Tracking polar solvation dynamics of a photoexcited organic chromophore with ultrafast X-ray scattering

Domain: chemistry
Published: 2026-04-13
Editorial outcome: major_revision

Abstract
Solvation plays a key role in many photochemical and biological processes. Yet, direct atomic-scale observation of solvent reorganization around photoexcited molecules containing only light atoms has so far remained elusive. Here, we use time-resolved X-ray scattering at an X-ray free-electron laser to track with a 120 fs time resolution the photoinduced ultrafast rearrangement of polar acetonitrile solvent molecules around an organic hemithioindigo chromophore. The experiments reveal that the solvation shell reorganizes with a 0.3 ps time constant driven by photoinduced charge transfer in the chromophore, and then reequilibrates over 3 ps as the excited state population returns to the ground state. These results demonstrate the viability of using ultrashort X-ray pulses to directly visualize the motion of light atoms in solution, paving the way for atomic-scale understanding and control of photoinduced processes in biological and synthetic photoactive organic molecules.

Introduction
Typically, photochemical and photophysical processes occur in a condensed-phase environment. In a liquid solution, the solvent molecules are not just spectators but play an active role, sometimes by directly participating in photochemical transformations, such as in photoinduced ligand substitution reactions of solvated metal complexes [1,2]. More generally, changes in the electronic and atomic distribution of a solute upon photoexcitation trigger the rearrangement of surrounding solvent molecules to minimize the overall free energy, thereby affecting the rates and outcomes of the excited state relaxation [3–6]. These nonequilibrium solvation dynamics are especially critical when they involve the response of a polar solvent to a photoinduced charge transfer. In this case, the solvent molecules adapt to the change in the local electric field of the excited solute by reorienting their dipole, a process known as polar solvation dynamics. This response in turn modulates the charge distribution, atomic structure and energetics of the photoexcited chromophore [5, 7–10]. Understanding polar solvation dynamics at the atomic level is essential, as photoinduced charge transfer is a key step in fundamental natural processes such as photosynthesis [11] and applications in, e.g., solar energy conversion [12] and optoelectronics [13].

Our current understanding of polar solvation dynamics stems largely from time-resolved optical spectroscopy experiments [5, 8, 14] and theoretical studies, particularly molecular dynamics (MD) simulations [15–24], conducted over the past few decades. The free energy minimization induced by the solvation-shell response gives rise to shifts of emission and absorption spectra. A common approach is to track the dynamic Stokes shift, i.e. the time-dependent redshift of fluorescence [25–27]. More recently, time-resolved terahertz spectroscopy and nonlinear spectroscopy techniques, particularly two-dimensional electronic and electronic-vibrational spectroscopies, have also been employed to investigate solvation dynamics triggered by photoinduced processes [28–30]. These approaches are important to gain insights into the excited state solute-solvent interactions driving solvation dynamics, but they do not directly provide an atomic-scale picture of the solvent rearrangement.

Ultrashort, highly brilliant X-ray pulses generated at X-ray free-electron lasers (XFELs) make it possible to track photoinduced processes at the atomic length scale. In particular, time-resolved X-ray solution scattering (TR-XSS) at XFELs [31, 32] can directly probe changes in atomic arrangements (time-resolved X-ray liquidography [32]), both in the solute [33–40] and surrounding solvent, which includes rearrangements of the solvation shell [17, 21, 41–43]. TR-XSS has been used to elucidate the solvation-shell rearrangement of photoexcited diatomic halogen molecules [44] and metal complexes [21, 43], the coherent solvent response to charge transfer in a bimetallic complex [17], the solvent mediated photodissociation and associated atomic charge redistribution of triiodide [39, 42], the solvation dynamics following photoabstraction of an electron from halogen ions [16, 41]. All these TR-XSS studies leveraged the presence of electron-rich atoms in the solute, which amplifies both the solute and the solvation-shell response signals relative to the large scattering contribution of the bulk solvent, facilitating signal acquisition and interpretation [45]. In references [17, 39], this made it possible to infer the charge redistribution within the solute through the solvent response.

The direct observation of atomic motions involving organic molecules, containing only light, weakly scattering atoms, has so far been limited to the gas phase [32, 46]. A recent study by Ihee and coworkers [47] used TR-XSS to probe the kinetics and transient intermediates of a photoexcited solvated p-hydroxyphenacyl organic molecule indirectly, by monitoring the solvent heating signal due to energy transfer from the solute. However, to our knowledge, no experimental study to date has directly captured atomic-scale solvation dynamics around an organic chromophore. This represents a significant gap, given the importance of organic photoactive molecules for energy conversion, optoelectronics, photopharmacology, and biological systems [14]. Direct structural insights into the solvent response are essential for understanding how the local environment governs photorelaxation pathways. Such knowledge could ultimately enable control over the lifetime of charge-separated states and help reduce energy loss due to thermal dissipation, thereby improving efficiency and stability in photosensitization and photoconversion.

Here, the ultrafast, atomic-scale solvation dynamics of the polar solvent acetonitrile triggered by charge transfer in a photoexcited hemithioindigo (HTI) organic chromophore are directly captured using TR-XSS measurements at the SwissFEL XFEL facility. HTIs [4, 48–51] consist of thioindigo and stilbene fragments connected by a double and a single carbon-carbon bond. Previously, it has been shown that the photorelaxation of HTIs with strong electron-donating dialkylamino substituents is dramatically influenced by the solvent: In apolar solvents, photoexcitation predominantly induces isomerization from a Z to an E isomer (see Suppl. Fig. 27) via double bond twisting, whereas in polar solvents, the formation of a twisted intramolecular charge transfer state has been proposed to dominate [4, 49]. Evidence for this charge transfer state in polar environments includes a reduced Z/E photoisomerization yield (< 1%), dual fluorescence, and red-shifted features in transient optical absorption, emission, and time-resolved vibrational spectra [4, 49]. A ground state geometry partially twisted about the connecting single bond (see Suppl. Note 9) has been proposed as a prerequisite for formation of the intramolecular charge transfer state [50], wherein the thioindigo and stilbene units may further twist, potentially adopting a near-perpendicular conformation. In the present work, measurements are performed on an HTI where the stilbene moiety is replaced by a strong electron electron donating julolidine group (hereafter referred to as HTI-J, see Fig. 1). A large increase in the dipole moment of HTI-J upon electronic excitation, from 5.3 to 27.3 D at the Franck-Condon geometry, is predicted by density functional theory (DFT) calculations (see Suppl. Note 9). Therefore, photoexcitation in acetonitrile is expected to induce a large reorganization of the solvent molecules around the solute.

Fig. 2 illustrates the experimental setup for the TR-XSS measurements, along with the dynamical processes following photoexcitation of the HTI-J molecule, as revealed by the TR-XSS and supporting transient absorption spectroscopy (TAS) experiments and DFT calculations. By analyzing the time-resolved X-ray scattering signal with the support of MD simulations, the solvation-shell response is disentangled from the dominant scattering contribution of bulk solvent heating and other structural changes arising from vibrational relaxation. It is found that reorganization of the acetonitrile molecules around the solute is driven by intramolecular charge transfer and occurs with a timescale of 0.3 ps. The solvation-shell then reequilibrates as the excited state population undergoes internal conversion returning to the ground electronic state on a 2.6 ps timescale.

This work demonstrates the feasibility of visualizing motions of light atoms in solution via ultrafast hard X-rays, highlighting their potential as a powerful tool for probing solvation phenomena in organic photochemistry at the atomic scale. By directly tracking the polar solvation dynamics following photoexcitation of HTI-J, it is shown that the solvent response is dominated by intramolecular charge transfer, which drives a pronounced reorganization of the surrounding solvation-shell, rather than by a structural change of the chromophore. Thus, our results provide structural evidence for the formation of a charge transfer excited state in electron donor substituted HTIs in polar solvent, and yield insights into the microscopic mechanism underlying polar solvation dynamics in these systems.

Results
Excited state lifetime from optical transient absorption spectroscopy

Femtosecond optical TAS provides information on the excited state population kinetics of HTI-J in acetonitrile, supporting the analysis and interpretation of the structurally sensitive TR-XSS measurements. Fig. 3a shows the transient absorption spectrum as a function of the probe wavelength and time delay, recorded by exciting the sample at 490 nm, close to a maximum of the static absorption spectrum of HTI-J in acetonitrile (see Suppl. Note 2).

Data below 530 nm are affected by scattering of the pump light and are not considered in the analysis. The transient spectrum displays a broad excited state absorption band from around 540 to 690 nm that decays within 10 ps, and a shorter lived stimulated emission feature from around 670 to 750 nm, the red edge of the probe range. These features are consistent with those previously observed by Wiedbrauk et al. [4, 50] in transient absorption spectroscopy of HTI-J in polar solvents. The TA spectrum is found to be well described by a kinetic model consisting of a sum of three exponential decay components convolved with the instrument response function (IRF) and a constant offset. Global fitting with this kinetic model yields two spectral components that decay rapidly with time constants τTAS = 0.5 ± 0.3 ps and τTAS = 1.2 ± 0.7 ps, and a slower component decaying with τTAS = 2.6 ± 0.5 ps that accounts for most of the excited state absorption feature dominating the spectrum (more details on the TAS analysis can be found in Suppl. Note 3). Fig. 3b shows a time trace of the spectrum for a wavelength of 585 nm, around the maximum of the excited state absorption, together with the corresponding trace of the fitted model. DFT calculations predict that excitation of HTI-J in acetonitrile occurs directly to the lowest excited state, which has a charge transfer character (see Suppl. Note 9.2). Based on this and following Wiedbrauk et al.’s assignment of TA spectral components of HTI compounds [4, 50], the fast kinetics are attributed to structural and vibrational relaxation of the solute in the excited state and decay of stimulated emission, while the 2.6 ± 0.5 ps decay is interpreted as the time τIC for internal conversion to the ground state, i.e. the excited state lifetime. The offset likely accounts for a minor population of the E isomer through photoisomerization [4, 50].

Structural dynamics revealed by time-resolved X-ray solution scattering

The time-resolved X-ray scattering signal of a solution of HTI-J in acetonitrile was measured in a laser pump X-ray probe experiment at the SwissFEL XFEL to directly track the structural dynamics after photoexcitation. In the TR-XSS experiments, the ensemble of HTI-J molecules in acetonitrile was probed with X-ray pulses of 11 keV mean photon energy, either with (laser-on) or without (laser-off) prior excitation at 470 nm, corresponding to the maximum of the absorption spectrum, and an excitation fluence of approximately 1.2 mJ/mm−2, corresponding to the upper end of the linear-response region determined via a laser fluence scan (see Suppl. Note 4.1). Scattering patterns were recorded on the 2D detector as a function of the magnitude of the scattering vector, q, and the azimuthal angle, ϕ. Difference scattering images ∆S(∆t,q,ϕ) = S_on(∆t,q,ϕ) − S_off(q,ϕ) were then obtained by subtracting laser-off from laser-on images at each time delay ∆t between pump and probe pulse and decomposed into isotropic and anisotropic components via Legendre decomposition, yielding 1D difference scattering curves [52, 53]. Further details on the reduction of the scattering data are provided in the Methods section and Suppl. Note 4.2, which includes plots of the reduced isotropic and anisotropic difference scattering signals.

The time-resolved isotropic difference scattering signal is analyzed using a stepwise global fitting approach that builds on global analysis strategies commonly used in time-resolved spectroscopy [50, 54]. The difference scattering data are fitted with a bilinear model of the signal plus background artifact components (see Suppl. Note 4.6):
∆Sfit(∆t,q) = ∆Smodel(∆t,q) + ∆Sbkg(∆t,q) (1)
= Σk A_k(∆t; {a_i, τ_i})C_k(q) + Σl B_l(∆t)D_l(q).
Here, A_k are kinetic profiles consisting of rising and decaying exponential functions parameterized with a set of amplitudes {a_i} and time constants {τ_i} and convolved with the IRF, C_k are the corresponding scattering q-profiles, while B_l and D_l are amplitudes and corresponding q-profiles of background (bkg) components. The global fitting approach is described in detail in the Methods section and Suppl. Note 6. Briefly, in sequential steps, the amplitudes of the background components, B_l, as well as the kinetic profiles and corresponding q-profiles, A_k and C_k, are optimized based on linear least squares minimization [54, 55].

Fig. 4a shows the isotropic difference scattering signal of HTI-J in acetonitrile, ∆Sdata, after subtraction of the background components obtained in the global fitting. The onset of the signal following photoexcitation at time zero can be clearly seen. The signal is noticeably dominated by two positive features, around 1.4 and 2.4 Å−1, and a negative feature, around 1.8 Å−1, which evolve on sub-picosecond and picosecond timescales. These features are consistent with the scattering changes arising from a temperature increase in bulk acetonitrile at constant volume, resulting from dissipation of energy from the photoexcited HTI-J solute [56]. However, the shape of the signal in the first picosecond after photoexcitation deviates from that of bulk solvent heating, indicating the presence of signal from additional structural processes.

Within the global fitting analysis, the measured difference scattering signal, ∆Sdata, is found to be well reproduced by a linear combination of four physically justified signal contributions (see Suppl. Note 5):
∆Smodel(∆t,q) = α(∆t,τ_OKE)∆S_OKE(q) (2)
+ β(∆t,τ_SSR1,τ_SSR2)∆S_SSR(q)
+ γ_VR(∆t,τ_VR1,τ_VR2)∆S_VR(q)
+ I_∆T(∆t,τ_∆T1,τ_∆T2)∆S_∆T(q).
According to the assignment described in detail below, these four contributions represent (i) solvent motions associated with the optical Kerr effect (OKE), (ii) solvation-shell structural rearrangement (SSR) driven by changes in the electronic structure of the excited solute, (iii) solvent rearrangements due to vibrational relaxation of the solute (TVR), and (iv) bulk solvent heating due to a temperature increase of ∆T. α, β, γ, and ∆T with associated time constants τ define the kinetic profiles of the four signal components. The resulting best-fit model scattering signal is shown in Fig. 4b, while the best-fit time constants are reported in Table 1. A plot of the optimized kinetic profiles is provided in Fig. 5d-f and Suppl. Note 6.2.

Solvent heating, vibrational relaxation, and optical Kerr effect

The contribution of the bulk solvent heating, ∆T · ∆S_∆T, is shown in Fig. 4c. It accounts for the majority of the observed signal after the first picosecond. The kinetics of this component are best described by a double exponential rise. The initial rise has a time constant of 0.2 ± 0.1 ps. Since the TAS measurements reveal an internal conversion time of τ_IC = 2.6 ± 0.5 ps, the rapid onset reflects energy transfer from the solute in the excited state. We conclude that this rapid solvent response arises from fast non-radiative relaxation following multiphoton excitation of a fraction of molecules to higher-lying states, as discussed in more detail in Suppl. Note 8. A similar fast rise of the solvent temperature was also observed in a recent TR-XSS study of a photoexcited metal complex in acetonitrile [38]. Except for the rapid onset of the heating signal (τ_∆T1 ≈ 0.2 ps) no contributions from the higher laser fluence in the TR-XSS experiments compared to the TAS could be discerned in our analysis. The second, slower exponential rise has a time constant of τ_∆T2 = 13.5 ± 0.5 ps. This component is attributed to bulk solvent heating resulting from vibrational cooling of the solute in the ground state and is consistent with the decay time of the vibrational relaxation component reported in Table 1 and discussed below. Nonequilibrium MD simulations of the vibrational cooling of the solute and heating of the solvent performed by vibrationally exciting the HTI-J molecule in the ground electronic state give a slow exponential rise of the solvent kinetic energy of ~14.2 ps (see Suppl. Note 10.2), in good agreement with the experimental observation.

Fig. 4d shows the time-resolved isotropic difference scattering signal after subtraction of the background artifact and bulk solvent heating components, highlighting the underlying signal contributions. This signal is compared to the sum of the remaining components of the model in Fig. 4e. The corresponding q-profiles for each of the remaining components are presented in Fig. 5a-c together with associated kinetic profiles in Fig. 5d-f and schematics of the assigned molecular processes in Fig. 5g-i. Heat-subtracted isotropic difference scattering traces at selected delay times, chosen to highlight where specific processes dominate, are shown in Fig. S23 together with the corresponding individual model components.

As can be seen in Figs. 4d-e and 5b, the time-resolved isotropic difference scattering signal contains a component with a negative feature around q = 1.5 Å−1 and positive features around q = 2 Å−1 and below q = 1 Å−1, which rises in the first few picoseconds and then decays on a longer timescale. Global fitting including a single-exponential rise and decay for this component yields time constants of τ_VR1 = 1.6 ± 0.3 ps and τ_VR2 = 15 ± 2 ps, respectively. Since the decay time is comparable to the rise time of the bulk solvent heating signal, this component is attributed to structural rearrangements due to vibrational relaxation of the photoexcited solute. While the solute is vibrationally hot, it displaces or excludes the solvent from a larger volume compared to thermal equilibrium. This leads to changes in the solvent-solvent atom distances, which are reflected in an increase of the scattering signal at low-q [41, 58]. The positive and negative peaks at higher q are assigned to a shift of the liquid peak to higher q, consistent with a local density increase of the acetonitrile solvent around the solute. The initial rise of this component reflects an increase of the excluded volume during internal conversion, consistent with vibrational heating of the solute and molecular dynamics simulations indicating a smaller excluded volume in the excited state relative to the ground state (see Suppl. Note 10.1). The decay time of 15 ± 2 ps is assigned to vibrational cooling in the ground state. This is in agreement with previous time-resolved optical and infrared transient absorption studies of organic molecules in acetonitrile, which report vibrational cooling times of 10–15 ps, largely independent of the solute [59–61].

The fast decaying component visible before 1 ps after subtraction of the background and bulk solvent heating component, with a prominent peak around q = 1.6 Å−1, has a shape (see Fig. 5a) similar to that attributed by Ki et al. [57] to structural dynamics in liquid acetonitrile triggered by the optical Kerr effect. The electric field of the pump laser induces photoalignment of solvent molecules, and the subsequent structural relaxation through translational motions to restore equilibrium produces an isotropic scattering signal [57]. Here, the OKE contribution to the time-dependent isotropic X-ray scattering signal of HTI-J in acetonitrile is found to decay with a time constant of τ_OKE = 0.4 ± 0.1 ps, in agreement with the value of 0.35 ± 0.21 ps reported by Ki et al. [57] for an azo dye solution.

Solvation dynamics

Fig. 5c shows the q-profile of the component of the global fitting of the TR-XSS data attributed to solvation-shell structural rearrangement (SSR) upon photoexcitation of HTI-J in acetonitrile. The presence of the SSR contribution can be inferred from the modulation of the shape of the difference scattering signal on the < 10 ps timescale (see, e.g., the negative feature around q = 1 Å−1 in Fig. 4d). Although small in amplitude, this contribution is found to be necessary for adequately reproducing the observed time-dependent scattering signal, as confirmed by analysis of the residual of a model including only contributions of bulk solvent heating and vibrational relaxation (see Suppl. Note 5.4).

The assignment of the SSR component is supported by DFT calculations and MD simulations (see Computational methods). The DFT calculations predict that excitation leads to a twist around the single bond that connects the thioindigo with the julolidine groups from ~60 to ~90°, and a change in the magnitude of the dipole moment of ~26.3 D, corresponding to the transfer of ~0.7 electrons from the julolidine to the thioindigo group (see Suppl. Note 9.2). Fig. 6a-b show representative radial distribution functions (RDFs) obtained from equilibrium MD simulations in both the ground and excited state, with force-field parameters for the solute derived from the DFT calculations. Fig. 6c shows isosurfaces of the change in number density of methyl and nitrogen sites of the solvent molecules obtained from the simulations. The plots show that upon photoexcitation the average density of solvent methyl sites increases around the thioindigo fragment, which gains electron density upon excitation. Conversely, around the julolidine fragment, there is an increase in the density of nitrogen atoms. These results illustrate how the dipoles of the acetonitrile molecules reorient to adapt to the changes in electric field arising from the intramolecular charge transfer. Excited state MD simulations where only the partial charges are changed in the excited state compared to the ground state give very similar results, indicating that the observed solvation-shell reorganization is dominated by the response to charge transfer, rather than rearrangements due to twisting of the chromophore. Key structural parameters of the solvation shell in the ground and excited states computed from the solute-solvent RDFs are reported in Suppl. Note 10.1.

Fig. 7 shows an average of the experimental data between 0 and 5 ps after subtracting contributions from background artifacts, bulk solvent heating, vibrational relaxation and the signal response to the optical Kerr effect obtained from global fitting with charge transfer and twist as initial guess for the SSR component (see Suppl. Fig. 24). This signal is compared to a difference scattering signal obtained from the MD simulations where both the molecular geometry and partial charges are changed in the excited state (Fig. 7a), and where only the partial charges are changed in the excited state while the molecular geometry is the same as in the ground state (Fig. 7b). The simulated signal in Fig. 7a consists of two contributions, one due to changes in intramolecular distances obtained from the intra-solute RDFs, and one due to changes in solute-solvent atom pair distances obtained from the solute-solvent RDFs. The latter, often referred to as cross term, represents solvent-shell rearrangements encoded in variations of solute-solvent distances. Over almost the entire q-range up to around 3 Å−1, the solute-solvent term dominates over the intra-solute term. The simulated signal shown in Fig. 7b includes only a solute-solvent term due to the solvent response to charge transfer. As expected based on the comparison of RDFs in Fig. 6, the solute-solvent simulated signal due to charge transfer and twisting and due to only charge transfer are very similar. The average experimental signal closely matches the simulated signal, with both models giving a similar quality of the fit (reduced χ2 = 4.4 and 4.5, respectively). For charge transfer and twisting, the fit is marginally improved between 2 to 3 Å−1, but the signal-to-noise ratio does not allow for quantitative discrimination between the two models, meaning that the current data can resolve only the transient solvation-shell structure, not the transient solute structural changes.

As the simulated signals for charge transfer with twisting and charge transfer only are similar (see Suppl. Note 10.1), comparable results are obtained when the latter is used as initial guess for the SSR component in the global fitting (see Suppl. Fig. 24). In contrast, simulations accounting only for the geometric rearrangement of the solute without charge transfer fail to reproduce the experimental SSR component, even when the simulated signal is used as an initial guess for the global fitting (Suppl. Fig. 25). Altogether, these results show the observed solvation-shell reorganization is primarily driven by charge transfer, rather than the rearrangement due to twisting of the chromophore. The structural rearrangements in the solvation shell underlying the observed scattering signal are analyzed in greater detail in Suppl. Note 10.1 through a quantitative analysis of the calculated solute–solvent RDFs.

Having established the origin of the SSR component, we finally turn to the interpretation of the observed kinetics. The kinetic model yields a single-exponential rise with best-fit time constant of τ_SSR1 = 0.3 ± 0.1 ps and a decay with τ_SSR2 = 3 ± 1 ps, respectively. The first time constant corresponds to the rise of the SSR signal due to solvent reorganization driven by photoinduced intramolecular charge transfer of the solute, lowering the free energy of solvation. Measurements of the dynamic Stokes shift [14, 25] and previous MD simulations [18, 23] found that the minimization of the free energy of solvation following intramolecular charge transfer in dipolar solvents has a biexponential time evolution. These timescales were attributed to an initial ultrafast response, driven by the torque exerted by charge redistribution on nearby solvent molecules, and a slower, diffusive reorganization of the solvent, respectively. In the case of acetonitrile, time-resolved emission measurements of coumarin 153, a molecule that also exhibits a significant change in dipole moment upon photoexcitation, yield timescales of approximately 0.09 ps for the initial, fast response and around 0.6 ps for the slower diffusive motion, with an average solvation time of around 0.25 ps [23, 25]. Due to a time resolution of ~120 fs full width at half maximum (FWHM) and limited q-range of the TR-XSS measurements, only a single time constant of 0.3 ± 0.1 ps for the solvation-shell rearrangement could be resolved in the present study, in agreement with the average timescale reported in the previous studies on coumarin 153 [23, 25]. The slower time constant of 3 ± 1 ps reflects the reequilibration of the solvation-shell structure as the excited state population undergoes internal conversion returning to the ground state. This is supported by the close agreement with the excited state lifetime of 2.6 ± 0.5 ps obtained from the TAS measurements.

Overall, the TR-XSS measurements capture with atomic resolution both the ultrafast reorganization of acetonitrile molecules around the photoexcited HTI-J compound and the subsequent reequilibration as the system relaxes to the ground state. The solvent shell reorganization is found to be governed by the response to intramolecular charge transfer, while the response to the structural change of the solute is minor. This polar solvation dynamics provide compelling evidence for the formation of a charge transfer excited state in electron donor substituted HTIs in polar solvents, which has previously been proposed based on the indirect observation of shifts in time-resolved optical and vibrational spectra [4, 49].

Methods
Sample synthesis and handling

HTI-J was synthesized as detailed in Suppl. Note 1. The synthesized compound was transported to the experimental facilities in powder form, where it was dissolved in acetonitrile to prepare sample solutions for the measurements. To prevent photoisomerization of HTI-J under ambient light, the solute samples were stored in the dark and handled exclusively under red light.

Optical transient absorption spectroscopy

Femtosecond optical transient absorption spectroscopy measurements were carried out at the Center for Nanoscale Materials at the Argonne National Laboratory using a commercially available femtosecond laser system (Spectra-Physics) and spectrometer (Ultrafast Systems). Pump and probe pulses were delivered by an amplified Ti:sapphire laser capable of providing pulses with a width of 80 fs. An excitation wavelength of 490 nm was achieved through the use of an optical parametric amplifier and a white-light continuum in the visible range was used to probe the sample solution of HTI-J in acetonitrile with a concentration of 0.3 mM. The measurements were performed with the sample contained in a 2 mm quartz cuvette and stirred continuously. The excitation power was set to 300 µW on a 200 µm spot size and the measurement was repeated with a rate of 2.5 kHz. 19 recorded spectra were averaged and corrected for chirp and time zero. Background signals were removed by subtracting the average before time zero. The borders and pump scatter region were cut and the remaining spectra were binned by a factor of two along the wavelength axis and globally fitted with a kinetic model including a sum of three exponential functions and an offset using the python package KiMoPack (version 7.12.14) [54]. Details on the analysis of the optical transient absorption spectra can be found in Suppl. Note 3.

Time-resolved X-ray solution scattering

Laser pump, X-ray probe experiments were conducted at the Alvra endstation of the SwissFEL X-ray free electron laser. A 3 mM sample solution of HTI-J in acetonitrile was supplied by a liquid jet with a diameter of 100 µm and a flow speed sufficient to replenish the sample for each pump-probe event at 100 Hz repetition rate. An optical laser with an excitation wavelength of 470 nm and focused to a spot size of 73x95 µm (FWHM) was used to excite HTI-J at the absorption maximum (Z-isomer) with a laser energy of 12 µJ per pulse (for a laser fluence scan see Suppl. Note 4.1). Probing of the sample solution after a variable time delay ∆t was achieved with X-ray radiation with a photon energy of 11 keV and a pulse energy of around 1 mJ/pulse focused to a spot size of 30x30 µm (FWHM). A 6:1 pulse scheme was used, meaning that every seventh measurement was performed without prior laser excitation. A Jungfrau 16M two-dimensional detector in fixed gain mode with medium gain for the center modules and high gain for the edge modules was used to record the forward scattering at a sample-detector distance of approximately 9.7 cm, which was calibrated using a LaB6 reference measurement. The arrival time of the X-ray pulse relative to the optical laser was monitored by a timing tool [77]. Suppl. Table 2 summarizes key experimental parameters. The acquired raw 2D scattering images were corrected for the setup geometry and masked. Images deviating from the median measurement conditions were rejected based on an intensity filter and the correlation between the measured intensity and an I0 intensity monitor, resulting in rejection of approximately 30% of the images. The remaining images were integrated in 13 azimuthal bins [52, 78], normalized and the difference scattering signal was calculated by subtracting laser-off signals obtained without prior laser excitation from each laser-on signal. The generated difference scattering signals were re-binned into equistatistical time bins based on the timing tool information and averaged. The averaged difference scattering signal was decomposed into its isotropic and anisotropic components [52, 53] and scaled to the smallest stoichiometrically representative unit, the liquid unit cell (LUC). A detailed description of the data reduction can be found in Suppl. Note 4. The analysis presented here focuses on the isotropic difference scattering signal, which is referred to as ∆S(∆t,q) throughout the article.

Global fitting of time-resolved X-ray solution scattering data

After reduction, the time-resolved isotropic difference scattering signal is given as a function of a set of M values of time delay {∆t_1, ∆t_2, ..., ∆t_m} and N values of scattering vector magnitude {q_1, q_2, ..., q_n}. A stepwise global fitting procedure was used to analyze the data, employing a bilinear model of the signal plus background (bkg) artifact components (see also eq. 1):
∆Sfit(∆t_m,q_n) = ∆Smodel(∆t_m,q_n) + ∆Sbkg(∆t_m,q_n) (3)
= Σk A_k(∆t_m; {a_i, τ_i})C_k(q_n) + Σl B_l(∆t_m)D_l(q_n).
In matrix form,
∆Sfit = ∆Smodel + ∆Sbkg = A · C + B · D, (4)
where A is an M × K matrix with columns representing K kinetic profiles, dependent on amplitudes {a_i} and time constants {τ_i} and convolved with the IRF, C is a K × N matrix with rows containing the corresponding scattering profiles, B is an M × L matrix of time-dependent amplitudes for L background components with q-profiles contained in the rows of the L × N matrix D. Suppl. Note 7 describes the kinetic functions used in the model, while no kinetic form is assumed for the amplitudes of the background components. In the global fitting, A, B and C are optimized by minimizing the square of the residual between the data and the fit function, while the background artifact profiles contained in D are fixed to left singular vectors of an SVD of the difference scattering data at negative time delays (see Suppl. Note 4.6).

First, both the kinetic profiles of the model and the amplitudes of the background components are optimized by solving
min_{A, B} ||∆Sdata − A · C_0 − B · D||^2_σ, (5)
where C_0 contains initial q-profiles that are kept fixed in this step and σ is the noise estimated using a piecewise polynomial fitting [62] (see Suppl. Note 4.7). ||·|| denotes the loss function. In the present global fitting, the more outlier-resistant arctan loss function was used. The model includes four signal components (see eq. 2). The initial q-profile for the component representing bulk solvent heating is obtained as an average of the experimental data after 55 ps, ∆S_∆T = < ∆Sdata >_{55<∆t<60ps}, when the signal has stabilized, indicating that the solute has dissipated all excess energy, reaching thermal equilibrium in the ground state. The initial q-profile of the other components is obtained either from an SVD of the data (optical Kerr effect and vibrational relaxation terms, ∆S_OKE and ∆S_VR), or as the difference scattering signal calculated from intra-solute and solute-solvent RDFs of equilibrium MD simulations (solvation-shell structural rearrangement term, ∆S_SSR), as described in more detail in Suppl. Notes 5 and 10.1.

In a second step, the q-profiles are optimized by solving
min_{C} {||∆S' − A · C||^2 + W ||C − C_0||^2}, (6)
where ∆S' = ∆Sdata − B · D are the data after subtraction of the background components obtained in the first step. The optimization can be regularized by penalizing deviations from the initial q-profiles through the term W ||C − C_0||^2, where W is a diagonal matrix with the square roots of the regularization parameters on the diagonal (see Suppl. Note 6.1 for more details). In the present analysis, the q-profile of the bulk solvent heating is fully constrained as the average at late time delays is expected to accurately represent this contribution, while no constraint was placed on the other components.

In the final step, the kinetic profiles are reoptimized by fitting the data after subtraction of the background components, while keeping the q-profiles fixed at the values obtained in the previous step:
min_{A} ||∆S' − A · C||^2_σ. (7)

Computational methods

The DFT calculations used to obtain equilibrium geometries and partial charges for the solute in the MD simulations employ the range-separated hybrid functional CAM-B3LYP [79–81] and an ma-def2-TZVP basis set [82, 83]. Excited state calculations are performed using linear-response time-dependent DFT (TDDFT) [84] as well as a time-independent density functional approach where the orbitals are variationally optimized for the excited state [85, 86]. The orbital-optimized calculations are performed within the spin-unrestricted formalism by optimizing the orbitals after promotion of an electron from an occupied orbital of the ground state to an unoccupied one. These orbitals are identified as the orbitals of the electron-hole pair that contributes most significantly to the lowest excited state with significant oscillator strength as obtained in the TDDFT calculations (in this case, this state also corresponds to the lowest excited state overall). All DFT calculations include implicit solvent effects through the conductor-like polarizable continuum model (CPCM) [87] for acetonitrile. Previous studies [19, 23] have shown that the excitation and solvation energy obtained with polarizable continuum models are consistent with the corresponding ensemble-averaged values from fully quantum-mechanical calculations including explicit solvent molecules. The structural parameters and partial charges of the HTI-J molecule in the excited state used in the MD simulations are obtained from the orbital-optimized calculations, since this approach has been shown to provide more accurate properties, such as excitation energy [88] and dipole moment [89], for intramolecular charge transfer excitations than TDDFT. To validate the DFT calculations, additional calculations were carried out using the domain based local pair-natural orbital (DLPNO) coupled cluster method with singles, doubles and perturbative triples (CCSD(T)) [90, 91], including implicit solvent effects through CPCM. All DFT and CCSD(T) calculations were performed using the ORCA software version 6.0 [92, 93]. Further aspects of the DFT and CCSD(T) calculations and the corresponding results are presented in Suppl. Note 9.

The MD simulations use the generalized Amber force field (GAFF) [94]. For the HTI-J molecule, equilibrium bond lengths, angles, and selected dihedral angles, as well as partial charges, are assigned based on the ground and excited state structures optimized relaxing the atomic forces described using DFT with the CAM-B3LYP functional and CPCM. The HTI-J molecule and the acetonitrile molecules are flexible in the simulations. To prevent non-bonding interactions to make the HTI-J single-bond twist angle deviate too much from the DFT equilibrium value, this angle is restrained by using larger force constants than the ones provided by GAFF. Other force constants are kept at the default GAFF values. The HTI-J partial charges are derived from fitting the molecular electrostatic potential obtained in the DFT calculations using the CHELPG method [95]. The MD simulation box includes one HTI-J molecule and 717 acetonitrile molecules. MD simulations with fixed-value partial charges have been shown to describe the orientational response of a polar solute to charge transfer in the solute [19, 23]. Moreover, using a flexible solute with fixed partial charges has been shown to capture some polarization effects, since the solute can distort in response to solute–solvent interactions and thereby change its instantaneous and average dipole moment relative to the equilibrium geometry in an implicit solvent [23].

Four sets of equilibrium MD simulations were performed, for the ground state and for three different models of the excited state: charge transfer and twist, only charge transfer, and only twist. In the first model, both the geometry and partial charges are changed in the excited state simulations. In the second, only the partial charges are changed while the ground state geometry is retained. In the third, only the geometry is changed while the ground state partial charges are retained. These simulations correspond to the HTI-J molecule equilibrated in the excited state with a fully formed solvation shell, and do not account for nonequilibrium solvent motions during the solvation shell rearrangement. First, a 5 ns NPT run at room temperature (298.15 K) and pressure of 1 atm was performed for the ground state to find the equilibrium volume of the box, resulting in a side length of approximately 40.1 Å. Using this box size, the system was further equilibrated in the NVT ensemble for 5 ns for the ground state and the three models of the excited state. For each system, after the equilibration, an NVT production run of 400 ns was performed, collecting one frame every picosecond, in total 400000 equidistant frames. All of these frames were used in the subsequent calculations of the equilibrium RDFs. The timestep used was 1 fs in all equilibration runs and equilibrium MD simulations.

For the nonequilibrium MD simulations of the vibrational cooling of the HTI-J and the heating of the solvent, approximately 4000 frames were selected from the equilibrated NVT trajectory. The velocities of HTI-J atoms were then rescaled according to a Maxwell-Boltzmann distribution to achieve a total kinetic energy increase of 2.64 eV, corresponding to the photon energy of the laser pump pulse used in the TR-XSS experiments. Each nonequilibrium trajectory was then propagated in the NVE ensemble for 60 ps with a timestep of 0.5 fs. All MD simulations were performed using the OpenMM 8.2.0 software package [96].

The RDFs were computed from the equilibrium and nonequilibrium NVT trajectories using the molecular graphics software VMD [97]. The scattering signals were then calculated from the RDFs as described in Dohn et al. [98]. Prior to calculation of the scattering signal, the RDFs are corrected for finite-size effects by applying the Ganguly and van der Vegt correction [58, 99] as well as a smooth step damping function with cutoff 17 Å as described by Zederkof et al. [15] (equilibrium RDFs) or a 20 Å Lorch-liked damping window function [100] (nonequilibrium RDFs). The difference scattering signal was obtained by subtracting the scattering signal of the ground state from that of the excited state as calculated from ground and excited state RDFs, respectively.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Summary\nThis manuscript reports a femtosecond time-resolved X-ray solution scattering (TR-XSS) study of hemithioindigo–julolidine\n(HTI-J) in acetonitrile, aiming to visualize charge-transfer-induced solvation dynamics at the atomic scale. By constructing a\nglobal fitting model that includes optical Kerr effect (OKE), vibrational relaxation (VR), solvation-shell rearrangement (SSR),\nand bulk heating (ΔT) components, the authors attempt to separate and interpret multiple solvent responses over sub- to\ntens-of-picosecond timescales. The work represents a meaningful attempt to extend TR-XSS to organic chromophores\ncontaining only light atoms and highlights the potential of model-based decomposition for disentangling complex solvent\nand solute responses.\nOverall assessment\nThe work tackles an important and technically demanding problem in ultrafast structural chemistry: distinguishing charge-\ntransfer-driven solvent reorganization from solute structural motion in a light-atom system. The experimental setup and\nanalysis framework are sophisticated, and the topic is timely. However, several central claims and figures appear to\noverstate what the data can conclusively demonstrate, which can mislead the readers. The following points must be\nthoroughly addressed before the manuscript can be considered for publication.\n1) On the generalization and claim of “atomic-scale observation”\nThe title and abstract emphasize “atomic-scale observation of polar solvation dynamics,” yet the analysis does not yield\nexplicit atomic coordinates. The observed effects are primarily inferred from solvent density changes in the scattering profiles\ninduced by charge transfer in the solute molecules, whose atomic-scale structural changes (twisting) are themselves not\ndirectly visible in their scattering signal. This phrasing may overstate what was actually observed with the technique. The\nauthors should clarify that the study provides indirect structural sensitivity to solvent reorganization rather than a direct\natomic reconstruction. A more accurate framing in the title or abstract, focusing on charge-transfer-driven solvent\nreorganization, would strengthen the manuscript’s credibility.\n2) On CT-only versus CT + twist models\nFigures 4–5 and Supplementary Figs. S19–S21 show that including a twist marginally improves the fit near 2–3 Å−1 but\nworsens agreement around 1 Å−1. However, the manuscript does not present quantitative criteria for assessing these\ndifferences. It would be helpful to include direct numerical comparisons (e.g., χ2 values) between the two models to support\nthe argument. If the signal-to-noise ratio prevents discrimination, the authors should state explicitly that the current data\ncannot resolve solute geometry changes. Displaying both fits side by side in a single panel would make this limitation\nvisually clear.\n3) On the definition and interpretation of the “SSR” component\nThe term “solute and solvation-shell structural rearrangement (SSR)” suggests resolvable solute motion, yet the fits use time-\ninvariant shape functions and provide no quantitative evidence of twisting. Because the observed features can be explained\nby charge redistribution alone, the terminology should be revised to “solvation-shell rearrangement” to avoid implying direct\nsolute structural change. Related schematics (e.g., Fig. 5c, 5f) should also be modified so that solute twisting is presented as\nhypothetical rather than confirmed via TR-XSS. The text should clearly state that the experimental data primarily support\nsolvent reorganization following charge transfer.\n4) On figure presentation and potential misinterpretation\nSome figures (notably Fig. 2b and Fig. 5f) depict a twisted geometry that may be interpreted as an experimentally observed\nstructure. These should be redrawn to emphasize charge-transfer-driven polarization and solvent motion instead of definite\ntwisting of the solute molecule. Figure captions should explicitly describe such illustrations as conceptual representations\nbased on DFT calculations and transient absorption measurement, not experimental determinations by TR-XSS.\n5) On the DFT assignment and relevance of the proposed twisting geometry\nThe proposed twisting is supported by DFT calculations on a julolidine-substituted HTI derivative. However, static DFT\ngeometries may not accurately reflect solvated structures under experimental conditions. The authors should justify how well\nthe computed geometries correspond to the actual solute configuration in acetonitrile and clarify whether solvent effects\nwere included in the calculations. It is recommended that the authors explore how the twisting geometry and the extent of\ncharge transfer depend on a broader range of quantum chemical methods.\n6) On excitation fluence and possible nonlinear effects\nThe excitation conditions correspond to a pulse energy of 12 µJ at 470 nm focused to a 73 × 95 µm (FWHM) spot, which\ngives an average fluence of approximately 1.1 mJ mm−2 within the FWHM area, assuming that half of the pulse energy lies\ninside the Gaussian FWHM circle. This fluence is relatively high for pump–probe experiments in solution and may enter the\nregime where multiphoton ionization or solvent plasma formation becomes relevant. Under such conditions, the number of\nsolvated electrons and ionized solvent molecules can approach significant concentrations, potentially modifying the solvent\nresponse and introducing additional anisotropy or background signals in the transient scattering data.\nIt would therefore be important for the authors to demonstrate that the TR-XSS signals scale linearly with excitation fluence\nand that the measurements were performed under single-photon excitation conditions. Power-titration tests or transient\nabsorption data verifying linear excited-state population would provide valuable support.\n7) On extraction of the heating contribution\nThe heating term is obtained by averaging the 55–60 ps trace under the assumption that non-thermal contributions have fully\ndecayed. While this is reasonable given the reported 2.6 ps electronic lifetime, the robustness of this assumption should be\ndemonstrated. Testing alternative time windows (e.g., 45–50 ps versus 55–60 ps) and reporting how the resulting heating\nprofile varies would provide useful validation. Any residual non-thermal contribution could also be estimated from the model\nor transient absorption data and summarized in the Supplementary Information.\n8) On the treatment of solvent effects in DFT calculations\nThe DFT calculations appear to be based on static geometries optimized within an implicit solvent model (CPCM). Such\ntreatment may not adequately capture the specific solute–solvent interactions that influence the charge distribution and\ndipole moment in the charge-transfer state. To ensure consistency between theory and experiment, the authors should\nperform additional excited-state calculations that include explicit solvation, for example using a QM/MM approach, to\nevaluate whether the computed charge distribution and dipole moments remain consistent with the experimental data.\nThis additional analysis would help determine whether the experimentally observed solvent response arises solely from\nelectronic polarization of the solvent environment or also involves genuine structural rearrangements within the first\nsolvation shell that are not represented in the implicit-solvent model.\n9) On the anisotropic signal and its physical interpretation\nThe Supplementary Information states that the anisotropic signal contains the response to the optical Kerr effect (OKE), and\nthat this fast OKE component is used to define time zero and determine the time resolution. However, the anisotropic signal\ncan also contain valuable information about the orientational dynamics of the photoexcited solute and the correlated motion\nof nearby solvent molecules. A more complete analysis of the anisotropic component would provide independent insight into\nthe solvation mechanism. In particular, the authors should clarify whether contributions from the photoexcited solute or its\nfirst solvation shell are distinguishable from the pure OKE response of the bulk solvent. Demonstrating this separation,\neven qualitatively, would substantially reinforce the interpretation of solvent reorganization dynamics.\nMinor comments\n- Terminology: The term solute–solute is used throughout the manuscript (e.g., Fig. 7) to denote intramolecular scattering\nwithin a single solute molecule. However, this usage is unconventional and potentially misleading, because solute–solute\ntypically refers to interference terms arising from scattering between different solute molecules in highly concentrated\nsolutions. The authors should rectify this usage by adopting a clearer label such as solute term or intra-solute scattering.\n- Experimental conditions: Include a compact table summarizing concentration, fluence, jet thickness, and pump–probe\ngeometry.\n- Captions and legends: Ensure that all figures specify whether heating and OKE contributions are included or subtracted.\nConclusion and recommendation\nThis manuscript presents an important step toward extending TR-XSS to light-atom organic chromophores and toward\ndissecting solvent reorganization following charge transfer. The experimental results are strong, and the analytical\nframework is promising. However, the current data do not provide clear evidence of solute twisting, and several textual and\nvisual elements do not clearly convey this point. Clarifying terminology, presenting CT-only versus CT + twist comparisons\nquantitatively, and harmonizing figure representations would substantially improve the clarity and rigor of the paper.\nRecommendation: Major revision.", "reviewer_2": "Please find a pdf of my review attached. Thank you.", "reviewer_3": "The manuscript presents an in-depth study of a nonequilibrium response of a polar solvent acetonitrile which is caused by\ncharge transfer in the small organic molecule hemithioindigo (HTI) chromophore following it’s photoexcitation by short\nfemtosecond laser pulse. The work consists of experiments in static and time-resolved pump-probe absorption (VIS)\nspectroscopy, time-resolved pump-probe X-ray solution scattering (Swiss XFEL) and extensive computational experiments,\nnamely density functional theory (DFT) and molecular dynamics (MD) simulations to probe the structural and electronic\nchanges in HTI and the subsequent response of acetonitrile polar solvent. The work utilizes advanced and robust\ntechniques for data processing, data analysis and statistical analysis of the data. The results presented in this manuscript\nare of interest to scientists working in the fields of chemistry, photonics, photobiology and solvation dynamics.\nSignificance.\nSolution X-ray scattering is a contrast X-ray imaging technique in which measured X-ray scattering signal is proportional to\ndifference of densities (contrast) of solute and solvent. Therefore, for small organic molecules, such as HTI chromophore, it\nis notoriously challenging to disentangle scattering from the solute molecule and its solvent, in this case acetonitrile. Time-\nResolved X-ray Solution Scattering (TRXSS) has capability to probe simultaneously structural properties of a solute\nmolecule and solvent molecules, and their interaction after the reaction initiation, in this case photoinitiation of the solute\nchromophore. The authors studied how photoexcitation of the HTI chromophore changes distribution of its intramolecular\ncharges and its structural geometry, and how acetonitrile solvent responds to the changes. The charge transfer properties of\nHTI were amplified by substituting stilbene fragments by julolidine group (HTI-J) which caused increase of dipole moment of\nthe molecule upon photoactivation from 5.3 D in ground state to 27.3 D in the excited state (DFT calculations) and therefore\namplified electrostatic interaction of polar solvent with the photoexcited molecule of HTI-J.\nThe authors observed experimentally, analyzed and determined the sequence of the processes which occur in the system\nupon HTI-J photoexcitation: polarization of the polar solvent by the electric field of the pump laser light (optical Kerr effect),\nchanging energy of HTI-J from the ground to exited state and charge redistribution in the solute, vibrational energy transfer\nfrom solute to a solvent, solvent response and electrostatic interaction of the solute molecule and the polar solvent. While\nchanging structure of HTI-J upon photoexcitation does not contribute significantly to scattering patterns observed in the\nexperiment, vibrational energy exchange and electrostatic interaction between the exited state of HTI-J and polar solvent\ndoes. The significance of this work is in observing structural changes in solute and solvent, encoded in the scattering\npatterns, identifying the nature of chemical processes governing photoexcitation of HTI-J and its energy exchange with polar\nsolvent and capturing time scales and sequences of those processes. This work contributes significantly to understanding\nmechanisms of solvation dynamics of polar solvents.\nNovelty and originality.\nThe novelty of this work is in observing experimentally a change is the scattering pattern due to the dipolar rearrangement of\nthe solvent molecules surrounding photoexcited HTI-J, capturing the formation of the transient solvation shell structure on a\n∼0.5 ps timescale and its re-equilibration as the system returns to the ground state with a time constant of ∼2.6 ps, This is\nthe first time when such structural dipolar rearrangement of the solvent molecules following HTI-J photoexcitation is\nexperimentally captured in X-ray solution scattering patterns and those experimentally observed structural changes are\nmodeled on the atomic scale.\nData and methodology. Validity of the results.\nThe manuscript presents state of the art research both experimentally, methodologically and computationally. It combines\ncutting edge technologies in TRXSS with static and time-resolved optical spectroscopy and extensive in-silico experiments.\nThe combination of all those tools is required to identify, assign and model physical processes which occur upon\nphotoexcitation of a small organic chromophore in a polar solute including energy dissipation, relaxation and charge re-\ndistribution processes. The disentanglement of those processes is accomplished by a robust stepwise global optimization\nprocedure guided by methods of singular vector decomposition, DFT and MD simulations. Methods section in the\nmanuscript, and Supplemental Material present in detail robustness of procedures of statistical analysis and errors handling,\ndata acquisition, data processing, data analysis and simulations and ensure validity of the results.\nComments and Suggestions.\n-Data presentation should be improved. The time-resolved scattering patterns are dominated by the solute heating signal\ncaused by vibrational cooling of solute molecules and heating of the solvent molecules. It is very important to see how data\nlooks after global fitting and subtraction of the solvent heating signal. In this regard map representation does not give much\ninformation about actual data and noise levels contained in the data. In addition to the maps (Figure 4) heating signal\nsubtracted data should be presented for selected time delays where a major contribution from specific processes is\nidentified. The data should show experimental errors, and the quality of the fits, discussed in the manuscript.\n- It is not clear why authors use acetonitrile patten from a ref [15] in the data analysis. The experimental data were\nnormalized according to established standards. 1 HTI-J molecule is surrounded by 6400 acetonitrile molecules\n(Supplemental Materials, Liquid unit cell scaling) and in the fitting procedure of scattering pattern from [15] authors dismiss\nvery small contribution for HTI-J molecule anyway. Why authors need fitting of the acetonitrile data, collected at different\nexperimental conditions previously, and then rescale already normalized data. Did authors try to process the data without\nthis additional scaling? Did they perform static measurements of the acetonitrile and temperature jump measurements in this\nsolvent along with HTI-J photoexcitation measurements?\n-The authors did laser power titration measurements for the laser wavelength 470nm and proved that the measurements are\nperformed in the linear regime. How many photons were delivered per HTI-J chromophore? Did authors try to estimate what\nadditional fitting procedure (discussed above) could contribute to errors in the heating signal increase (temperature jump)\nand authors conclusions about multiphoton excitation in the experiment ?\n- Multiphoton absorption in organic chromophores may significantly impact energy transfer and relaxation processes and\ntrigger different pathways and rates. Therefore, it may alter the photophysical properties of the system. The authors should\ndiscuss in the main text what changes (if any) in the identified in the manuscript relaxation processes this may cause\nbesides mentioned in the manuscript fast non-radiative relaxation from higher excited state causing fast solvent heating\n(0.2ps, Table1).\n-DFT calculations and MD simulations are very important for the atomic-scale interpretation of the data . How MD\nsimulations are used for the determination of the final structures, however, requires additional explanations. The manuscript\npresents exiting results of interplay between molecule of solute and surrounding molecules of solvent (solvation dynamics),\nincluding solvent excluded volume changes, changes in its interatomic distances and changes in polarization of the solvent.\nIn Methods it is emphasized that for equilibrium MD simulations, 400000 equidistant frames were collected over 400 ns. It is\nnot clear how these 400000 trajectories were used in the final selection of the structures. How many runs were performed,\nwhat were criteria for the final structure selections? This should be explained, and appropriate data should be presented.\n- It is confusing to read sections of the main text of the manuscript describing solvent heating, vibrational relaxation, and\noptical Kerr effect and solvation dynamics with the detailed explanations of physics, energy flow and timing of multiple\nprocesses. It would be much easier for a reader if this information is also presented in a visual schematic or a diagram which\na reader can follow in addition to the text description.\n-Sentence lines 95-98 is not clear, it read like” ..Knowledge … could reduce energy loss through thermal dissipation..’,\nperhaps it should be rephrased.\nReferences:\nThe manuscript references previous literature adequately.\nClarity:\nThe manuscript is written in clear language, understandable to an expert and a general audience. Argumentation is clear\nwith the appropriate references when required. Abstract, Introduction and Conclusions are appropriate. Methods and\nSupplemental materials are very helpful.\nReviewer expertise: My expertise is in time-resolved X-ray solution scattering and supporting experiments (for example\noptical spectroscopy) and analysis of time-resolved and static X-ray solution scattering data, in particular for biomolecules in\nsolutions. The topic of this manuscript does align partially with the field of my expertise.\nMy assessment that the manuscript can be published in Nature Communications with revisions, which address above\ncomments", "reviewer_4": "This is an important experimental advance – obtaining time-resolved solution scattering on an organic molecule. Time-\nresolved X-ray solution scattering on the femto-picosecond timescale is now a well-established technique at X-FELs. To\ndate, these experiments have been performed on solute-solvent systems where the solute contains high Z atom(s), which\nhave strong scattering cross-sections.\nBefore the work is published the authors should address the following comments:\n1. The issue of high optical pump fluence 12 microJ/pulse needs to be addressed. From Figure S8, this reviewer does not\nagree that the data was collected in the linear regime. The data could be easily fit by higher order terms.\na. What was the optical pump fluence used in the optical transient absorption experiments? If different from the X-ray\nexperiments, then it should be rationalized, or the TA experiments must be performed with the same fluence for a 1:1\ncomparison.\nb. The pump fluence dependent study for the transient X-ray was conducted at 20 ps. At that time delay, there is very little\ndifference signal from the solute. It seems that the pump-fluence is measuring the solvent heating response. Is that correct?\nc. At some point the in the main text of SI, the authors point to multi-photon effects in the solvent. How does that match with\nthe difference signal being linear in pump-fluence.\n2. The authors have detailed their data analysis and data reduction. However, one is left wondering if the data is over-fit. The\nmultiple timescales and nomenclature used in Table 1 of the main text are confusing. It is also odd that the transient\nabsorption data does not measure the longer timescales for solute vibrational cooling or the slow solvent heating. Why are\nthese not the same thing?\n3. The authors claim that their work provides atomic scale resolution of polar solvation dynamics. However, they do not\nprovide any non-equilibrium RDFs of the solute-solvent interactions via MD simulations. Their MD simulations should\ninclude non-equilibrium solute-solvent interactions because they fit these timescales in their data. If non-equilibrium MD\nsimulations are not included, then there should be a discussion in the main text about what is missing.\nIn conclusion, while this is an important experimental demonstration, the data analysis and quality of the data do not provide\nany new physically relevant information."}, "author_response": "Response to Reviewers of Manuscript NCOMMS-25-77889\nBelow, we provide a point-by-point response to the reviewers’ comments. Changes made to the\nmanuscript in response to the reviewers’ comments are highlighted in blue in the annotated\nversion of the revised manuscript provided with this resubmission. Each revision is marked\nwith the label “RXCY”, where X is the reviewer number and Y is the number of the reviewer’s\ncomment.\nReviewer 1\nSummary\nThis manuscript reports a femtosecond time-resolved X-ray solution scattering (TR-\nXSS) study of hemithioindigo–julolidine (HTI-J) in acetonitrile, aiming to visualize\ncharge-transfer-induced solvation dynamics at the atomic scale. By constructing a\nglobal fitting model that includes optical Kerr effect (OKE), vibrational relaxation\n(VR), solvation-shell rearrangement (SSR), and bulk heating (∆T) components, the\nauthors attempt to separate and interpret multiple solvent responses over sub- to\ntens-of-picosecond timescales. The work represents a meaningful attempt to extend\nTR-XSS to organic chromophores containing only light atoms and highlights the po-\ntential of model-based decomposition for disentangling complex solvent and solute\nresponses.\nOverall assessment\nTheworktacklesanimportantandtechnicallydemandingprobleminultrafaststruc-\ntural chemistry: distinguishing charge-transfer-driven solvent reorganization from\nsolute structural motion in a light-atom system. The experimental setup and anal-\nysis framework are sophisticated, and the topic is timely. However, several central\nclaims and figures appear to overstate what the data can conclusively demonstrate,\nwhich can mislead the readers. The following points must be thoroughly addressed\nbefore the manuscript can be considered for publication.\nR1C1 On the generalization and claim of “atomic-scale observation”\nThe title and abstract emphasize “atomic-scale observation of polar solvation dy-\nnamics,” yet the analysis does not yield explicit atomic coordinates. The observed\neffects primarily are inferred from solvent density changes in the scattering profiles\ninduced by charge transfer in the solute molecules, whose atomic-scale structural\nchanges (twisting) are themselves not directly visible in their scattering signal. This\nphrasing may overstate what was actually observed with the technique. The authors\nshould clarify that the study provides indirect structural sensitivity to solvent reor-\nganization rather than a direct atomic reconstruction. A more accurate framing in\nthetitleorabstract,focusingoncharge-transfer-drivensolventreorganization,would\nstrengthen the manuscript’s credibility.\nResponse R1C1\nIndeed, the present TR-XSS analysis does not yield the time evolution along specific solute\n1\nor solute-solvent atomic coordinates because the solvation-shell rearrangement is modelled as\nβ(∆t)∆S (q). Nevertheless, the transient changes in solvation-shell structure encoded in\nSSR\n∆S (q) are resolved at the atomic length scale because the difference scattering signal is\nSSR\ndirectly sensitive to changes in interatomic pair-distance distributions, as exemplified in Figs.\n6 and 7 (in the manuscript, we use “atomic-scale” in the standard sense of atomic-length-scale\nstructuralsensitivity). Tomakeitclearthattheexperimentscapturethetransientsolvation-shell\nstructure formed upon intramolecular charge transfer and its decay, rather than specific solvent\nmotions or solute atomic coordinates, we have added the following sentence to the Conclusions\nsection: “The experiments provide a clear atomic-scale signature of the transient solvation shell.\nHowever, neither the time evolution of specific solvent motions underlying the solvation-shell\nrearrangement nor any accompanying solute structural change could be resolved.”. The text in\nseveral other parts in the manuscript has also been modified to make it clear that the current\nexperiments are not able to resolve solute structural changes, as described in more detail in the\nreplies to the next comments.\nR1C2 On CT-only versus CT + twist models\nFigures4-5andSupplementaryFigs. S19–S21showthatincludingatwistmarginally\nimproves the fit near 2–3 ˚A-1 but worsens agreement around 1 ˚A-1. However, the\nmanuscript does not present quantitative criteria for assessing these differences. It\nwould be helpful to include direct numerical comparisons (e.g., χ2 values) between\nthe two models to support the argument. If the signal-to-noise ratio prevents dis-\ncrimination, the authors should state explicitly that the current data cannot resolve\nsolute geometry changes. Displaying both fits side by side in a single panel would\nmake this limitation visually clear.\nResponse R1C2\nFigure 7 has been modified to display side-by-side fits of an average of the data after subtraction\nof all contributions apart from ∆S (q) with the two models (charge transfer and twist, and\nSSR\ncharge transfer only). the value of reduced χ2 has also been included for each fit. The reduced\nχ2 is found to be 4 in both cases. This indicates that the signal-to-noise ratio prevents discrim-\nination between the charge transfer and twist and charge transfer only models. To make this\nclear, we have added the following sentence in the Solvation dynamics section: “The average ex-\nperimentalsignalcloselymatchesthesimulatedsignal, withbothmodelsgivingasimilarquality\nof the fit (reduced χ2 = 4). For charge transfer and twisting, the fit is marginally improved\nbetween 2 to 3 ˚A−1, but the signal-to-noise ratio does not allow for quantitative discrimination\nbetween the two models, meaning that the current data can resolve only the transient solvation-\nshell structure, not the transient solute structural changes.”. Moreover, the new caption of Fig.\n7 includes the following sentence: “The quality of the fit is similar in both cases, indicating that\nthe current signal-to-noise ratio prevents a quantitative determination of the solute structural\nchange.”.\nR1C3 On the definition and interpretation of the “SSR” component\nThe term “solute and solvation-shell structural rearrangement (SSR)” suggests re-\nsolvable solute motion, yet the fits use time-invariant shape functions and provide no\nquantitative evidence of twisting. Because the observed features can be explained by\ncharge redistribution alone, the terminology should be revised to “solvation-shell re-\narrangement” to avoid implying direct solute structural change. Related schematics\n(e.g., Fig. 5c, 5f) should also be modified so that solute twisting is presented as hy-\npothetical rather than confirmed via TR-XSS. The text should clearly state that the\nexperimentaldataprimarilysupportsolventreorganizationfollowingchargetransfer.\nResponse R1C3\nWe agree with the reviewer that the terminology “solute and solvation-shell structural rear-\nrangement (SSR)” could be read as implying that the analysis of the TR-XSS data provides\n2\nclear evidence of a solute structural change. Therefore, we have revised the terminology of\nthis component to “solvation-shell rearrangement (SSR)”. Related figures and discussions were\nrevised accordingly. The twisting has been removed from Fig. 5 (see also answer to the next\ncomment), whilethetextofthemanuscripthasbeenmodifiedinseveralpartstoemphasizethat\nthe observation of solvent-shell reorganization at atomic length scale following charge transfer\nis the primary conclusion supported by the TR-XSS data (see also answers to the previous\ncomments).\nR1C4 On figure presentation and potential misinterpretation\nSome figures (notably Fig. 2b and Fig. 5f) depict a twisted geometry that may be\ninterpreted as an experimentally observed structure. These should be redrawn to\nemphasize charge-transfer-driven polarization and solvent motion instead of definite\ntwisting of the solute molecule. Figure captions should explicitly describe such il-\nlustrations as conceptual representations based on DFT calculations and transient\nabsorption measurement, not experimental determinations by TR-XSS.\nResponse R1C4\nWe agree that the depiction of a twisted geometry in Figs. 2 and 5 poses a risk of misinterpre-\ntation. We have updated the caption of Fig. 2 to state explicitly that the twisting of HTI-J is\nsupported solely by the DFT calculations, and is not experimentally determined by TR-XSS.\nAs also explained in the answer to the previous comment, we revised Fig. 5 by removing the\ndepiction of twisting to emphasize the observation of the solvation-shell rearrangement from the\nTR-XSS measurements.\nR1C5 On the DFT assignment and relevance of the proposed twisting geometry\nThe proposed twisting is supported by DFT calculations on a julolidine-substituted\nHTI derivative. However, static DFT geometries may not accurately reflect solvated\nstructures under experimental conditions. The authors should justify how well the\ncomputedgeometriescorrespondtotheactualsoluteconfigurationinacetonitrileand\nclarify whether solvent effects were included in the calculations. It is recommended\nthatthe authorsexplore how thetwistinggeometry andthe extentof charge transfer\ndepend on a broader range of quantum chemical methods.\nResponse R1C5\nSolvent effects are included in the DFT calculations through the implicit conductor-like polar-\nizable continuum model (CPCM) for acetonitrile, as described in the Methods section.\nTo validate the DFT results, we have performed additional coupled cluster calculations at\nDLPNO-CCSD(T) level. These calculations are performed for the triplet state (T1). Since\nT1 is the lowest triplet state, a ground state method can be used, which makes it possible to\nincludesolvationeffectsincoupledclustercalculations. T1andthesingletexcitedstate(S1)differ\nonly in the exchange interaction, so they are expected to exhibit similar electronic character and\npotential energy surface (see, e.g., J Mol Model 18, 2479 (2012) DOI:10.1007/s00894-011-1270-\n6). The CCSD(T) calculations are mentioned in the Methods section and the results presented\nin Supplementary Note 9.3. The results confirm the predictions of DFT for the S1 state, as\nthey show that the excited state has a 90 degree twisted geometry and has a large dipole mo-\nment in acetonitrile (magnitude of the dipole moment of 33.9 D for CCSD(T) vs. 31.6 D for\nDFT/CAM-B3LYP).\nR1C6 On excitation fluence and possible nonlinear effects\nThe excitation conditions correspond to a pulse energy of 12 µJ at 470 nm focused\nto a 73 × 95 µm (FWHM) spot, which gives an average fluence of approximately 1.1\nmJ mm-2 within the FWHM area, assuming that half of the pulse energy lies\n3\ninside the Gaussian FWHM circle. This fluence is relatively high for pump–probe experi-\nments in solution and may enter the regime where multiphoton ionization or solvent\nplasma formation becomes relevant. Under such conditions, the number of\nsolvated electrons and ionized solvent molecules can approach significant concentrations, po-\ntentially modifying the solvent\nresponse and introducing additional anisotropy or background signals in the transient scattering data.\nIt would therefore be important for the authors to demonstrate that the TR-XSS signals scale linearly with\nexcitation fluence and that the measurements were performed under single-photon\nexcitation conditions. Power-titration tests or transient absorption data verifying\nlinear excited-state population would provide valuable support.\nResponse R1C6\nTo investigate the signal response to laser pump fluence, laser fluence scans were carried out\nduring the TR-XSS experiment and the results were included in Supplementary Note 4.1 and\nSupplementary Fig. S8 in the original submission. A linear as well as second order polynomial\nfit suggest that the dependency of the signal response on the laser fluence starts deviating from\nlinearity between 10–13 µJ/pulse. An excitation energy of 12 µJ/pulse as used to collect the\ndata is in the upper end of the linear regime and we observe a higher energy deposition into the\nsolvent than expected for one-photon excitation (discussed in Supplementary Note 8). However,\nas detailed below, we exclude photoionization of the solvent or the solute, and attribute the\nhigher-than-expected increase of temperature to multiphoton excitation to higher-energy states.\nFirst of all, we exclude direct excitation and photoionization of the acetonitrile solvent. Ki et\nal. (J. Am. Chem. Soc. 143, 14261 (2021) DOI:10.1021/jacs.1c06088) report no evidence of\nsuch effects in acetonitrile when using a 400 nm laser with a fluence ∼50% higher than used in\nthe present study. This is not unexpected, as the ∼12 eV (CRC Handbook of Chemistry and\nPhysics95edn(2014))ionizationthresholdforacetonitrilewouldentaila4-photonprocessat400\nnm (3.1 eV). Secondly, we also exclude direct photoionization of the HTI-J solute molecules.\nRelated stilbene and indigo compounds exhibit photoionization thresholds of ∼7.5 eV (CRC\nHandbook of Chemistry and Physics 95 edn (2014)) and ∼6.9 eV (Chem. Phys. Lett. 449, 11–\n17(2007)DOI:10.1016/j.cplett.2007.10.015), respectively. Thissuggeststhatat470nm(2.6eV)\nphotoionization would require a 3-photon process. Since the laser fluence scans show only weak\nnon-linearbehaviorat12µJ/pulse, weconsidersignificantphotoionizationofthesoluteunlikely.\nGiven the near-linear fluence dependence at 12 µJ/pulse, we conclude that a fraction of the\nexcited state population is excited into higher-lying excited states. These states are expected\nto rapidly relax non-radiatively to the lowest excited state, depositing the excess energy into\nthe solvent. This causes the rapid increase (τ1 ∼ 0.2 ps) in the magnitude of the solvent tem-\n∆T\nperature component seen in this study. Similar fluence-dependent solvent-heating contributions\nhave been reported in TR-XSS studies performed under comparable experimental conditions\n(Phys. Rev. Lett. 122, 63001 (2019) DOI:10.1103/PhysRevLett.122.063001, Chem. Sci. 14,\n2572 (2023), DOI:10.1039/d2sc06600a).\nTo reflect these important considerations better in the manuscript, we have added the following\nto the main text: “... and an excitation fluence of approximately 1.2 mJ/mm−2, corresponding\nto the upper end of the linear-response region determined via a laser fluence scan (see Sup-\nplementary Note 4.1).”, and “We conclude that this rapid solvent response arises from fast\nnon-radiative relaxation following multiphoton excitation of a fraction of molecules to higher-\nlying states, as discussed in more detail in Supplementary Note 8. ... Except for the rapid\nonset of the heating signal (τ1 ∼ 0.2 ps) no contributions from the higher laser fluence in\n∆T\nthe TR-XSS experiments compared to the TAS could be discerned in our analysis.”. We have\nadditionally expanded Supplementary Note 8 to incorporate a more thorough discussion of this\n4\npotential concern.\nR1C7 On extraction of the heating contribution\nThe heating term is obtained by averaging the 55–60 ps trace under the assumption\nthat non-thermal contributions have fully decayed. While this is reasonable given\nthe reported 2.6 ps electronic lifetime, the robustness of this assumption should be\ndemonstrated. Testingalternativetimewindows(e.g., 45–50psversus55–60ps)and\nreporting how the resulting heating profile varies would provide useful validation.\nAny residual non-thermal contribution could also be estimated from the model\nor transient absorption data and summarized in the Supplementary Information.\nResponse R1C7\nWe have added a new figure, Fig. S17, to Supplementary Note 5.1 showing that the difference\nbetween the isotropic difference scattering data averaged between 45-50 ps and between 55-\n60 ps can be explained by contributions from background artifact components alone. Apart\nfrom these, no residual contribution was observed in the scattering data at long time delays.\nR1C8 On the treatment of solvent effects in DFT calculations\nThe DFT calculations appear to be based on static geometries optimized within an\nimplicit solvent model (CPCM). Such treatment may not adequately capture the\nspecific solute–solvent interactions that influence the charge distribution and\ndipole moment in the charge-transfer state. To ensure consistency between theory and ex-\nperiment, the authors should perform additional excited-state calculations that include\nexplicit solvation, for example using a QM/MM approach, to evaluate whether\nthe computed charge distribution and dipole moments remain consistent with the\nexperimental data. This additional analysis would help determine whether the ex-\nperimentally observed solvent response arises solely from electronic polarization of\nthe solvent environment or also involves genuine structural rearrangements within\nthe first solvation shell that are not represented in the implicit-solvent model.\nResponse R1C8\nWe agree that a static optimization in an implicit (CPCM) solvent cannot by itself describe\nsolvent structural rearrangement in the solvation shell. However, our interpretation of the\nstructural solvent response does not rely on the implicit solvent model but on equilibrium MD\nsimulations with explicit acetonitrile molecules for both ground and excited state solute mod-\nels (see Figs. 6 and 7 and related text in the manuscript). The DFT/CPCM calculations are\nused primarily to obtain the ground and excited state solute geometry and partial charges for\nthe parametrization of the solute in the MD simulations. To make this clear, we have added\nthe following to the Computational methods section: “The DFT calculations used to obtain\nequilibrium geometries and partial charges for the solute in the MD simulations employ the\nrange-separated hybrid functional CAM-B3LYP ...”.\nRegarding the adequacy of an implicit solvent model, previous studies (see, e.g., J. Phys.\nChem. C 123, 2056 (2019) DOI:10.1021/acs.jpcc.8b10768, and J. Phys. Chem. B 122, 2975\n(2018) DOI:10.1021/acs.jpcb.7b12560) have shown that the excitation energy and the solvent-\nstabilization (solvation) energy obtained with polarizable continuum models are consistent with\nthe corresponding ensemble-averaged values from fully quantum-mechanical calculations that\ninclude explicit solvent molecules. In the Methods section, we have added “Previous studies [19,\n23] have shown that the excitation and solvation energy obtained with polarizable continuum\nmodels are consistent with the corresponding ensemble-averaged values from fully quantum-\nmechanical calculations including explicit solvent molecules.”, where [19, 23] are references to\nthe above mentioned articles.\nulations based on fixed-value partial charges from DFT calculations with implicit solvent have\nbeen shown to describe the orientational response of the solvent molecules to charge transfer of\nthe solute (J. Phys. Chem. C 123, 2056 (2019) DOI:10.1021/acs.jpcc.8b10768, J. Phys. Chem.\nB 109, 3553 (2005) DOI:10.1021/jp0456032). Moreover, a flexible solute can account for some\npolarization effects as the solute geometry adjusts to stabilize the solute-solvent interactions,\nwhich can change the instantaneous and average dipole moment relative to that of the equilib-\nrium geometry in an implicit solvent environment, as shown in J. Phys. Chem. B 109, 3553\n(2005) DOI:10.1021/jp0456032. To make this clear, we have added the following in the Methods\nsection, page 19: “MD simulations with fixed-value partial charges have been shown to describe\nthe orientational response of a polar solute to charge transfer in the solute [19, 23]. Moreover,\nusing a flexible solute with fixed partial charges has been shown to capture some polarization\neffects, since the solute can distort in response to solute–solvent interactions and thereby change\nits instantaneous and average dipole moment relative to the equilibrium geometry in an implicit\nsolvent [23].”.\nR1C9 On the anisotropic signal and its physical interpretation\nThe Supplementary Information states that the anisotropic signal contains the re-\nsponse to the optical Kerr effect (OKE), and that this fast OKE component is used\nto define time zero and determine the time resolution. However, the anisotropic\nsignal can also contain valuable information about the orientational dynamics of the\nphotoexcited solute and the correlated motion of nearby solvent molecules. A more\ncomplete analysis of the anisotropic component would provide independent insight\ninto the solvation mechanism. In particular, the authors should clarify whether\ncontributions from the photoexcited solute or its first solvation shell are distinguishable\nfrom the pure OKE response of the bulk solvent. Demonstrating this separation,\neven qualitatively, would substantially reinforce the interpretation of solvent reorga-\nnization dynamics.\nResponse R1C9\nWe thank the reviewer for this suggestion. We have added a new section in the Supple-\nmentary Information, Supplementary Note 4.3, with an analysis of the anisotropic difference\ndata based on singular value decomposition (SVD). The SVD shows that the anisotropic sig-\nnal is dominated by two components with a short and a long decay time constants. Similar\nanisotropic signals with timescales of ∼60 fs and 1.5 ps have been observed for an acetonitrile\nsolution in time-resolved X-ray scattering by Ki et al. (J. Am. Chem. Soc. 143, 14261 (2021)\nDOI:10.1021/jacs.1c06088), where they were assigned to orientational rearrangement of the sol-\nvent associated with the optical Kerr effect (OKE). Related anisotropic OKE signals have also\nbeen reported by Montoya-Castillo et al. for chloroform (Phys. Rev. Lett., 129, 056001 (2022)\nDOI:10.1103/PhysRevLett.129.056001). On this basis, we attribute the two leading SVD com-\nponents of the anisotropic data to the bulk-solvent OKE.\nThe OKE contributions dominate the anisotropic signal. Apart from them, the SVD contains\nan additional weaker component. Fitting its temporal evolution gives a decay constant of 2.5\n± 0.8 ps. This value is consistent, within uncertainty, with (i) the solvation-shell structural\nrearrangement (SSR) time constant obtained from the global analysis of the isotropic difference\nscattering data (3±1 ps) and (ii) the excited state lifetime from the TAS data (2.6±0.5 ps).\nHowever, theoverlapwiththetimescaleoftheslowcomponentoftheOKEsignalandsignificant\nartifacts affecting the anisotropic data limit robust quantitative separation of solvation shell\ncontributions from the bulk-solvent OKE compared to the isotropic signal, where the OKE and\nSSR components have significantly different decay rates.\nR1 Minor comments\n6\n- Terminology: The term solute–solute is used throughout the manuscript (e.g.,\nFig. 7) to denote intramolecular scattering within a single solute molecule. How-\never, this usage is unconventional and potentially misleading, because solute–solute\ntypically refers to interference terms arising from scattering between different solute\nmolecules in highly concentrated solutions. The authors should rectify this usage by\nadopting a clearer label such as solute term or intra-solute scattering.\n- Experimental conditions: Include a compact table summarizing concentration,\nfluence, jet thickness, and pump–probe geometry.\n- Captions and legends: Ensure that all figures specify whether heating and OKE\ncontributions are included or subtracted.\nResponse R1 Minor comments\n- Terminology: We thank the reviewer for pointing out this potentially misleading use of\nthe terminology “solute-solute scattering” to describe the scattering arising from changes of\nintramolecular atomic distances. We have decided to adopt the terminology “intra-solute scat-\ntering”.\n- Experimental conditions: We added a new table in the beginning of Supplementary Note\n4, Table S2, summarizing key experimental parameters of the TR-XSS measurements.\n- Captions and legends: In the revised manuscript, we ensure that all figures specify whether\nheating and OKE contributions are included or subtracted.\nConclusion and recommendation\nThis manuscript presents an important step toward extending TR-XSS to light-\natom organic chromophores and toward dissecting solvent reorganization following\nchargetransfer. Theexperimentalresultsarestrong, andtheanalyticalframeworkis\npromising. However,thecurrentdatadonotprovideclearevidenceofsolutetwisting,\nand several textual and visual elements do not clearly convey this point. Clarifying\nterminology, presenting CT-only versus CT + twist comparisons quantitatively, and\nharmonizing figure representations would substantially improve the clarity and rigor\nof the paper.\nR1 Additional comment\nIn the discussion of charge-transfer–induced structural responses, the authors cite\nBiasin et al. (Nat. Chem. 2021) as the only representative study. To provide a more\ncomprehensive view of the field, it would be beneficial to also cite Heo et al. (Nat.\nCommun., 2022, 13, 522). This work used anisotropic TR-XSS to resolve atomic\ncharge redistribution accompanying the bond cleavage of the I3- system in solution,\nwhich offers a relevant and complementary context to the solvation dynamics and\nstructural reorganization discussed in the current manuscript. I believe citing this\nwork would benefit readers and the community as a whole.\nResponse R1 Additional comment\nWe thank the reviewer for pointing us to the work of Heo et al. (Nat. Commun. 13, 522 (2022)\nDOI:10.1038/s41467-022-28168-0). We agree that this work is highly relevant, and have added\na citation in the Introduction.\nReviewer 2\nPhotochemical and photophysical processes in molecules can play out quite differ-\nently in solution compared to the gas-phase. The reasons for this include steric\neffects, with solvent molecules hindering geometric changes in the solute, and the\nability of the solvent to quench excited vibrations, allowing energy to dissipate from\nthe solute into the solvent. This can have significant effect on the final quantum\nyields of reactions and makes fragmentation a much more important channel in the\n7\ngas-phase since the excess energy in the ground electronic state is often sufficient\nto break chemical bonds. Another important example of solvent effects appears if\nthe solvent is polar and the excited electronic state has charge transfer character, in\nwhich case the solvent can rearrange to stabilise the charge separation. This effect\nis predicted by theory and has been observed spectroscopically.\nIn the current manuscript by Mitterer et al, the authors provide convincing evidence\nfor the rearrangement of a polar solvent in response to an excited charge-transfer\nstate. They carry out timeresolved X-ray solution scattering experiments at the\nSwissFEL, supported by transient absorption measurements and simulations. By\ndoing this, the team is able to infer the real-space rearrangements of atoms in the\nsolute and the solvent following excitation by a 470 nm pulse. This is a significant\nachievement by itself, but the experiment also constitutes a notable demonstration\noftheimprovementinsolution-phaseX-rayscatteringexperimentsovertime, aswell\nas a textbook example of the current state-of-the-art in this domain (especially in\nterms of the analysis).\nWhiletime-resolvedgas-phaseX-rayscatteringonlyappearedin2015(10.1103/Phys-\nRevLett.114.255501), solution-phase measurements have a longer history with early\nexperimentsdonewiththehelpofamechanicalchopperatMichaelWulff’sbeamline\nID09attheESRFalreadycarriedoutintheearly2000s(10.1103/PhysRevLett.87.195508).\nThecitedpioneeringpaperbyWulffetalinvestigatesI2dissociationinsolution, and\ncatches a glimpse of the caging effect of the solvent. However, what this early so-\nlution phase paper has in common with later papers, despite very significant later\nimprovements in the data analysis and improvements in the time-resolution follow-\ning the arrival of XFELs, is the reliance on targets with strongly scattering atoms\nto achieve sufficient contrast to the solvent. Later examples include an impres-\nsive range of studies in organometallics from the current authors (eg [Fe(bmip)2]2+\nin 10.1038/s41467-020-14468-w and Pt2POP4 in 10.1103/PhysRevLett.122.063001,\netc) and work in systems such as [Au(CN)2–]3 by Ihee et al (see eg 10.1038/s41586-\n020-2417-3 and 10.1038/nature14163). However, the current work stands out by\nperforming a study in an organic chromophore. Although this is arguably a some-\nwhat technical point, it is nevertheless an important one, as it demonstrates the\noverall quality of the measurements that can now be achieved. In my view this is an\nimportant aspect of the current manuscript.\nOverall, the paper is very well written and presents impressive results. I partic-\nularly appreciate the systematic and thorough analysis of the data - it is rare that\nthe supplementary information is enjoyable to read, but in this instance, I gained a\nlot from reading the SI. I find that the paper is suitable for publication in Nature\nCommunications, but, as always, there are some points that I would like the authors\nto clarify and/or expand upon first.\nQuestions and comments:\nR2C1 The kinetic model used to interpret the experimental scattering data benefits\ngreatly from the complementary transient absorption spectroscopy (TAS) measure-\nments. However,fromwhatIcantelltheTASmeasurementsusea490nmexcitation\npulse, while the X-ray scattering experiment uses a 470 nm pulse. Could the authors\nplease explain what consequences this discrepancy may have, and to what degree it\nmay affect the obtained rate constants (or their assignment)?\nResponse R2C1\nBoth wavelengths lie within the same lowest-energy absorption band and therefore populate the\nsame excited electronic state. The main difference is that 470 nm deposits slightly more excess\n8\nvibrational energy in the solute. The difference in photon energy between 470 and 490 nm is\nabout 0.1 eV, which is modest compared to the width of the absorption band. In solution, this\nsmall additional excess energy is expected to be at least in part dissipated to the solvent before\nthe passage of the wave packet through the conical intersection with the ground state. As a\nresult, the subsequent population decay and solvation structural dynamics that determine the\nfitted kinetic time constants are not expected to depend strongly on whether excitation is at\n470 or 490 nm.\nR2C2 When reading the manuscript, I was somewhat concerned that the X-ray\nsignal may contain contributions from higher excited states or even ionisation. How-\never, when looking in the SI, I saw that the authors performed a ’power titration’ to\nestablish the highest power commensurate with linear excitation. This is excellent,\nand I wonder if it may be worth including a (brief) reference in the main manuscript\npointing readers to the SI on this topic?\nResponse R2C2\nWe thank the reviewer for the suggestion. We have included a reference in the Methods section\nof the main manuscript that points to Supplementary Note 4.1, reporting a laser fluence scan\n(formerly “power titration”).\nWe would like to mention that in response to reviewer comments R1C6, R3C4, and R4C1, we\nhave expanded the discussion about nonlinearity and multiphoton excitation and would like to\nrefer the reviewer to our responses to R1C6 and R4C1.\nR2C3 The theoretical description of charge transfer states by DFT is not entirely\nunproblematic, unless one uses range-separated hybrid functionals, as the authors\nhave indeed done here. Nevertheless, I wonder if the authors have carried out any\nbenchmarking beyond what is already presented in the SI, which contains a test of a\nsequence of basis sets with the CAM-B3LYP functional. I appreciate that the size of\nthe molecular target is somewhat prohibitive for higher level methods, but it would\nbe encouraging to see some reference data obtained with a non-DFT method.\nResponse R2C3\nTo validate the DFT results, we have performed higher-level coupled cluster calculations using\nthe DLPNO-CCSD(T) method. In order to include implicit solvent effects, these calculations\nare performed for the lowest triplet state instead of the singlet excited state (S1) state (see also\nthe reply to R1C5 of Reviewer 1). Under the typical assumption that T1 and S1 have similar\ngeometry and electronic character, the CCSD(T) calculations support the DFT predictions, as\nboth methods yield a 90 degree twisted structure and a strong charge transfer character for the\nexcitedstate(magnitudeofthedipolemomentof33.9DforCCSD(T)vs. 31.6DforDFT/CAM-\nB3LYP). The new CCSD(T) calculations are briefly described in the Methods section and the\nresults presented in Supplementary Note 9.3.\nR2C4 On page 14 in the SI, the authors state that approximately 30% of the images\nare rejected from the data. This is quite high. The process for excluding data is\nwell described, but could the authors explain better why so much data had to be\nexcluded to begin with? It would be good to make this as clear as possible given\nthat this seems to be an example of rather aggressive curating of experimental data.\nResponse R2C4\nWe agree with the reviewer that a 30% outlier rejection can be considered fairly aggressive, and\nhave added the following new text to the Supplementary Note 4.2 to explain the rationale for\nthis in more detail: “The stochastic nature of the self-amplified spontaneous emission (SASE)\nprocess at XFELs induces substantial shot-to-shot fluctuations in beam intensity, photon\n9\nenergy, and pointing. Combined with sample delivery instabilities and variations in experimental\nchamber conditions, these effects may alter the spatial distribution of scattered X-rays on the\ndetector. Although mitigation strategies exist [9, 10], XFEL data reduction typically begins by\ndiscarding frames most affected. In the present work, images lacking beamline diagnostics (e.g.,\nintensity monitors or timing tool data) or deviating by more than 10% from the median total\nscatteringintensitywithinascanwererejected(Fig.S10a). Tocompensateforbeam–jetoverlap\nvariations, we applied a correlation filter between summed scattering intensity and the I0 diode\nsignal, removing shots in the lowest and highest deciles (Fig. S10b). The filtering resulted in a\nrejection of approximately 30% of the images. A more relaxed outlier filtering with 20% of the\nimages rejected was also tested during data reduction, but significantly increased the magnitude\nofbackgroundartifactcomponentsinthecalculateddifferencescatteringsignal. Backgroundar\ntifact components in the TR-XSS data are discussed in Supplementary Note 4.6.”. Here, [9, 10]\nare references to Faraday Discuss. 177, 443 (2015) DOI:10.1039/c4fd00203b and J. Synchrotron\nRad. 33, 1, (2026), DOI:10.1107/S1600577525009737.\nR2C5 The authors make a strong point in the manuscript about ’atomistic reso-\nlution’. Although I fully appreciate that the experimental signal is reflective of the\nspatial arrangement of the atoms, I feel that with such a narrow q-range (essentially\n1-3.5 ˚A-1) and an instrument response function of 120 fs, perhaps one should be\nmore careful in the manuscript to not overstate this, or at least acknowledge the\nlimitations of the current data more clearly. I would also be curious to know what\nlimited the q-range in the current measurements. Looking elsewhere in the litera-\nture, the authors’ own measurements on Pt2POP4 at LCLS achieved qmax = 4.5\n˚A-1(10.1103/PhysRevLett.122.063001)andIhee’smeasurementson[Au(CN)2–]3at\nSACLA achieved qmax = 6 ˚A-1 (10.1038/s41586-020-2417-3). What were the spe-\ncific limitations here, and can these be overcome in the future? Could the authors\nperhaps include theoretical signals for a wider range of q, say up to 10 ˚A-1, in the\nSI to help interested readers think about how much information may be present at\nthose higher q-ranges?\nResponse R2C5\nRegarding the atomic resolution, the present TR-XSS analysis does not yield the time evolution\nalong specific solute or solute-solvent atomic coordinates because the solvation-shell rearrange-\nment is modelled as β(∆t)∆S (q) (see also our response to R1C1). Nevertheless, the tran-\nSSR\nsient changes in solvation-shell structure encoded in ∆S (q) are resolved at the atomic length\nSSR\nscale, because the difference scattering signal is directly sensitive to changes in interatomic pair-\ndistance distributions (see Figs. 6 and 7). To clarify this, we have added the following sentence\nto the Conclusions section: “The experiments provide a clear atomic-scale signature of the tran-\nsient solvation shell. However, neither the time evolution of specific solvent motions underlying\nthe solvation-shell rearrangement nor any accompanying solute structural change could be re-\nsolved.”.\nScattering signals were recorded over a q-range of approximately 0.4–5 ˚A−1. Supplementary\nFig. S11 shows the isotropic and anisotropic difference scattering signals within this range. At\nhigh q values (above ∼3.8 ˚A−1), background artifact contributions influenced the modeling of\nthe data; therefore, a smaller q-range was selected for the final global fitting of the X-ray scat-\ntering data. A discussion of this is provided at the end of Supplementary Note 5.2. We point\nout that this work focuses on the solvation-shell rearrangement in response to charge transfer\nin an organic molecule, for which the amplitude of this signal is largest at low q.\nSupplementary Fig. S30 was updated to display the simulated difference scattering signals up to\nq = 10 ˚A−1. While the solute–solvent term dominates the total simulated signal at lower q, the\ntotalsimulatedsignalatlargerq mainlyreflectscontributionsfromthestructuralresponseofthe\nsolute. However, the presence of background artifacts at higher q and the limited signal-to-noise\n10\nratio of the data did not allow for a determination of a solute structural change. Future studies\naiming to resolve solute structural changes in photoexcited organic molecules in solution may\nbenefit from a larger q-range.\nR2C6 In a similar vein to question 5 above, but somewhat speculative, do the\nauthors think that it might eventually become possible to measure the actual rear-\nrangement of electrons that corresponds to the charge transfer in the excited state?\nThere are examples in the gas-phase. Perhaps first to come to mind is the study of\ncharge transfer in a molecule by Weber et al (10.1073/pnas.2021714118), which has\nsome similarities to the current study in terms of the analysis and which succeeds to\nisolate the signature of the charge transfer in the scattering signal. Arguably other\nexamplesinthegas-phaseincludemorerecentpaperssuchas10.1103/53h3-vykland\n10.1038/s41467-020-15680-4 (although not involving charge transfer per se).\nResponse R2C6\nIn principle, X-ray scattering is sensitive to purely electronic rearrangements, as demonstrated\nfor isolated molecules in the works cited by the reviewer. Extending this capability to solution-\nphase systems is more challenging because the signal associated with changes in the electron\ndensityistypicallymuchsmallerthanthecontributionsfromsoluteandsolventatomicreorgani-\nzation. Nevertheless, weexpectthatdirectobservationsofexcited-stateelectronicredistribution\nin solution will become increasingly feasible as XFEL sources advance, particularly through im-\nproved time resolution, higher photon energy and repetition rates, and continued progress in\nanalysis and simulation frameworks needed to separate electronic and nuclear components. To\nreflectthisperspective,wehaveaddedthefollowingsentencetotheConclusionssectionafterour\ndiscussion of future experimental and theoretical developments: “Moreover, while in the present\nstudy the photoinduced charge transfer is inferred indirectly through the solvent response, we\nanticipate that the aforementioned advancements will make it possible to achieve direct X-ray\nscattering observations of pure electronic rearrangements in solution, as already demonstrated\nfor isolated molecules [74-76].”, where [74-76] are references to the articles mentioned by the\nreviewer.\nR2C7SpeakingoftheglobalanalysisoftheexperimentaldatapresentedinSection6\nof the SI, could the authors comment further on the limitations of the ’staged’ fitting\nalgorithm that they have implemented, whereby some constraints are determined in\nthe first step before the final global fit? What is the risk that this introduces bias in\nthe fit? That being said, I found the overall analysis to be compelling. In particular,\nI like how Figures S18, S21 and S22 make it quite clear that both the twisting\nand the charge transfer in the solute molecule come into play to explain the observed\nsignal.\nResponse R2C7\nWe tested the influence of the regularization applied to the q-profiles of the optical Kerr effect,\nsolvation shell rearrangement, and vibrational relaxation components by repeating the global\nanalysis without regularization. The results were unchanged within uncertainty. Moreover, we\nfoundthatthefinalglobalfitissufficientlystabletoconvergereliablywithoutregularization. For\nthisreason,wehaveremovedtheregularizationforthesecomponentsfromtheanalysispresented\nin the manuscript and the Supplementary Information. The q-profile of the bulk solvent heating\nremains fully constrained as the average at late time delays is expected to accurately represent\nthis contribution. We have updated the Methods section and Supplementary Note 6.1 to reflect\nthese changes to the analysis.\nR2C8 From what I understand, following photoexcitation the system eventually\nreturns to the electronic ground state where the solvent quenches the vibrational\n11\nexcitation. In terms of the final yields of the photoexcitation, do the authors know\n(or can estimate) the final quantum yield of the Z versus the E conformation of the\nmolecule?\nResponse R2C8\nThe Z/E photoisomerization yield of HTI-J has been measured by Wiedbrauk et al. in various\nsolvents (J. Am. Chem. Soc., 138, 12219 (2016) DOI:10.1021/jacs.6b05981). For polar solvents,\nthey report quantum yields of < 1%. We have added this information in the Introduction.\nR2C9 The authors assign the ∆S2 anisotropic component in the scattering to the\noptical Kerr effect and use this to confirm time zero. This all good and well, but is\nthere no structural information that could be extracted from the anisotropic signal\nforthealignedmolecules? Itseemstomethatitwouldbeinterestingtoconsiderthis,\nin addition to the ∆S0. In this context, can the authors confirm that the isotropic\n∆S0 signal is obtained via Legendre decomposition, rather than overall averaging of\nthe total signal?\nResponse R2C9\nWe thank Reviewer 2 for the suggestion of investigating the anisotropic signal, which is in line\nwith comment 9 by Reviewer 1. As also explained in our response to R1C9, we have performed\na singular value decomposition (SVD) of the anisotropic difference data, which is presented in a\nnewsectionoftheSupplementaryInformation,SupplementaryNote4.3. TheSVDconfirmsthat\ntheanisotropicsignalisdominatedbytheultrafastopticalKerreffect(OKE)ofthebulksolvent\nused to define time zero. In addition, it identifies a much weaker non-OKE component with a\ndecay time consistent within uncertainty with the time constant of the solvation-shell structural\nrearrangement (SSR) extracted from the isotropic TR-XSS analysis and with the excited state\nlifetime from TAS. This suggests that the anisotropic data contain a solvation-related contribu-\ntion. However, overlap with the timescale of slow OKE component and significant artifacts in\nthe anisotropic data preclude a robust quantitative separation of solvation-shell dynamics from\nthe bulk-solvent OKE, compared to the isotropic data.\nWe can confirm that the isotropic ∆S signal is obtained via Legendre decomposition (not by\n0\nsimple averaging), and have specified this in the main article and Supplementary Note 4.2.\nReviewer 3\nThe manuscript presents an in-depth study of a nonequilibrium response of a polar\nsolvent acetonitrile which is caused by charge transfer in the small organic molecule\nhemithioindigo (HTI) chromophore following it’s photoexcitation by short femtosec-\nond laser pulse. The work consists of experiments in static and time-resolved pump-\nprobe absorption (VIS) spectroscopy, time-resolved pump-probe X-ray solution scat-\ntering(SwissXFEL)andextensivecomputationalexperiments,namelydensityfunc-\ntional theory (DFT) and molecular dynamics (MD) simulations to probe the struc-\nturalandelectronicchangesinHTIandthesubsequentresponseofacetonitrilepolar\nsolvent. The work utilizes advanced and robust techniques for data processing, data\nanalysisandstatisticalanalysisofthedata. Theresultspresentedinthismanuscript\nareofinteresttoscientistsworkinginthefieldsofchemistry,photonics,photobiology\nand solvation dynamics.\nSignificance.\nSolution X-ray scattering is a contrast X-ray imaging technique in which measured\nX-ray scattering signal is proportional to difference of densities (contrast) of solute\nand solvent. Therefore, for small organic molecules, such as HTI chromophore, it\n12\nis notoriously challenging to disentangle scattering from the solute molecule and its\nsolvent, in this case acetonitrile. Time-Resolved X-ray Solution Scattering (TRXSS)\nhas capability to probe simultaneously structural properties of a solute molecule\nand solvent molecules, and their interaction after the reaction initiation, in this case\nphotoinitiation of the solute chromophore. The authors studied how photoexcita-\ntion of the HTI chromophore changes distribution of its intramolecular charges and\nits structural geometry, and how acetonitrile solvent responds to the changes. The\ncharge transfer properties of HTI were amplified by substituting stilbene fragments\nby julolidine group (HTI-J) which caused increase of dipole moment of the molecule\nuponphotoactivationfrom 5.3Dingroundstate to27.3Dinthe excitedstate(DFT\ncalculations) and therefore amplified electrostatic interaction of polar solvent with\nthe photoexcited molecule of HTI-J. The authors observed experimentally, analyzed\nand determined the sequence of the processes which occur in the system upon HTI-J\nphotoexcitation: polarization of the polar solvent by the electric field of the pump\nlaser light (optical Kerr effect), changing energy of HTI-J from the ground to exited\nstate and charge redistribution in the solute, vibrational energy transfer from solute\nto a solvent, solvent response and electrostatic interaction of the solute molecule and\nthe polar solvent. While changing structure of HTI-J upon photoexcitation does not\ncontributesignificantlytoscatteringpatternsobservedintheexperiment,vibrational\nenergy exchange and electrostatic interaction between the exited state of HTI-J and\npolar solvent does. The significance of this work is in observing structural changes\nin solute and solvent, encoded in the scattering patterns, identifying the nature of\nchemical processes governing photoexcitation of HTI-J and its energy exchange with\npolar solvent and capturing time scales and sequences of those processes. This work\ncontributessignificantlytounderstandingmechanismsofsolvationdynamicsofpolar\nsolvents.\nNovelty and originality.\nThe novelty of this work is in observing experimentally a change is the scattering\npattern due to the dipolar rearrangement of the solvent molecules surrounding pho-\ntoexcited HTI-J, capturing the formation of the transient solvation shell structure\non a ∼0.5 ps timescale and its re-equilibration as the system returns to the ground\nstate with a time constant of ∼2.6 ps, This is the first time when such structural\ndipolar rearrangement of the solvent molecules following HTI-J photoexcitation is\nexperimentally captured in X-ray solution scattering patterns and those experimen-\ntally observed structural changes are modeled on the atomic scale.\nData and methodology. Validity of the results.\nThe manuscript presents state of the art research both experimentally, methodolog-\nically and computationally. It combines cutting edge technologies in TRXSS with\nstatic and time-resolved optical spectroscopy and extensive in-silico experiments.\nThe combination of all those tools is required to identify, assign and model physical\nprocesses which occur upon photoexcitation of a small organic chromophore in a\npolar solute including energy dissipation, relaxation and charge re-distribution pro-\ncesses. The disentanglement of those processes is accomplished by a robust stepwise\nglobal optimization procedure guided by methods of singular vector decomposition,\nDFT and MD simulations. Methods section in the manuscript, and Supplemental\nMaterial present in detail robustness of procedures of statistical analysis and errors\nhandling,dataacquisition,dataprocessing,dataanalysisandsimulationsandensure\n13\nvalidity of the results.\nComments and suggestions.\nR3C1 Data presentation should be improved. The time-resolved scattering pat-\nterns are dominated by the solute heating signal caused by vibrational cooling of\nsolute molecules and heating of the solvent molecules. It is very important to see\nhow data looks after global fitting and subtraction of the solvent heating signal. In\nthis regard map representation does not give much information about actual data\nand noise levels contained in the data. In addition to the maps (Figure 4) heating\nsignal subtracted data should be presented for selected time delays where a major\ncontributionfromspecificprocessesisidentified. Thedatashouldshowexperimental\nerrors, and the quality of the fits, discussed in the manuscript.\nResponse R3C1\nWe have added a new figure (Fig. S23) in Supplementary Note 6.2 showing the background\nartifact- and heat-subtracted isotropic difference scattering signal at selected time delays to-\ngether with the model from global fitting as well as the model contributions attributed to the\noptical Kerr effect (OKE), the solvation-shell rearrangement (SSR), and vibrational relaxation\n(VR). The selected time delays are chosen as representative times where these components\ncontribute most strongly. Experimental uncertainties obtained as error bars from polynomial\nnoise estimation are also included. A reference to this figure has been added to the main text:\n“Heat-subtracted isotropic difference scattering traces at selected delay times, chosen to high-\nlight where specific processes dominate, are shown in Fig. S23 together with the corresponding\nindividual model components.”.\nR3C2 It is not clear why authors use acetonitrile patten from a ref [15] in the data\nanalysis. Theexperimentaldatawerenormalizedaccordingtoestablishedstandards.\nterials, Liquid unit cell scaling) and in the fitting procedure of scattering pattern\nfrom [15] authors dismiss very small contribution for HTI-J molecule anyway. Why\nauthors need fitting of the acetonitrile data, collected at different experimental con-\nditions previously, and then rescale already normalized data. Did authors try to\nprocess the data without this additional scaling? Did they perform static measure-\nments of the acetonitrile and temperature jump measurements in this solvent along\nwith HTI-J photoexcitation measurements?\nResponse R3C2\nThe reference acetonitrile signal from Kjær et al. (Phys. Chem. Chem. Phys. 15, 15003 (2013)\nDOI:10.1039/C3CP50751C) corresponds to a known temperature increase of 1 K in the bulk\nsolvent. By scaling our bulk solvent heating contribution to this reference, we can express the\ntime-dependent temperature change, ∆T(∆t), in units of Kelvin. Omitting this calibration step\ndoesnotchangethequalityofthefitortheextractedtimeconstants. Wehaveclarifiedthispoint\nin the text of Supplementary Note 5.1 and the caption of Fig. S18. We did not perform static\nor temperature jump measurements of acetonitrile during the HTI-J experiment at SwissFEL.\nR3C3 The authors did laser power titration measurements for the laser wavelength\n470 nm and proved that the measurements are performed in the linear regime. How\nmany photons were delivered per HTI-J chromophore? Did authors try to estimate\nwhat additional fitting procedure (discussed above) could contribute to errors in the\nheatingsignalincrease(temperaturejump)andauthorsconclusionsaboutmultipho-\nton excitation in the experiment?\nResponse R3C3\nWe have included a second order polynomial fit to the laser fluence scan (Supplementary Note\n4.1), which suggests that a laser excitation energy of 12 µJ/pulse employed in the experiments\n14\nis at the upper end of the linear-response region. Using the pump laser energy of 12 µJ and a\nconcentration of the solution of 3 mM, we estimate that approximately 5 photons were delivered\nper HTI-J chromophore. Given this and the near-linear fluence dependence at 12 µJ/pulse,\nwe attribute the higher-than-expected increase in temperature to excitation of a fraction of the\nexcited state population into high-energy states via multiphoton excitation. We would like to\nrefer the reviewer to our response to R1C6 and R4C1 for further discussion on this.\nWhen using the average between 55 and 60 ps of the isotropic difference scattering signal as the\nbulk solvent heating component, ∆S , without the scaling to e.u./LUC/K, the kinetic profile\n∆T\nof this component exhibits a similar bi-exponential increase, but the amplitude, ∆T(∆t), does\nnot reflect a temperature increase in Kelvin in this case.\nR3C4 Multiphoton absorption in organic chromophores may significantly impact\nenergy transfer and relaxation processes and trigger different pathways and rates.\nTherefore, it may alter the photophysical properties of the system. The authors\nshoulddiscussinthemaintextwhatchanges(ifany)intheidentifiedinthemanuscript\nrelaxation processes this may cause besides mentioned in the manuscript fast non-\nradiative relaxation from higher excited state causing fast solvent heating (0.2 ps,\nTable 1).\nResponse R3C4\nThe weak non-linear behavior at the employed laser fluence shown by the laser fluence scans\nsuggests that multiphoton excitation only leads to excitation of a fraction of the excited state\npopulation into higher-lying excited states. Consistent with this, the only observable conse-\nquence in our TR-XSS data is the additional ultrafast heating component assigned to rapid\nnon-radiative relaxation from higher-lying states. As also discussed in detail in our response to\nR1C6 and R4C1, we find no evidence for additional pathways that would alter the relaxation\ndynamics. In particular, photoionization of either solvent or solute is unlikely under our condi-\ntions, as it would require 4- or 3-photon excitation based on known ionization thresholds and\nbecausepreviousstudieswithsimilarexperimentalconditions,reportnoevidenceofsucheffects.\nWe have expanded Supplementary Note 8 to include a more detailed discussion of multiphoton\neffects and added a short discussion to the main text.\nR3C5DFTcalculationsandMDsimulationsareveryimportantfortheatomic-scale\ninterpretationofthedata. HowMDsimulationsareusedforthedeterminationofthe\nfinal structures, however, requires additional explanations. The manuscript presents\nexciting results of interplay between molecule of solute and surrounding molecules of\nsolvent (solvation dynamics), including solvent excluded volume changes, changes in\nits interatomic distances and changes in polarization of the solvent. In Methods it\nis emphasized that for equilibrium MD simulations, 400000 equidistant frames were\ncollected over 400 ns. It is not clear how these 400000 trajectories were used in the\nfinal selection of the structures. How many runs were performed, what were criteria\nfor the final structure selections? This should be explained, and appropriate data\nshould be presented.\nResponse R3C5\nWe performed four sets of equilibrium MD simulations in the NVT ensemble for HTI-J in ace-\ntonitrile. OneinwhichHTI-JismodelledinitsgroundstateandthreewhereHTI-Jisdescribed\nwith different excited state models: charge transfer and twist, only charge transfer, and only\ntwist. Ineachcase, theequilibriumgeometryandpartialchargesofthesoluteareobtainedfrom\nDFT calculations in the ground or excited state. For each system, we performed an NVT pro-\nduction run of 400 ns, from which one frame every ps is sampled. The resulting 400000 frames\nrepresented an equilibrum ensemble from which the radial distribution functions (RDFs) are\ncalculated. No additional structure selection was performed beyond equilibration and uniform\n15\nsampling.\nWe have extensively rewritten the part of the Computational methods section presenting the\nequilibrium MD simulations to make this workflow more clear, clarifying the number of simula-\ntions and sampling procedure.\nR3C6 It is confusing to read sections of the main text of the manuscript describing\nsolvent heating, vibrational relaxation, and optical Kerr effect and solvation dynam-\nics with the detailed explanations of physics, energy flow and timing of multiple\nprocesses. It would be much easier for a reader if this information is also presented\nin a visual schematic or a diagram which a reader can follow in addition to the text\ndescription.\nResponse R3C6\nTo make the sequence and relative timing of the different processes easier to follow, we have\nadded a plot of the kinetic profiles of the individual signal components to Fig. 5. This provides\na visual representation of their temporal evolution, and we refer to this figure explicitly in the\nrelevant part of the main text.\nR3C7 Sentence lines 95–98 is not clear, it reads like “ ..Knowledge … could reduce\nenergy loss through thermal dissipation..’, perhaps it should be rephrased.\nResponse R3C7\nWe have rewritten this sentence, which now reads: “Such knowledge could ultimately enable\ncontrol over the lifetime of charge-separated states and help reduce energy loss due to thermal\ndissipation, thereby improving efficiency and stability in photosensitization and photoconver-\nsion.”.\nReferences:\nThe manuscript references previous literature adequately.\nClarity:\nThe manuscript is written in clear language, understandable to an expert and a general audience. Argumentation is clear\nwith the appropriate references when required. Abstract, Introduction and Conclusions are appropriate. Methods and\nSupplemental materials are very helpful.\nReviewer expertise:\nMy expertise is in time-resolved X-ray solution scattering and supporting experiments (for example\noptical spectroscopy) and analysis of time-resolved and static X-ray solution scattering data, in particular for biomolecules in\nsolutions. The topic of this manuscript does align partially with the field of my expertise.\nMy assessment that the manuscript can be published in Nature Communications with revisions, which address above\ncomments\nReviewer 3 (Remarks on code availability):\nI reviewed the code but did not run it. The code provides step-by step guide for\ndataanalysisofTRXSSandTRA.Fitsclearlyshowgoodquality. Thecodeisusable\nresource for the community and nice training tool for those who start their career in\nthe field. README file provides lists of tutorials on how to run the code. It would\nbe helpful if authors also provided Conda environment (instructions) as running the\ncode requires multiple python-based packages installed.\nResponse Reviewer 3 (Remarks on code availability)\nWe have added an ‘environment.yml’-file for conda to handle the required dependencies, and\nupdated the README.md of the code to provide conda-specific installation instructions to\ncomplement the pip/venv-based instructions we had initially supplied.\nReviewer 4\nThis is an important experimental advance – obtaining time-resolved solution scat-\nteringonanorganicmolecule. Time-resolvedX-raysolutionscatteringonthefemto-\n16\npicosecond timescale is now a well-established technique at X-FELs. To date, these\nexperiments have been performed on solute-solvent systems where the solute con-\ntains high Z atom(s), which have strong scattering cross-sections.\nBefore the work is published the authors should address the following comments:\nR4C1 The issue of high optical pump fluence 12 µJ/pulse needs to be addressed.\nFromFigureS8, thisreviewerdoesnotagreethatthedatawascollectedinthelinear\nregime. The data could be easily fit by higher order terms.\n(a) What was the optical pump fluence used in the optical transient absorption\nexperiments? If different from the X-ray experiments, then it should be rationalized,\northeTAexperimentsmustbeperformedwiththesamefluencefora1:1comparison.\n(b) The pump fluence dependent study for the transient X-ray was conducted at 20\nps. At that time delay, there is very little difference signal from the solute. It seems\nthat the pump-fluence is measuring the solvent heating response. Is that correct?\n(c) At some point in the main text or SI, the authors point to multi-photon effects\nin the solvent. How does that match with the difference signal being linear in pump-\nfluence.\nResponse R4C1\nWe have added a second-order polynomial fit to the laser fluence scan (Fig. S8, Supplementary\nNote 4.1), which suggests that the laser fluence dependency begins to deviate from the linear\nregime around 10–13 µJ/pulse. With an excitation energy of 12 µJ/pulse, we are at the upper\nend of the linear regime and we observe a higher energy deposition into the solvent than ex-\npected for one-photon excitation (discussed in Supplementary Note 8), indicating multiphoton\nexcitation of the solute.\n(a) The optical pump fluence used in the transient absorption spectroscopy (TAS) experiments\nwas approximately 2.6 µJ/mm2, which is lower than the ∼1.2 mJ/mm2 fluence of the pump\nlaser in the time-resolved X-ray solution scattering (TR-XSS) experiment. A higher pump flu-\nence of around 1 mJ/mm2 is typically necessary due to the repetition rate of these experiments\n(approximately 100 Hz), whereas TAS experiments generally operate at repetition rates of a few\nkHz, allowing sufficient signal-to-noise ratios to be achieved at lower pump power. While the\ninternal conversion time obtained from TAS was used as an initial guess for the global fitting of\nthe TR-XSS signal for the solvation-shell rearrangement (SSR) component, the right singular\nvector of the second SVD component, after subtracting bulk solvent heating and vibrational\nrelaxation from the difference scattering signal, shows a time evolution on a similar timescale as\nobtained in the final global fit. The corresponding left singular vector resembles the simulated\nsignal for a solvation-shell rearrangement in response to charge transfer in the solute, as shown\nin Fig. S21. Given the similar timescales observed for the excited state lifetime in TAS at lower\nexcitation fluence and the solvation-shell rearrangement in TR-XSS, we conclude that multipho-\ntonexcitationofthesoluteonlyleadstopopulationofhigher-energystates, followedbyultrafast\ninternal conversion to the lowest excited state, which then decays on a timescale similar to the\nexcited-state lifetime observed in TAS. Photoionization processes that would alter significantly\nthe relaxation pathways are excluded as discussed extensively in our reply to R1C6. This is con-\nsistent with (1) the regime of weak non-linearity at the employed laser energy of 12 µJ/pulse,\nand (2) the observed fast component of the rise of the heating signal (τ1 ∼ 0.2 ps). To better\n∆T\nreflect these important considerations, we have substantially revised Supplementary Note 8 and\nadded a discussion in the main text (see also our response to R1C6).\n(b) Yes, this is correct. Although desirable, analyzing and interpreting an energy-fluence scan\nfor the solute response during data acquisition is presently unfeasible given the small magnitude\nof the solute-related signal.\n(c) After revising the laser fluence scan discussed in Supplementary Note 4.1 and adding the\nsecond-orderpolynomialfit,itbecomesevidentthatthechosenlaserfluencewasattheupperend\n17\nof the linear-response regime. In combination with the discussion in Supplementary Note 8, this\nsupports the interpretation that multiphoton excitation of the solute occurred as a consequence\nof the applied laser energy.\nR4C2 The authors have detailed their data analysis and data reduction. However,\noneisleftwonderingifthedataisover-fit. Themultipletimescalesandnomenclature\nused in Table 1 of the main text are confusing. It is also odd that the transient\nabsorption data does not measure the longer timescales for solute vibrational cooling\nor the slow solvent heating. Why are these not the same thing?\nResponse R4C2\nThe scattering data were globally fitted using 11 parameters associated with four physically\njustified processes, with q-profiles and contributions to the scattering signal identified through a\nstepwiseanalysis,asdetailedinSupplementaryNote5. AsdescribedinSupplementaryNotes5.3\nand5.4, modelingthedatawithonlythebulksolventheatingandvibrationalrelaxationcompo-\nnents and subtracting these contributions from the signal leaves residual features that appear at\ntime zero and evolve on a picosecond timescale. As shown in Fig. S20, singular value decompo-\nsition (SVD) of this residual signal reveals two components with clear temporal evolution. The\ncomponent with largest amplitude is assigned to the optical Kerr effect, while the second com-\nponent strongly resembles the simulated signal for solvation-shell rearrangement due to charge\ntransfer in the solute, calculated from MD simulations (Fig. S21). The corresponding right\nsingular vector evolves on timescales similar to those obtained in the global fit of the complete\ndataset. For these reasons, we do not believe that the data are overfitted.\nTo make Table 1 easier to read, we added spacing to group parameters related to each of the\nfour physically justified signal components. In the caption, we also clarified that the internal\nconversion time from optical transient absorption spectroscopy is included alongside the time\nconstants obtained from the global fit of the TR-XSS data. Additionally, we added kinetic pro-\nfiles to Fig. 5 to visualize the temporal evolution of the signal components in the main article,\nwhich will help readers to better follow the discussion in the text.\nWe agree that observing vibrational cooling in the ground state in the optical transient ab-\nsorption data would provide additional support to our assignment of the vibrational relaxation\ncomponent in the TR-XSS data. However, in our TAS measurements, the laser excitation en-\nergy of 490 nm lies close to the absorption maximum, and pump scatter overlaps the spectral\nregion where the ground state bleach is expected. As a result, the ground state bleach cannot\nbe observed, and we cannot extract a vibrational cooling time from the TAS data. Therefore,\nwe relied on reference studies reporting vibrational cooling times in acetonitrile in the range of\n10–15 ps, as discussed in the paragraph on vibrational relaxation in the main article. To make\nthis limitation of the TAS data clear, we have added the following statement to the caption of\nSupplementary Fig. S5: “Due to pump scatter within the range of the absorption spectrum\nof HTI-J (see Fig. S4), the ground state bleach is not observed, and vibrational cooling in the\nground state could not be inferred from the TAS data.”.\nR4C3 The authors claim that their work provides atomic scale resolution of polar\nsolvation dynamics. However, they do not provide any non-equilibrium RDFs of the\nsolute-solventinteractionsviaMDsimulations. TheirMDsimulationsshouldinclude\nnon-equilibrium solute-solvent interactions because they fit these timescales in their\ndata. If non-equilibrium MD simulations are not included, then there should be a\ndiscussion in the main text about what is missing.\nResponse R4C3\nWhile the transient solvation-shell structure is resolved at the atomic length scale, the present\nTR-XSS analysis does not yield the time evolution along specific solute or solute-solvent atomic\ncoordinates (see also our response to R1C1), which would require simulating nonequilibrium ra-\ndialdistributionfunctions. WehaveaddedthefollowingsentencetotheComputationalmethods\n18\nsection to make it clear that nonequilibrium MD simulations are not included: “... These sim-\nulations correspond to the HTI-J molecule in the excited state with a fully formed transient\nsolvation shell, and do not account for nonequilibrium solvent motions during the solvation shell\nrearrangement.”.\nIn conclusion, while this is an important experimental demonstration, the data anal-\nysis and quality of the data do not provide any new physically relevant information.\nOther changes not directly connected to the reviewers’ comments\n1. IntheIntroduction, wehaveaddedcitationstothefollowingarticles, whichwehadmissed\nin the original version of the manuscript:\n• Krell-Jørgensen et al. Chem. Commun. 59, 563 (2023) DOI:10.1039/d2cc05548a. A\npaper reporting on recently developed hemithioindigo derivatives closely related to\nthe one investigated in the present manuscript.\n• Vester et al. J. Chem. Phys. 157, 224201 (2022). DOI:10.1063/5.0107224. A recent\ntime-resolved X-ray spectroscopy and scattering study of the solvent reorganization\nfollowing photoabstraction of an electron from a halogen ion.\n2. In Fig. 7, we replaced the optimized q-profile of the ∆S component from global fitting\nSSR\nwith an average of the data for 0 < ∆t < 5 ps after subtracting contributions from\nbackground artifact components, heat, vibrational relaxation and the optical Kerr effect.\n3. We added a specification of the loss function to the description of the global fitting algo-\nrithm in the Methods section in the main article and in Algorithm 1 in the Supplementary\nInformation.\n4. We renamed “power titration” to “laser fluence scan” and replaced “laser power” with\n“laser energy” when referring to the pump laser energy of the TR-XSS experiment.\n5. We fixed typos and made other minor changes to the text.\nBelow, we provide a point-by-point response to the reviewers’ comments. Changes made to the\nmanuscript and Supplementary Information (SI) in response to the reviewers’ comments are\nhighlighted in blue in the annotated version of the revised manuscript and SI provided with this\nresubmission.\nReviewer 1\nRequest for quantitative analysis of solvation shell (final action item)\nWhile the authors have clarified in the text that the experiment tracks the solvation-\nshell rearrangement signal rather than specific atomic trajectories, the support for\n“atomic-scaleobservation”currentlyreliesonqualitativecomparisonsofMD-derived\nRadial Distribution Functions (RDFs) in Figure 6. To bridge the gap between the\nqualitative visual evidence and the quantitative claim of “atomic-scale” sensitivity,\na specific breakdown of the structural changes is necessary.\nSuggestion:\nI suggest including a table (in the Main Text or SI) summarizing the quantitative\nstructural parameters extracted from the MD simulations for the first and second\nsolvation shells. Specifically:\n- Peak positions (rmax) for relevant atom pairs (e.g., Solute-N, Solute-Methyl) in\nthe ground state vs. charge-transfer state.\n- Coordination numbers (CN) for these shells.\n- The quantitative magnitude of the change (∆r, ∆CN) upon excitation.\nProviding these numbers will concretely define what “atomic-scale” changes are re-\nsponsible for the observed scattering signal.\nAuthors’ response\nWe thank the reviewer for this suggestion. We have added a new quantitative analysis of the\ncalculated solute-solvent radial distribution functions (RDF) in Suppl. Note 10.1. In particular,\nwe have included two new figures, one showing representative solute–solvent RDFs from the\nmoleculardynamicssimulationsofHTI-Jinthegroundandexcitedstates,andasecondshowing\nthe ratio of the cumulative coordination numbers for the nitrogen and methyl carbon sites of\nacetonitrile, computed from the corresponding solute–nitrogen and solute–methyl RDFs. In\naddition, we have added a new table summarizing key structural parameters extracted from\nthe RDF analysis: (i) the position of the first RDF maximum, r , (ii) the mean distance\ngmax\nwithin the first solvation shell, ⟨r⟩, and (iii) the coordination number of the first solvation\nshell, CN. These additions provide a quantitative measure of the structural changes in the\nfirst solvation shell upon excitation and clarify which atomic-scale rearrangements underlie the\nobserved scattering signal. References to the quantitative RDF analysis presented in Suppl.\nNote 10.1 are included in the main text of the manuscript when introducing the results of the\nmolecular dynamics simulations (Fig. 6) and after comparing the simulated X-ray scattering\nsignal to the experimental data in Fig. 7.\nSeveral reviewers have pointed out that this work does not provide an ”atomic-scale\nobservation” of polar solvation dynamics. The authors have chosen to address this\nby adding following statement in the conclusion: “The experiments provide a clear\natomic-scale signature of the transient solvation shell. However, neither the time\nevolution of specific solvent motions underlying the solvation-shell rearrangement\nnor any accompanying solute structural change could be resolved.” The authors\nshould consider changing the title of the paper and the abstract to clear indicate the\nlimitations of this work.\nAuthors’ response\nThetitleandabstractofthemanuscripthavenowbeenrewritten, addressingtheconcernsofthe\nreviewer. The new title reads “Tracking Polar Solvation Dynamics of a Photoexcited Organic\nChromophore with Ultrafast X-ray Scattering”."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Summary and Recommendation\nThe authors have submitted a substantially revised manuscript that successfully addresses the critical technical and\ninterpretational concerns raised in the first round of review. The revision demonstrates a high degree of transparency,\nparticularly regarding the excitation fluence conditions and the limitations in distinguishing solute structural models, and\nincorporates rigorous theoretical validation (DLPNO-CCSD(T)). The authors have adequately addressed the comments in\ntheir point-by-point responses, and the corresponding changes to the manuscript are appropriate. The work now stands as a\nrobust contribution to the field. I recommend acceptance of the manuscript, subject to one final addition described below to\nfully substantiate the “atomic-scale” claims made in the title and abstract.\n1) Request for quantitative analysis of solvation shell (final action item)\nWhile the authors have clarified in the text that the experiment tracks the solvation-shell rearrangement signal rather than\nspecific atomic trajectories, the support for “atomic-scale observation” currently relies on qualitative comparisons of MD-\nderived Radial Distribution Functions (RDFs) in Figure 6. To bridge the gap between the qualitative visual evidence and the\nquantitative claim of “atomic-scale” sensitivity, a specific breakdown of the structural changes is necessary.\nSuggestion:\nI suggest including a table (in the Main Text or SI) summarizing the quantitative structural parameters extracted from the MD\nsimulations for the first and second solvation shells. Specifically:\n- Peak positions (r ) for relevant atom pairs (e.g., Solute-N, Solute-Methyl) in the ground state vs. charge-transfer state.\nmax\n- Coordination numbers (CN) for these shells.\n- The quantitative magnitude of the change (Δr, ΔCN) upon excitation.\nProviding these numbers will concretely define what “atomic-scale” changes are responsible for the observed scattering\nsignal.\n2) Assessment of Resolved Issues\nAll other comments from the previous round have been satisfactorily addressed:\n- Model Comparison (Point 2): The new side-by-side comparison (Fig. 7) and the admission that the models are statistically\nindistinguishable (χ2 ≈ 4) are appropriate and scientifically rigorous.\n- Terminology (Point 3): The adoption of “Solvation-Shell Rearrangement (SSR)” eliminates ambiguity.\n- Figures (Point 4): Revised figures and captions correctly attribute twisted geometries to theoretical predictions rather than\ndirect observation.\n- Theoretical Validation (Points 5, 8): The inclusion of DLPNO-CCSD(T) calculations and clarification of the explicit solvent\nMD workflow provide a solid theoretical foundation.\n- Fluence/Heating (Points 6, 7): The power titration analysis and the validation of the heating signal extraction by comparing\ndifferent time delay windows (Fig. S17) effectively resolve concerns about signal contamination.\n- Anisotropic Signal (Point 9): The SVD analysis added to the SI adequately addresses the potential of the anisotropic data.\n- Literature Context: The authors have appropriately cited a key reference, providing the necessary context on anisotropic\nTR-XSS and atomic charge redistribution.\nRecommendation: Accept (after minor revision regarding the RDF quantification).", "reviewer_2": "I have read carefully the response of the authors to the referee reports, which all four raise interesting questions and\ncomments. The authors' responses, in some instances including additional work, and finally the commensurate changes in\nthe manuscript (or the SI), are satisfactory and improve the manuscript further. My overall assessment is that the resulting\nmanuscript warrants publication in Nature Communications.", "reviewer_3": "My comments were addressed in the revisions of the manuscript NCOMMS-25-77889A \"Atomic-Scale Observation of Polar\nSolvation Dynamics Triggered by Photoinduced Charge Transfer in an Organic Chromophore\". My assessment is that the\nrevised version of the manuscript can be published in Nature Communications.", "reviewer_4": "This review is on the revised manuscript and response to reviewer comments of Levi et al on the ultrafast X-ray solution\nscattering (XSS) study of an organic chromophore in solution.\nI would like to start by commending the authors for taking the time to respond to all the reviewer comments carefully. The\ncomments from four independent reviewers were quite consistent.\nAfter careful review, my overall impression of the manuscript remains unchanged.\n- This is an important demonstration experiment of the potential of ultrafast XSS on organic chromophores in solution.\nHowever, the lack of signal-to-noise, prevents the discovery of new molecular-level information.\n- This reviewer continues to remain concerned about the high excitation fluence. I appreciate the non-linear fit of the laser\nfluence scan in Fig. S8, which clearly shows that the data was measured in the non-linear regime excitation regime. This\nmakes it challenging to understand the ensuing photochemical pathways taken. The authors have discussed this in the\ncontext of there being higher electronic states which are accessed and that they decay to the lowest excited electronic state\non a short timescale. From an experimental standpoint, it is incorrect to interpret ultrafast photochemical experiments where\nthe pump pulse is in the non-linear excitation regime. If the goal of this paper is simply to demonstrate the feasibility of\nultrafast XSS on organic chromophores then, it has achieved its goal. If the goal is to identify specific physical phenomena\n(including solvation) following photoexcitation, then the higher fluence presents a problem.\n- Several reviewers have pointed out that this work does not provide an \"atomic-scale observation\" of polar solvation\ndynamics. The authors have chosen to address this by adding following statement in the conclusion: \"The experiments\nprovide a clear atomic-scale signature of the transient solvation shell. However, neither the time evolution of specific solvent\nmotions underlying the solvation-shell rearrangement nor any accompanying solute structural change could be resolved.\"\nThe authors should consider changing the title of the paper and the abstract to clear indicate the limitations of this work.\nWhile I have outlined my concerns about the manuscript above, I do believe that with advances at X-FELs we will be able to\nobserve time-dependent, atomic-scale displacements in photoexcited solute molecules and the surrounding solvent and be\nable to provide an atomic description of organic photochemistry in solution. This paper is an early demonstration about the\npotential of this technique."}, "author_response": "Response to Reviewers of Manuscript NCOMMS-25-77889A\nBelow, we provide a point-by-point response to the reviewers’ comments. Changes made to the\nmanuscript and Supplementary Information (SI) in response to the reviewers’ comments are\nhighlighted in blue in the annotated version of the revised manuscript and SI provided with this\nresubmission.\nReviewer 1\nRequest for quantitative analysis of solvation shell (final action item)\nWhile the authors have clarified in the text that the experiment tracks the solvation-\nshell rearrangement signal rather than specific atomic trajectories, the support for\n“atomic-scaleobservation”currentlyreliesonqualitativecomparisonsofMD-derived\nRadial Distribution Functions (RDFs) in Figure 6. To bridge the gap between the\nqualitative visual evidence and the quantitative claim of “atomic-scale” sensitivity,\na specific breakdown of the structural changes is necessary.\nSuggestion:\nI suggest including a table (in the Main Text or SI) summarizing the quantitative\nstructural parameters extracted from the MD simulations for the first and second\nsolvation shells. Specifically:\n- Peak positions (rmax) for relevant atom pairs (e.g., Solute-N, Solute-Methyl) in\nthe ground state vs. charge-transfer state.\n- Coordination numbers (CN) for these shells.\n- The quantitative magnitude of the change (∆r, ∆CN) upon excitation.\nProviding these numbers will concretely define what “atomic-scale” changes are re-\nsponsible for the observed scattering signal.\nAuthors’ response\nWe thank the reviewer for this suggestion. We have added a new quantitative analysis of the\ncalculated solute-solvent radial distribution functions (RDF) in Suppl. Note 10.1. In particular,\nwe have included two new figures, one showing representative solute–solvent RDFs from the\nmoleculardynamicssimulationsofHTI-Jinthegroundandexcitedstates,andasecondshowing\nthe ratio of the cumulative coordination numbers for the nitrogen and methyl carbon sites of\nacetonitrile, computed from the corresponding solute–nitrogen and solute–methyl RDFs. In\naddition, we have added a new table summarizing key structural parameters extracted from\nthe RDF analysis: (i) the position of the first RDF maximum, r , (ii) the mean distance\ngmax\nwithin the first solvation shell, ⟨r⟩, and (iii) the coordination number of the first solvation\nshell, CN. These additions provide a quantitative measure of the structural changes in the\nfirst solvation shell upon excitation and clarify which atomic-scale rearrangements underlie the\nobserved scattering signal. References to the quantitative RDF analysis presented in Suppl.\nNote 10.1 are included in the main text of the manuscript when introducing the results of the\nmolecular dynamics simulations (Fig. 6) and after comparing the simulated X-ray scattering\nsignal to the experimental data in Fig. 7.\nSeveral reviewers have pointed out that this work does not provide an ”atomic-scale\nobservation” of polar solvation dynamics. The authors have chosen to address this\nby adding following statement in the conclusion: “The experiments provide a clear\natomic-scale signature of the transient solvation shell. However, neither the time\nevolution of specific solvent motions underlying the solvation-shell rearrangement\nnor any accompanying solute structural change could be resolved.” The authors\nshould consider changing the title of the paper and the abstract to clear indicate the\nlimitations of this work.\nAuthors’ response\nThetitleandabstractofthemanuscripthavenowbeenrewritten, addressingtheconcernsofthe\nreviewer. The new title reads “Tracking Polar Solvation Dynamics of a Photoexcited Organic\nChromophore with Ultrafast X-ray Scattering”."}]

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