The authors state their measurements provide 'the basis for establishing a litany of currently unknown parameters for aqueous oxygen atoms including reaction rates, chemical lifetimes and Henry`s Law constant'. One would expect that the manuscript would at least report one of these parameters to show the impact of the measurement but no other data or analysis than Figure 3 is reported. The reporting of such parameters requires a detailed uncertainty analysis of the measurements and how this impact the determination of the parameters.\nResponse: Although this experimental setup is not explicitly designed for determining these parameters, given the steady-state nature of the O delivery, we believe that an order of magnitude estimate of a Henry’s law constant for O can be provided and appreciate the reviewer’s encouragement to do so. This is now included in the text, along with the appropriate caveats, and is in reasonable agreement with recent literature37 falling squarely between the dimensionless Henry’s law constants of O and OH (0.0317 and 926, respectively). It also resides well within the limits of Henry’s law constants available for other atomic reactive nonmetals, as included in the text.\nOld text (pg. 10): This paragraph was included in the “Spatial mapping of O in water” section to give an estimate of a Henry’s law constant for O, explain how it should be interpreted, and contextualize it with other literature.\nNew text (pg. 11, paragraph 2): While this experiment is not explicitly designed to determine a Henry’s law constant, one can be extracted if it is assumed that equilibrium between the gas and liquid phases is reached in the vicinity of the water surface13. The continuous and stable flux of O and a chemical lifetime in water on the order of 10s of µs should allow this. Densities of O close to the water are challenging to accurately assess due to the optical effects that accompany imaging gas-phase fluorescence through a deformed liquid interface. Here, maximum densities appear to reside from 1.5–2´1015 cm-3, with higher than usual uncertainty25 owing to the varying transmittance of 844.9 nm fluorescence through the deformed air/water interface. This would give a dimensionless Henry’s law constant for O – defined as 𝐻!! = 𝑛 /𝑛 – of between 15–20. While this should be taken as an upper bound approximation given the caveats included in the absolute calibration, an estimate on this order is plausible. Other reactive nonmetals with available data have dimensionless Henry’s law constants that fall between hydrogen (HCC = 6.4×10-3) and chlorine (HCC = 57), including fluorine (HCC = 0.50), iodine (HCC = 2.0), and bromine (HCC = 30)13. In addition, a recent study modeled the effect HCC(O) would have on phenol consumption in plasma-treated solution37. By evaluating in tandem with experimental data, it was reported that a wide range of Henry’s law constants could lead to the observed trends in phenol consumption, with model and experiment diverging when HCC(O) approached 20. This can still be qualified as fair agreement if the Henry’s law constant presented here is appropriately interpreted as an upper bound limit.\nAfter addressing the above comments in detail, I would highly recommend the work for publication in Nature Communications.\nReviewer #2 (Remarks to the Author):\nThe authors report on imaging of O atoms by means of femtosecond laser and two-photon absorption laser-induced fluorescence (fs-TALIF) in the process, where effluent of a microscale atmospheric pressure plasma jet (μ-APPJ) is in the contact with water surface and the TALIF signal originates both from the gas phase and from the liquid water. The authors provide an evidence that the O fluorescence signal from the water is not a reflection or result of laser light interaction with water and suggest a calibration method for O atom density under the liquid surface based on the comparison of the gas phase O density and quenching rates above and under the liquid surface. The collisional rate of O atoms with water molecules and the O diffusion velocity in water are estimated with the help of ab initio molecular dynamics (AIMD) simulations.\nThe fs-TALIF is the diagnostics of choice for quantitative laser-based O atom detection in plasmas (e.g. much better performance compared to ns-TALIF) and it has a potential to detect and quantify O atoms in the liquid phase as well, bringing significant progress into to understanding of O behavior in aqueous solutions. The measurements the authors performed and the data they are trying to obtain are of high importance and can bring the field significantly further. However, there are too many critical issues regarding the presented data, their analysis, and AIMD simulations, which have to be clarified before any publication. I am, therefore, not recommending this manuscript for publication.\n1) The main concern I have is the unrealistic density distribution of O atoms above and below gas-water interface.\nResponse: This is addressed in the responses to the following comments.\n1a) O atoms are generated inside the plasma jet and transported mainly through convection with the He gas flow towards the liquid. The O density decreases with increasing distance from the jet (increasing residence time in the gas phase outside of the jet) due to three body recombination reaction with O2 molecules or due to losses to the liquid once the gas flow reaches the water surface. The TALIF signal above the water surface as shown in figure 3a) should represent this O atom spatial distribution above the water, with highest density “coming from above” from the jet nozzle 5 mm far away and decreasing monotonously as O atoms are transported along the gas flow streamlines in the direction of the water surface and then along this surface. Instead, an egg-shaped region of the fluorescence signal is observed with very sharp boundary between regions with 0 counts/s and 30 counts/s. This is not the expected O atom distribution and it looks more like a “saturated” detector signal in the region with the highest signal intensity. Or was the representation of the measured signal limited only to the 30 counts/s? Still, I would expect that the O atoms will follow the streamlines and their density should change more smoothly above the liquid. The authors say that O density determined in the gas phase agrees with the literature values, but they are not showing any figure with calibrated gas phase densities. I would find it useful to show these results as well and to compare them to existing spatial distributions available in the literature.\nResponse: The authors appreciate the reviewer’s concerns regarding the distribution of O fluorescence and believe they can be addressed as follows: Here, two things are responsible for the observed shape. First, the color scale is capped at 30 counts/s to emphasize the signal in the liquid and would reveal a more nuanced shape in the gas phase if that was not the case. Second, the light is distorted traversing through the “bubble” created by the gas flow, warping the signal. The figure below is Figure 3a but with the color scale set to emphasize the shape and intensity of the gas-phase signal. You can clearly see the individual spatial measurements that constitute Figure 3a and the distortion of the would-be straight lines by the deformed air-water interface. For the gas-phase O densities, spatial and in situ effective lifetime measurements were completed at the 1 slm flow rate used in our previous work25 to demonstrate agreement. As this is a different flow rate than was used for experiments attempting to detect O , this data was not included to avoid confusion. Recording absolutely calibrated O densities in the deformation region would be incredibly helpful but the optical effects involved would make doing so exceedingly difficult.\n1b) The O density distribution under the water surface has unexpected spatial distribution as well. Why is the O signal measurable just in the very narrow region between -0.2 and 0.3 mm on the horizontal axis in the figure 3? Why is it not detectable everywhere there, where the high measured gas phase O density is facing the liquid? The asymmetric signal distribution makes a bit the impression that there is a turbulent flow above the surface, which is also influencing the stability of the gas-water interface. The presented signal in fig. 3a and 3b can still be fluorescence of O dissolved in water, but not in the depth of 0.4 mm, but just at the surface of some small temporarily formed bubbles. The signal integration over many laser pulses results then in a “diffuse-like” signal distribution. The asymmetry is then caused by the tilted position of the jet. This impression is corroborated by estimation of the O diffusion length based on its diffusion constant and life time. Using the mentioned life time of 64 μs and the O diffusion constant of ~ 2.8 10-9 m2/s (OH diffusion constant in water) one obtains a diffusion length of L ~ √Dτ ~ 0.4 μm, three orders of magnitude smaller than measured. This also indicates that it is not straightforward to extrapolate the diffusion transport of O atoms in the water bulk based on the AIMD simulation with just 40 water molecules. The authors have mentioned that they have determined the diffusion coefficient of O atoms from the simulation as well, but the value is not given. Is this value similar to the known OH diffusion constant?\nResponse: This comment brings up some very valid points that we feel are critical to address. The reviewer is absolutely correct that very limited penetration of O would occur in a strictly diffusive environment. Here, however, the plasma jet induces a flow in the liquid. Near the surface, existing literature has shown this may exceed 2 m/s29,30. While our angled configuration is not entirely comparable, we feel this is a reasonable estimate within a half mm of the surface. If we can comfortably measure 2 chemical lifetimes of O, a 2.5 m/s flow with a 64 µs lifetime would give a penetration depth of 320 microns. Our measurements are therefore contingent on the liquid flow and it is then no coincidence that we detect the deepest penetration of O right where the angled jet “digs” into the liquid. This is further supported by the fact that we see no solvated O where the gas flow is parallel to the liquid surface between around 0.5 and 1.5 mm on the x-axis. Here, O penetration perpendicular to the surface would be mediated by diffusion. Extensive edits have been made along with the addition of several references in the text to address this.\nThe diffusion coefficient from the AIMD simulations was similar to that of OH (1.5´10-9 m2/s) and the velocity of O in water was determined correctly for the collisional frequency but misapplied to calculating the penetration depth in the initial version of the manuscript. The additional velocity due to the induced flow has a negligible effect on the collisional frequency. We apologize for this error.\nOld text (pg. 7, paragraph 1): The on-resonance signal is displayed in Fig. 2a after the contribution from the laser background is subtracted. The signal below the liquid interface peaks at around 30 counts s-1 and is discernible several hundred micrometres from the surface (depicted in Fig. 2b). Atomic O TALIF signal at this depth is consistent with O extinction being mediated by O production via solvated O from the gas phase12, a reasonable assumption given the energy barrier preventing ground-state O from reacting with water molecules27. Extrapolating the solvated O density from the He/O feed gas using the Henry’s Law constant for O , O is predicted to have a chemical lifetime of 64 µs. How the recombination of O reduces the chemical lifetime is unknown, given the absence of a reaction rate constant, but it may be considerable. At the diffusion velocity estimated by AIMD simulations (24 m/s), a chemical lifetime of 64 µs would correspond to a 1.5 mm penetration depth. This is in very good agreement with TALIF observations in liquid and may be further refined if a rate constant for O self-reaction can be quantified.\nNew text (pg. 7, paragraph 1): The on-resonance measurement is displayed in Fig. 2a after the contribution from the laser background is subtracted, while Figs. 2b and 2c confirm that the signal originating in the water is not observed after a short time delay (3 ns) or off-resonance (224.7 nm). In Fig. 2a, the signal below the liquid interface peaks at around 30 counts s-1 and is discernible several hundred micrometres from the surface. For this to be the case, O extinction must be primarily mediated by O formation via solvated O from the gas phase12, a sound assumption given the energy barrier preventing ground-state O from reacting with water molecules27,28 and the absence of other reaction partners in He/O plasma-treated DI water9,22. With the extended duration of the He/O flow, as well as the small surface area of the water in the cuvette (1 cm2), it is expected that the solvated O concentration reaches equilibrium with the gas phase in accordance with Henry’s law near the water surface. Extrapolating the solvated O concentration from the He/O feed gas using the appropriate Henry’s law constant13, O is predicted to have a chemical lifetime of 64 µs16,28. Assuming enough O is entering the water to be detectable after several chemical lifetimes, a flow velocity of a couple m/s in the closest half mm to the surface would allow O to be measured in the spatial extend observed. Fluid modeling with similar APPJs have indicated this is quite reasonable near the water surface considering the angled orientation of the gas delivery, small water volume, and 2 slm flow rate used here29,30.\n2) The AIMD result are also used for determination of the O density in water and the resulting absolute O density just below water surface is given as ~3.3x1016 cm-3 (see fig. 3c). This is ten times larger than the O density above the water. O atom as a nonpolar species is expected to have low Henry’s law constant and usually the value known for O2 is used for it (dimensionless O2 Henry’s law constant is around 3x10-2). This would in this case result in the O density in water of just 9x1013 cm-3. Are there any arguments for much larger Henry’s low constant of O atoms? This discrepancy indicates that the estimated effective branching ratio is not correct or not every O collision with water results in non-radiative quenching.\nResponse: This is also a worthwhile critique and the reviewer is correct that the Henry’s law constant for O is most frequently applied for O in literature. Looking at Sander’s compilation of Henry’s law constants (ref. 13), other atomic reactive nonmetals with a value listed are as follows, in dimensionless units (HCC): H – 6.4x10-3, F – 0.50, Cl – 57, Br – 30, I – 2.0. Although many of the reactive nonmetals do not have established Henry’s law constants, this demonstrates that a Henry’s law constant of around 10–20 for O is well within the bounds of similar atomic species. Furthermore, a recent study investigated the degradation of phenol in He/O plasma-treated solution37. As a part of this, the authors simulated the effect that differing Henry’s law constants of O would have on their phenol consumption. They used Henry’s law constants ranging from O (3.17x10-2) to OH (926) and compared the simulated and experimental results. They found that their simulated and experimental results diverged when HCC(O) exceeded 100 times HCC(O ). Given the assumptions made in our measurements and their model, along with the aforementioned Henry’s law constants for other reactive nonmetals, we do not believe a value of HCC(O)=10–20 is unreasonable. With increased confidence that our continuous delivery of O affords a steady-state configuration close to the air/water interface, we have also included our estimate of the Henry’s law constant in the manuscript. Given the complete absence of data for HCC(O), we believe our estimate is still worth reporting even with the high uncertainty which is also discussed at length.\nOld text (pg. 10): Much of the “Spatial mapping of O in water” section was rewritten to better address the behavior of solvated O and explain our measurements and their limitations.\nNew text (pg. 11, paragraph 2): While this experiment is not explicitly designed to determine a Henry’s law constant, one can be extracted if it is assumed that equilibrium between the gas and liquid phases is reached in the vicinity of the water surface13. The continuous and stable flux of O and a chemical lifetime in water on the order of 10s of µs should allow this. Densities of O close to the water are challenging to accurately assess due to the optical effects that accompany imaging gas-phase fluorescence through a deformed liquid interface. Here, maximum densities appear to reside from 1.5–2´1015 cm-3, with higher than usual uncertainty25 owing to the varying transmittance of 844.9 nm fluorescence through the deformed air/water interface. This would give a dimensionless Henry’s law constant for O – defined as 𝐻!! = 𝑛 /𝑛 – of between 15–20. While this should be taken as an upper bound approximation given the caveats included in the absolute calibration, an estimate on this order is plausible. Other reactive nonmetals with available data have dimensionless Henry’s law constants that fall between hydrogen (HCC = 6.4×10-3) and chlorine (HCC = 57), including fluorine (HCC = 0.50), iodine (HCC = 2.0), and bromine (HCC = 30)13. In addition, a recent study modeled the effect HCC(O) would have on phenol consumption in plasma-treated solution37. By evaluating in tandem with experimental data, it was reported that a wide range of Henry’s law constants could lead to the observed trends in phenol consumption, with model and experiment diverging when HCC(O) approached 20. This can still be qualified as fair agreement if the Henry’s law constant presented here is appropriately interpreted as an upper bound limit.\nFurther studies employing fs-TALIF should make a concerted effort to refine the collisional quenching coefficients of solvated laser-excited atomic species, as the uncertainty in the branching ratio propagates to all other quantities derived from the density calibration. An accurate assessment of the collisional quenching coefficient would allow many unknown parameters to be quickly established including Henry’s law and reaction rate constants, chemical lifetimes, and diffusion coefficients for atomic species of interest. This would aid in the implementation and optimization of a host of novel applications predicated on solvated reactive atomic species.\n3) Regarding the concerns given in points above the authors have to provide much more information to make sure that their measurements, simulations, and data analysis are correct. First, they do not comment on the stability of the gas-water interface. Its ICCD image in figure 1 shows some complex “shadow” structure above the surface. Is this interface stable in time and is the gas flow laminar? The used jet can be operated at smaller gas flows, which would result in less strong deformation of the interface and maintain laminar flow with higher certainty. Schlieren imaging could provide some additional information about the gas flow. Second, the authors have to document all data processing steps by showing all the 2D images, which are used in the equation (1) before their subtraction. It is not clear to me, why there is still the large red area above the water surface in the figure 2b). The signal above the water should disappear or at least get much weaker after the subtraction of the signal measured at 3 ns after the laser pulse.\nResponse: This concern was consistently raised by reviewers and is important to clarify. The interface does shift very slowly over time and the rate of this shift has been included in the Methods section. As noted, the rate of evaporation is very slow due to the 10 L external reservoir and almost imperceptible over the measurement time of ~15 minutes (includes an initial measurement, laser background, and time-delayed). Over multiple measurements, the water surface was imaged periodically to update the location of the interface, relevant for Figure 3(a). The shift in the interface recorded before and after two complete measurements of solvated atomic oxygen is shown below. The measurements of the surface were taken 37 minutes apart and show a 22 micron drop in the water level (green line t=0, red line t=37 mins). This also exemplifies the consistency in the shape of the interface over long periods of time, which would not be possible in a turbulent flow regime. In addition, the FWHM of the interface has been determined in Matlab and error bars have been included in figs. 1, 2d, and 3a. The HWHM extending into the water was consistently around 100 microns throughout the region of interest. For a better idea of the image processing steps and to further convince readers beyond a reasonable doubt that O is being detected, additional images have been included as a part of Fig. 2 along with some corresponding text. The additional images include the background-subtracted time-delayed measurement (2.b) and the off-resonance measurement taken on a different day an O signal was also observed (2.c). In the former Fig. 2b, now Fig. 2d, the signal above the water does get considerably weaker after subtracting the time-delayed measurement as the reviewer suggests. However, the scaling is applied to highlight the signal in the water, which would be difficult to see otherwise. In addition, the laser-excited O has a lifetime of around 7 ns, so the initial t=0 ns signal is more intense than the t=3 ns signal leading to an imperfect subtraction.\nOld text (pg. 14, paragraph 2): For the measurements of O in liquid, as shown in Fig. 1, the laser was focused on the surface of the water vertically along the Z-axis and the plasma jet was directed to the interface at 22.5 degrees in the X-Z plane.\nNew text (pg. 16, paragraph 1): For the measurements of O in liquid, as presented in Fig. 1, the effluent of the COST jet was directed onto a volume of deionized (DI) water in a quartz cuvette. The cuvette was linked to a large external reservoir of DI water (~10 litres) to limit the effect of evaporation on the water level over the course of the measurement. Even so, slow evaporation does occur, lowering the water level about 35 micrometres an hour. This is derived from a shift of 22 micrometres in the water level between two measurements 37 minutes apart. This shift is almost imperceptible over the time scales of a single measurement (~15 minutes), is considerably less than the uncertainty in the location of the interface (discussed at the end of this paragraph), and is accounted for by recording the location of the surface periodically over composite measurements, such as Fig. 3a.\nOld text (pg. 7, Figure 2 caption): Figure 3: (a) The on-resonance, laser background-subtracted O fluorescence signal, recorded with the ICCD gated to capture the laser pulse and TALIF signal in water. (b) Absolute densities of atomic O with the location of the water surface annotated. Note that the density calibration shown here is only accurate below the water surface where the appropriate branching ratio applies. In (b), a delayed gate measurement 3 ns after the laser pulse has been subtracted from the initial measurement (see Methods), as indicated by the distorted shape of the TALIF signal in air. Due to the aggressive quenching of laser-excited O in the liquid environment, no O fluorescence is visible after this time delay. This then accounts for the contribution of isotropic scattering from the TALIF signal originating in the gas phase, highlighting the fluorescence from solvated O atoms.\nNew text (pg. 8th, Figure 2 caption): Figure 4: (a) The on-resonance, laser background-subtracted O fluorescence signal, recorded with the ICCD gated to capture the laser pulse and TALIF signal in water. The water surface is annotated by the red line. (b) The time-delayed, on-resonance O fluorescence signal recorded 3 ns after (a). Due to the aggressive quenching of laser-excited O in the liquid environment, no O fluorescence is visible after this time delay. (c) The off-resonance (wavelength centred at 224.7 nm), laser background-subtracted signal, gated to include the laser pulse. (d) Absolute densities of solvated atomic O. Note that the density calibration applied here is only accurate below the water surface where the appropriate branching ratio applies. Here, (b) has been subtracted from (a) to account for scattering from the intense gas-phase fluorescence, resulting in the distorted shape of the signal above the water surface.\nThere are some other issues, which needs to be checked and addressed in any future manuscript:\n4) The shape of the gas-water interface is inconsistent in figures 2b) and 3a), see the figure attached separately to my comments, where both 2D images are set roughly to the same scale. The parallel red dashed lines show that the steep increase of the water surface has a different slope.\nResponse: This may be due to differences in how the interface was plotted between figures or the spatial dimensions of the figures. Regardless, all figures now use the same interface function established by a Matlab code that also provides the uncertainty of the interface location, as seen in Figs. 1, 2d, and 3a. Figs 2a, 2b, and 2d were also resized to give a square 2 mm x 2 mm frame on the ICCD image. Note, the off-resonance measurement presented in Figure 2c was taken on a different day where an O signal was recorded, hence the different interface shape.\n5) The figure 1 should provide the photograph of the much larger surface area as well, including the jet nozzle. It should also make clear whether the 5 mm distance are measured to the undisturbed water level, or to the bottom of the deformed water surface. Additionally, I expect that the 22.5 degrees are measured to the normal, but it is not 100% sure from the text.\nResponse: Language has been included in the manuscript to enhance clarity. Although the ICCD image of the larger surface would add value, it was decided that emphasizing the error calculation in the interface detection would be a better use of Figure 1.\nOld text (pg. 12, paragraph 1): For these experiments, the effluent of the jet impinged on the liquid at a distance of 5 mm from the nozzle.\nNew text (pg. 14, paragraph 1): For these experiments, the effluent of the jet impinged on the liquid at a distance of 5 mm from the nozzle, not accounting for the deformation introduced by the gas flow.\nOld text (pg. 14, paragraph 2): For the measurements of O in liquid, as shown in Fig. 1, the laser was focused on the surface of the water vertically along the Z-axis and the plasma jet was directed to the interface at 22.5 degrees in the X-Z plane.\nNew text (pg. 16, paragraph 1): The laser was focused on the surface of the water vertically along the Z-axis and the plasma jet was positioned towards the interface at an angle 22.5 degrees from normal.\n6) How is the depth in figure 3b determined? Is it just a vertical distance to the line representing the surface or it is really a depth measured in the perpendicular direction to this line?\nResponse: This can be somewhat confusing given the wording. The depth is measured as a perpendicular distance from the surface, not as a vertical distance. Some clarifying language is included.\nOld text (pg. 10, Figure 3 caption): Figure 3: (a) A composite of O fluorescence in the gas and liquid phase for a series of scans across the water interface. (b) O TALIF signal from the region just below the water surface, rotated to the vertical to emphasize the spatial extent of O. (c) O densities as a function of depth in the liquid, taken from the zero position on the x-axis in (b).\nNew text (pg. 11, Figure 3 caption): Figure 3: (a) A composite of O fluorescence in the gas and liquid phase for a series of scans across the water interface. (b) O TALIF signal from the region just below the water surface where the depth denotes the distance to the air/water interface. (c) O densities as a function of depth in the liquid, taken from the zero position on the x-axis in (b).\n7) Water parameters (pro analysis?, purity?) are not mentioned in the manuscript.\nResponse: DI water was used to ensure water purity for the positive detection of solvated oxygen atoms. The water composition should change very little given the large volume available for treatment (~10 L) and the microscale nature of the plasma source used. The use of DI water is consistent with our previous gas and liquid-phase studies of plasma-generated O.\nOld text (pg. 4, paragraph 1): As a continuous wave source, the plasma delivers a nearly constant flux of O atoms to the liquid.\nNew text (pg. 4, paragraph 1): As a continuous wave source, the plasma delivers a nearly constant flux of O atoms to a volume of deionized water held in a quartz cuvette.\nOld text (pg. 14, paragraph 2): For the measurements of O in liquid, as shown in Fig. 1, the laser was focused on the surface of the water vertically along the Z-axis and the plasma jet was directed to the interface at 22.5 degrees in the X-Z plane.\nNew text (pg. 15, paragraph 4): For the measurements of O in liquid, as presented in Fig. 1, the effluent of the COST jet was directed onto a volume of deionized (DI) water in a quartz cuvette. The cuvette was linked to a large external reservoir of DI water (~10 litres) to limit the effect of evaporation on the water level over the course of the measurement.\nReviewer #3 (Remarks to the Author):\nThe manuscript “Imaging solvated oxygen atoms with a femtosecond laser” by B. Myers et al. demonstrates nuances of application of femtosecond two-photon absorption laser-induced fluorescence (TALIF) for direct imaging of ground-state oxygen atoms in water. It looks like the authors are the first examining this technique for this particular purpose. The current manuscript builds on their previous work that showed fluency of using femtosecond version of TALIF technique for imaging oxygen atoms in plumes of atmospheric pressure plasma jets (APPJ). Nanosecond version of the method has been applied about 15 years ago to quantify absolute atomic oxygen densities in analogous type of APPJs. It was not clear whether oxygen atoms can penetrate and survive in aqueous phase long enough to get involved in any relevant chemical reactions without reacting with water first. In recent years, however, it has been unequivocally demonstrated by multiple studies of product generated in aqueous solutions exposed to oxygen APPJs that hydrated oxygen atoms survive long enough to freely diffuse and react with the aqueous phase solutes. The authors have cleverly recognized that, with the expansion of TALIF technique into femtosecond domain they can succeed in probing oxygen atoms, which cross plasma/water interface into aqueous medium and become hydrated. It can only occur if population of the upper excited state is produced faster than its heavy collisional quenching in condensed phase. Authors demonstrate that indeed they can clearly observe atomic oxygen TALIF signals under the surface of water at the area exposed into plasma jet containing small percentage of oxygen atoms. Based on the spatial distribution of intensities of the hydrated atomic oxygen TALIF signals researches are able to estimate that O atoms can diffuse up to 400 microns deep under the liquid surface, yet with the distance the signal intensity substantially drops. Varying the delay between plasma exposure and TALIF signal detection, using gated intensified CCD, the authors estimated that lifetime of the observed fluorescence signal originating in liquid phase is no longer than few nanosecond which is more than one order of magnitude shorter than in the plasma phase. Based on their ab-initio molecular dynamics simulations authors projected that hydrated oxygen atoms can migrate up to 1.5 mm if not scavenged by any solutes or involved in mutual recombination.\nI believe that the manuscript deserves publication in Nature Communications as is after the authors consider answering optional comments indicated below.\nComment1:\nThe authors estimated chemical lifetime of hydrated O at about 64 microseconds assuming that the main path of Oaq decay is pseudo first order reaction of Oaq + O2aq giving ozone. The concentration of oxygen in bulk water authors estimated based on Henry’s constant for molecular oxygen in the plasma gas phase. Do authors have evidence that indeed molecular oxygen concentration in their cell was at the level of single micromoles? Was the water outgassed before the exposure to APPJ? Chemical physics intuition would suggest that if authors used cuvette filled with air saturated water the concentration of molecular oxygen in bulk water would be in the order of 260 micromoles which would drop the lifetime of Oaq in water to submicrosecond range due to very same reaction O + O2 =O3.\nResponse: The reviewer is correct that solvated O concentrations are much higher in water exposed to open atmosphere and this would likely quench any O entering the water quickly. For our purposes, however, the robust He gas flow incident on the small surface area of the cuvette (1 cm2) effectively outgases the water, resulting in the much lower O concentration in equilibrium with the He 0.3% O gas flow.\nOld text (pg. 7, paragraph 1): Atomic O TALIF signal at this depth is consistent with O extinction being mediated by O production via solvated O from the gas phase12, a reasonable assumption given the energy barrier preventing ground-state O from reacting with water molecules27. Extrapolating the solvated O density from the He/O feed gas using the Henry’s Law constant for O , O is predicted to have a chemical lifetime of 64 µs.\nNew text (pg. 7, paragraph 1): For this to be the case, O extinction must be primarily mediated by O formation via solvated O from the gas phase12, a sound assumption given the energy barrier preventing ground-state O from reacting with water molecules27,28 and the absence of other reaction partners in He/O plasma-treated DI water9,22. With the extended duration of the He/O flow, as well as the small surface area of the water in the cuvette (1 cm2), it is expected that the solvated O concentration reaches equilibrium with the gas phase in accordance with Henry’s law near the water surface. Extrapolating the solvated O concentration from the He/O feed gas using the appropriate Henry’s law constant13, O is predicted to have a chemical lifetime of 64 µs16,28.\nComment2:\nWas the water ultrapure and free of any impurities that could react with Oaq and decrease its apparent lifetime?\nResponse: DI water was used to ensure water purity for the positive detection of solvated oxygen atoms. The water composition should change very little given the large volume available for treatment (~10 L) and the microscale nature of the plasma source used. The use of DI water is consistent with our previous gas and liquid-phase studies of plasma-generated O.\nOld text (pg. 4, paragraph 1): As a continuous wave source, the plasma delivers a nearly constant flux of O atoms to the liquid.\nNew text (pg. 4, paragraph 1): As a continuous wave source, the plasma delivers a nearly constant flux of O atoms to a volume of deionized water held in a quartz cuvette.\nOld text (pg. 14, paragraph 2): For the measurements of O in liquid, as shown in Fig. 1, the laser was focused on the surface of the water vertically along the Z-axis and the plasma jet was directed to the interface at 22.5 degrees in the X-Z plane.\nNew text (pg. 15, paragraph 4): For the measurements of O in liquid, as presented in Fig. 1, the effluent of the COST jet was directed onto a volume of deionized (DI) water in a quartz cuvette. The cuvette was linked to a large external reservoir of DI water (~10 litres) to limit the effect of evaporation on the water level over the course of the measurement.\nComment3:\nIf the response to above 2 comments indicate that indeed water was ultrapure and free of oxygen from air than single nanosecond decay of aqueous phase TALIF signals would indicate Oaq involvement in second order reactions with other intermediate species like solvated electrons or OH radicals etc. Is that a plausible explanation of the apparent observation of the very short lifetimes of Oaq or is the concentration of these transient species too low at the near surface depths to become relevant?\nResponse: Respectfully, it appears the reviewer is conflating the laser-excited lifetime of O aq and the chemical lifetime. The former is very fast due to collisions with water molecules leading to quenching of the laser-excited state. Our MD simulations estimate a laser-excited lifetime on the order of 10s of ps assuming all collisions result in de-excitation, although the ICCD does not allow us to measure this directly. The latter is mediated by chemical reactions in the water, primarily with solvated O as previously discussed, which occur on the time scale of 10s of µs.\nComment4:\nIs it possible to introduce Oaq scavenging solutes (phenol, chloride ions etc.) to APPJ exposed aqueous solutions and measure TALIF signals dependence on their concentration and penetration depth to unequivocally prove these signals indeed originate from Oaq?\nResponse: As the reviewer correctly suggests, this would reaffirm the presence of solvated O. However, the authors believe that the off-resonance measurement, in conjunction with the temporal evolution of the signal in liquid vs. air conclusively demonstrates that O is being observed. To further convince readers of this, additional figures (2b and 2c) have been added, along with some corresponding text.\nOld text (pg. 6, paragraph 3): The on-resonance signal is displayed in Fig. 2a after the contribution from the laser background is subtracted. The signal below the liquid interface peaks at around 30 counts s-1 and is discernible several hundred micrometres from the surface (depicted in Fig. 2b).\nNew text (pg. 6, paragraph 3): The on-resonance measurement is displayed in Fig. 2a after the contribution from the laser background is subtracted, while Figs. 2b and 2c confirm that the signal originating in the water is not observed after a short time delay (3 ns) or off-resonance (224.7 nm). In Fig. 2a, the signal below the liquid interface peaks at around 30 counts s-1 and is discernible several hundred micrometres from the surface.\nOld text (pg. 7, Figure 2 caption): Figure 5: (a) The on-resonance, laser background-subtracted O fluorescence signal, recorded with the ICCD gated to capture the laser pulse and TALIF signal in water. (b) Absolute densities of atomic O with the location of the water surface annotated. Note that the density calibration shown here is only accurate below the water surface where the appropriate branching ratio applies. In (b), a delayed gate measurement 3 ns after the laser pulse has been subtracted from the initial measurement (see Methods), as indicated by the distorted shape of the TALIF signal in air. Due to the aggressive quenching of laser-excited O in the liquid environment, no O fluorescence is visible after this time delay. This then accounts for the contribution of isotropic scattering from the TALIF signal originating in the gas phase, highlighting the fluorescence from solvated O atoms.\nNew text (pg. 8, Figure 2 caption): Figure 6: (a) The on-resonance, laser background-subtracted O fluorescence signal, recorded with the ICCD gated to capture the laser pulse and TALIF signal in water. The water surface is annotated by the red line. (b) The time-delayed, on-resonance O fluorescence signal recorded 3 ns after (a). Due to the aggressive quenching of laser-excited O in the liquid environment, no O fluorescence is visible after this time delay. (c) The off-resonance (wavelength centred at 224.7 nm), laser background-subtracted signal, gated to include the laser pulse. (d) Absolute densities of solvated atomic O. Note that the density calibration applied here is only accurate below the water surface where the appropriate branching ratio applies. Here, (b) has been subtracted from (a) to account for scattering from the intense gas-phase fluorescence, resulting in the distorted shape of the signal above the water surface."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed many of the comments of the reviewers although one key issue (and the most important one) remains. Authors should perform O-scavenger measurements to show without doubt that O radicals are measured in the liquid phase. This has also been stressed by another referee. Off resonance measurements do not provide any evidence of the origin of the fluorescence signal measured as claimed by the authors.\nThe reviewer is not fully convinced about the very limited explanation of the liquid dynamics provided. Typically, an asymmetric 'bubble' with a gas jet impinging on the liquid surface will oscillate (authors only seem to address slow dynamics on second/minute time scales). This is important because the O TaLIF signal claimed to originate from the liquid phase could be explained alternatively by gas phase O TaLIF signal at the time that the bubble has the largest depth. This concern can be addressed with the O-scavenger measurement.", "reviewer_2": "see the attached text file and video.", "reviewer_3": "I thank the authors for their additional comments. In my opinion the paper can be accepted for publication as is."}, "author_response": "Reviewer 1\n(1) The authors have addressed many of the comments of the reviewers although one key issue (and the most important one) remains. Authors should perform O-scavenger measurements to show without doubt that O radicals are measured in the liquid phase. This has also been stressed by another referee. Off resonance measurements do not provide any evidence of the origin of the fluorescence signal measured as claimed by the authors.\nResponse: As mentioned in the cover letter, multiple attempts were made with known O scavengers to address the comments from reviewers 1 and 2. Both phenol and the spin trap TEMP exhibited strong fluorescence of their own at the excitation wavelength. Considering the very limited number of documented O scavengers in liquid, additional experiments instead focused on establishing the temporal nature of the reported signal. These new measurements, shown in figure 2e, clearly demonstrate the distinct quenching environments of the gas and liquid phases, with the latter exhibiting a laser-excited lifetime of <250 ps. This eliminates any possibility that the signal observed below the water surface originates from the gas phase. When combined with the ultra-narrowband filter at the fluorescence wavelength and the absence of an off-resonance signal, O is the only explanation for the fluorescence below the water surface. Along with the new temporal measurements, an additional figure was included to emphasize the intensity of the O signal. The new figure (2c) displays the signal-to-background ratio of the O fluorescence. We feel that the inclusion of this data showing signal-to-background ratios in excess of 5 for the region where solvated O is expected, highlights that the signal below the water surface cannot originate from any other source than O fluorescence. In other words, the observed signal is not the result of an errant background subtraction or some other artifact.\nWe also want to note that we do not agree with the reviewer’s characterization that “off-resonance measurements do not provide any evidence of the origin of the fluorescence signal”. The absence of a signal when tuning the laser slightly off resonance is a clear indication that O fluorescence is the source of the signal, especially considering the use of an ultra-narrowband filter centered around the fluorescence wavelength. Reviewer 2 agrees with this assessment, stating, “I agree with the authors that their measurements provide the evidence that the signal originates from O atoms,” when referring to the off-resonance measurements (see comment 3 from Reviewer 2).\n(2) The reviewer is not fully convinced about the very limited explanation of the liquid dynamics provided. Typically, an asymmetric 'bubble' with a gas jet impinging on the liquid surface will oscillate (authors only seem to address slow dynamics on second/minute time scales). This is important because the O TALIF signal claimed to originate from the liquid phase could be explained alternatively by gas phase O TALIF signal at the time that the bubble has the largest depth. This concern can be addressed with the O-scavenger measurement.\nResponse: This comment was brought up by the second reviewer as well, who actually included a video documenting the induced flow with the same reference plasma source. Even in the self-described “quick and dirty” experiment, the video from Reviewer 2 shows a clean, laminar, induced flow in the liquid up to 1.8 slm. While oscillations start at 2 slm in the video, with a more careful setup, a laminar surface is also achievable at 2 slm. Furthermore, the aggressive vibrations shown at 2 slm would be clearly visible in the long timescale measurements used to track the location of the surface (as shown in figure 1 in the manuscript). Below are two stills from the video provided by Reviewer 2. The first shows the laminar surface at 1.8 slm, while the second shows the vibrating surface at 2 slm.\nReviewer 2\n(1.1) The most important information for me was the additional image on the page 8 of the response letter, which is shown here together with the figure 3(a) from the manuscript. The authors write that they have performed measurements at 15 locations along the surface, but only 8 can be recognized in this figure (where are the remaining ones?). Additionally, all significant O fluorescence in the water belongs only to one (first from the left) position of the laser beam, which is also shown in figure 2 as the only example. This is also the laser location with the strongest fluorescence in the gas phase above the liquid and one of the shortest ones (vertical length). All other positions show weaker gas phase fluorescence with different relative position to the liquid surface (see the strange “dark space” between the gas phase fluorescence and water surface for the next two locations as indicated in the image). The fact, that the measurement of O TALIF signal below the water surface is observed at only one location could also be the artefact caused by the sudden movement of the liquid surface during this single measurement. Having this anomaly just 1 % of the acquisition time of 15 minutes (9 seconds) would result in the averaged signal intensity of 10 counts out of 1,000. Or some other anomaly of the laser position (seems to be vertically lower than the neighboring TALIF signal) or laser power (the most intense one) could be the cause of the observed effect. Have been the measurements repeated on several different days and with different O concentrations or gas flows (see also my comments later)?\nResponse: We understand why this may be a source of confusion. To clarify, additional images are included below. Clockwise from top left, the first image is the one referenced by the reviewer included in the last rebuttal letter. It is the same as figure 3a in the manuscript, just with a scale that highlights the gas-phase fluorescence. It is composed of 13 spatial measurements, although 14 total were collected.\nThe second image (top right), shows spatial measurements 1–5. Here, the color scale better highlights the fluorescence below the water surface. Two things to note: First, this is the region, to the left of the apex of the deformation zone, where O is expected based on the induced flow. Indeed, we find a robust signal below the surface, especially for spatial measurements 3–5. In this way, the reviewer is incorrect that O fluorescence is only observed for one spatial measurement. The second thing to note is the errant signal on the far left side of the frame below the water surface. This is from spatial measurement 1 and due to the laser background not aligning with the measurement. This occurs at this location because of the laser diffraction from the steep edge of the deformation zone. As a result, even the very slight water evaporation between the two measurements results in a misalignment between measurement and background. Since this signal is clearly erroneous, it was not included in figure 3a (hence 13 spatial measurements instead of 14).\nThe third image (bottom left), shows spatial measurements 6–10. In this region, to the right of the apex of the deformation zone, the induced flow is parallel to the gas flow and the penetration depth of oxygen atoms should be mediated by diffusion. As a result, no fluorescence signal should be evident. This is exactly what is observed, with only measurement noise below the water surface.\nThe fourth and final image (bottom right), shows spatial measurements 11–14. Similar to the previous image, solvated O should not be detectable in this region. Again, this is what is found in these measurements. In addition, they highlight the lensing of the gas-phase fluorescence due to the deformed air-liquid interface, resulting in the overlap of the gas-phase signals. This also occurs for spatial measurements 1–5, although to a lesser extent, explaining why the reviewer only counted 8 spatial measurements in the figure provided in the last rebuttal letter.\nTo respond to the reviewer’s final question, yes, in addition to the O signal appearing in multiple spatial measurements, it appeared in other measurements over several days. At the time, practical constraints (a visiting Ph.D. student at a collaborative facility) precluded a more expansive series of measurements. Now, with additional experiments having been performed, the O signal has also been observed in two different laboratories with different personnel.\n(1.2) This image also brings another question related to the details of the measurement, which are not provided in the manuscript. How long is the effective area with TALIF signal (depth-of-field of the laser waist?). Is it roughly the length seen in the image here (< 0.8 mm)? And was any attempt performed to scan this position vertically, to “submerge” this area more into the water to see, whether there is any dependence of the depth profile from this radial position (it shouldn’t be the case)? The precise size and the location of the laser “spot” relative to the surface of the liquid (for example half above the liquid/half below?) should be provided in the figure 1. Was the vertical relative position adjusted to be the same during the scan along the water surface? Any change in this relative position can influence the result because the TALIF signal is very steeply decaying at the edge of the depth-of-field. The test with the vertical variation of the laser position should be part of the manuscript to make sure that the observed penetration depth is not an artefact of the laser light interaction with the liquid (see my comments at point 2) below).\nResponse: While this is a valid concern, the use of a spherical lens with a 20 cm focal length brings the laser to a uniform beam waist over a large area relative to the expected penetration depth of atomic oxygen. The calibration measurements with xenon demonstrate this nicely, as shown below. Here, even with the Xe flowing from the jet (not a homogenous Xe distribution), the fluorescence appears uniform over a wide area. Note that these measurements were performed with the plasma source angled, so the laser was passed perpendicular to the gas flow.\n(1.3) As you can see, the additional image, provided only in the response letter, is an important source of information about the experiment and it should be part of the manuscript or at least part of the supplementary information file (SIF). I would also appreciate to have figure 2 for all 15 locations in the SIF. Additionally, showing the TALIF measurements in the gas phase without the presence of the cuvette could clearly show the size of the area effectively scanned by the laser at one given location. The previous measurements of the authors above the liquid (reference 25) have been performed with the ps laser for the jet with just 1 slm gas flow. The results with fs laser, at the larger gas flow, and without the liquid should be provided in the manuscript or at least in SIF for this article to demonstrate that the gas phase measurements are consistent with the previous results and can be used for the density calibration of the O density in the liquid.\nResponse: We would be happy to provide these measurements in a supplementary information file (for both the gas and liquid phases) if the editor thinks it would add clarity to the results and/or increase transparency. Regardless of a supplementary information file, all results will be made openly available following manuscript acceptance, as indicated in the data availability statement.\n(2.1) The authors have explained the observed large penetration depth of O atoms of 400 micrometers by the possible convection transport, citing two simulation works with liquid velocities of 1-2 m/s. It should be, however, noted that this large velocity is usually observed at the surface and parallel to the surface. The liquid velocity perpendicular to the surface is much smaller. Additionally, the liquid flow at the position with the measured highest O concentration aq is from the bulk towards the surface, in the opposite direction than needed to explain the observed density distribution. We have performed “quick and dirty” experiment with 10 x 10 mm2 cross section cuvette filled with distilled water with dispersed small particles illuminated from the side by laser light. Helium gas flow at the same angel of 22.5° to the normal from a rectangular tube with the inner cross section of 1 x 1 mm2 and distance of 5 mm to the undisturbed liquid surface have been used to simulate the experimental conditions used in the manuscript. The image here shows the position of the illuminated particles, where the exposure time of 30 ms results in elongated traces there, where the particles are moving fast. The arrow indicates the rotation of the vortex observed on the left-hand side of the image. The liquid (the particles) is moving upwards in the direction of the surface underneath the observed dip and cannot, therefore, explain the penetration depth of 400 micrometers observed by the authors.\nResponse: It is true that the flow induces a large-scale clockwise circulation in the liquid, as the reviewer suggests. However, on the right-hand side of the deformation zone, the gas flow accelerates the water parallel to the surface. Because of the acute angle of the deformation zone, the water that was previously moving parallel to the surface has a velocity component perpendicular to the surface at the apex. Just to the left of the apex is where we would expect to (and do) see O transported to some depth. The video included by the reviewer actually gives an excellent depiction of this. In the series of frames included below, taken with a 1.6 slm gas flow, a large, illuminated particle moves towards the surface in the broader clockwise circulation. It then comes to a near stop as it approaches the interface near the apex of the deformation zone. Subsequently, it is quickly accelerated by the induced flow to the left — and critically — downward, from the surface. The particle then traverses the exact region where we observe the O fluorescence.\nIt should also be noted that, with a 30 ms exposure, this particle moves about 2 mm in the final frame shown above. This would correspond to an average velocity of 0.07 m/s. Considering the size of the particle, the fact that it was accelerated from rest, and the lower gas flow rate used, these images also support water velocities on the order of 1 m/s (as the referenced simulations have shown in the literature).\n(2.2) The attached video (Video30ms_exposure_time.mp4) shows the dynamic behavior of the dispersed particles for different He flows up to the used value of 2,000 sccm. Again, the particle movement can be clearly seen in this video. As discuss above, this movement is not compatible with the explanation provided in the manuscript. Additionally, the highest flow used (2,000 sccm), which is also used by the authors, results in our case to visible dynamic movement “vibration” of the liquid surface. Was such a movement observed by the authors and was it considered in the error estimation in their analysis of the surface location? They have focused mainly on the problem related to the evaporation, but this dynamic behavior is a potential source of systematic errors especially for the measurements with long integration time. Interestingly, lower flows of for example 1,000 or even 1,600 sccm are not resulting in this dynamic behavior in our case and measurements with this lower gas flows could get rid of this problem. Is there any particular reason to use the high gas flow of 2,000 sccm? I recommend the authors to perform the measurement with some lower gas flow again. Our “quick and dirty” experiment is probably not reproducing perfectly the experimental conditions used by the authors. But similar experiment performed at their setup will provide the necessary information about the possible transport of O atoms in the water, helping them to find the best explanation for their results.\nResponse: As mentioned in the response to Reviewer 1, the attached video provided by Reviewer 2 shows a laminar surface at 1.8 slm for a self-described “quick and dirty” experiment. With some additional time, we were able to achieve this surface behavior with a 2 slm flow. We wanted to maximize the O flux at the surface, so we performed experiments with this flow rate, the highest we could use and still maintain a laminar surface. Considering the importance of the induced flow for detection (which we did not realize at the time) and the reduced flux, it seems possible that solvated O would not be discernable at lower flow rates. As also mentioned in the response to Reviewer 1, the robust vibrations/oscillations documented in the video would have been very evident in the long timescale images we used to track the surface location (shown in figure 1 in the manuscript).\n(3) My last comment is related to the on-resonance measurement, time-delayed on-resonance measurement, off-resonance measurement and measurement without plasma as an evidence of O atoms being measured in the liquid, which are related to the plasma and which are not generated by the laser pulse in the water with dissolved O . I agree with the authors that their measurements provide the evidence that the signal originates from O atoms. One issue, which was not considered yet, is the possibility that the laser light is dissociating the weakly bonded ozone molecules, which are generated in the water in the O + O recombination reaction. The O atoms generated in such a way by the laser light could be than measured in the very same pulse as dissolved O atoms, providing on- resonance TALIF signal only when the plasma is running. Measurements with different O admixtures (e.g. 0.3% O admixture for large amount of dissolved O and low amount of generated dissolved O and 1% O admixture or more for much larger amount of dissolved O with low density of dissolved O) and measurements with vertical scan of the laser spot (O has very larger penetration depth) could clearly resolve this issue.\nResponse: The possibility of photodissociation of ozone and TALIF of the resulting O to explain our signal in water can be ruled out for several reasons including the relative densities of O and O (DOI: 10.1088/1367-2630/12/1/013021) and the fs pulse duration. However, perhaps the most definitive is that this would effectively make the measurement a three-photon process. This would add an additional cross section term ( 𝜎 ) to the measured signal (S), where n is the density, a the branching ratio, and 𝜎2 the two-photon cross section:\n𝑆 ∝ 𝑛 𝜎2 𝑎 → 𝑆 ∝ 𝑛 𝜎 𝜎2 𝑎\nAt 225.6 nm, on the edge of the Hartley band, this value is 𝜎 ≈ 5×10-18 cm2 (DOI: 10.1016/j.chemphys.2007.08.024), while the branching fraction for the production of ground-state O is ~30% (DOI: 10.1063/1.469341). Considering the existing difficulties of measuring O , the contribution from photodissociation of O to the TALIF signal in water would be negligible."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The additional measurements included in the manuscript provides stronger confidence in the origin of the O radicals in solution and the signal does not finds its origin somehow in the gas phase. Hence my major concern is removed.\nI do urge the authors to remain factual as the approach and analysis still has significant limitations. A few examples:\n* Reaction rates, chemical lifetimes and Henry`s law constants have at best been estimated (not measured as suggested in the abstract).\n* The diagnostic is not new, it is applied to a new condition/environment.\n* Unprecedented and groundbreaking seem inappropriate adjectives when describing ones own work.", "reviewer_2": "I have carefully read the authors’ response and the manuscript again. The main source of my doubts was the “seemingly unnatural” depth and localization of the observed O density. The arguments based on the series of frames with an illuminated particle moving close to the apex of the deformation zone are, however, convincing and I can accept them. The fast flow movement “detaching” from the curved surface is specific for their studied case and can indeed explain the localization of the signal and also the measured depth. I also went through all my other previous and current doubts and concerns and I have to realize that the authors have provided plausible explanations and answers for all of them now. I am, therefore, happy to congratulate them on the obtained results and recommend strongly the manuscript for publication in the current form."}, "author_response": "Reviewer 1\n(1) I do urge the authors to remain factual as the approach and analysis still has significant limitations. A few examples:\n* Reaction rates, chemical lifetimes and Henry`s law constants have at best been estimated (not measured as suggested in the abstract).\n* The diagnostic is not new, it is applied to a new condition/environment.\n* Unprecedented and groundbreaking seem inappropriate adjectives when describing ones own work.\nResponse: Language has been adjusted throughout the manuscript to remain completely factual regarding the work and its implications. This includes (as was stated in the author checklist) removal of instances of new/novel/first or other exaggerated language.\nReviewer 2\n(1) I have carefully read the authors’ response and the manuscript again. The main source of my doubts was the “seemingly unnatural” depth and localization of the observed O density. The arguments based on the series of frames with an illuminated particle moving close to the apex of the deformation zone are, however, convincing and I can accept them. The fast flow movement “detaching” from the curved surface is specific for their studied case and can indeed explain the localization of the signal and also the measured depth. I also went through all my other previous and current doubts and concerns and I have to realize that the authors have provided plausible explanations and answers for all of them now. I am, therefore, happy to congratulate them on the obtained results and recommend strongly the manuscript for publication in the current form.\nResponse: The authors appreciate the reviewer’s kind comments."}]

Decision letter
[Review submitted as PDF attachment - text not found in document]