Single droplet displacement infrared action spectroscopy

Domain: biology
Published: 2026-03-26
Editorial outcome: minor_revision

Abstract
Aqueous microdroplets have fascinated scientists with unique properties that deviate from bulk solutions. Understanding microdroplet chemistry has been limited by few spectroscopic measurements on single well-defined microdroplets. Here we present a new technique to record an IR spectrum of a single droplet called Single Droplet Displacement Infrared Action Spectroscopy (SiDDIRAS) with potential broad applications in physical, analytical, and environmental chemistry. The droplet was trapped in a linear quadrupole electrodynamic balance. When IR radiation at resonant frequency was introduced, mass loss from IR-induced evaporation caused the droplet to displace upwards. Mapping the displacement as a function of IR frequency yields an IR spectrum of a single microdroplet. As a demonstration, the SiDDIRA spectrum of an ~8 µm droplet containing NaCl and NaN3 shows that the azide asymmetric stretch blueshifts by 5 cm-1 relative to bulk with significant peak broadening, evidence that this droplet is supersaturated with salts compared to bulk solutions.

Introduction
Electrospray ionization (ESI) remains a popular method to introduce ions and charged biomolecules to a mass spectrometer for analysis since its invention in the 1960s.1 Yet the complex ionization process from charged droplets to charged ions continues to be a topic of active research.2, 3 More recently, using ESI and pneumatic sprays, studies by several groups have reported enhanced chemical reactivity in microdroplets.4, 5, 6, 7, 8 More broadly, chemistry occurring in droplets and aerosol particles in the environment has great impact on atmospheric chemistry and climate.9

The electrodynamic balance is a class of instruments that have been used to study microdroplets for decades. In recent years, the linear quadrupole electrodynamic balance (LQ-EDB) has become popular.10 The LQ-EDB laterally confines charged droplets by applying AC voltages to four cylindrical rods held in a quadrupole configuration (Figure 4A, vide infra) while balancing gravity on the droplet with a DC voltage (Figure 4B, vide infra). The LQ-EDB is held in an environmentally controlled chamber and can be coupled with various optical techniques to obtain physical information such as radius,11 refractive index (RI),12 hygroscopicity,13 and viscosity.14 Specifically, in our set up, a broadband LED is used to collect back-scattered light with characteristic whispering gallery modes (WGMs) from the droplet to determine its radius and RI simultaneously (left in Figure 4A, vide in fra), as described previously12 and in more detail in Section S1.2 in the Supporting Information (SI). The chemical composition of the droplet can also be obtained by coupling the LQ-EDB with Raman13, 15 or fluorescence16 spectroscopies and mass spectrometry,17, 18, 19 further highlighting the versatility of the instrument.

Among these techniques, the use of IR spectroscopy for measuring single droplets has been limited, despite its potential for revealing the unique chemical microenvironment of the droplets, especially its sensitivity to changes in the hydrogen bonding network of water. Traditional FTIR measurements are not possible with trapped micron-sized droplets due to scattering and the small optical path length. IR spectroscopy studies based on optical photothermal effects have been reported for droplets deposited on a substrate,20, 21, 22 but the droplet-substrate interface might interfere with droplet chemistry. Additionally, the charge state and radius of such droplet is difficult to determine. To overcome these challenges, we report a new type of IR “action” spectroscopy, the principle of which has provided, and continues to provide, deep chemical and physical insights in the gas-phase community for the past few decades.23, 24, 25, 26, 27

Action IR spectroscopy on aerosol particles trapped in an EDB were first introduced by several groups in the 1980s. Arnold et al.28, 29 described a technique called Structure Resonance Molecular Spectroscopy (SRMS), where the authors induced an ammonium sulfate droplet’s evaporation at a certain IR frequency using an SiC Glo-Bar. The fractional change in a droplet’s scattering pattern upon resonant IR-induced evaporation is then fitted with Mie theory and mapped as a function of IR frequency to produce an IR spectrum. Lin30 followed with a method for measuring an action IR spectrum of solid particles, where the photophoretic force acting on a trapped solid ammonium sulfate particle upon absorbing IR photons from a CO laser displaced the particle from its original position. Thus, by measuring the fractional change in balancing voltage required to bring the particle back to its original position (ΔV/V) and mapping it to the IR frequency, an action spectrum was obtained.

Based on these seminal works, we described below an action IR technique for aqueous microdroplets in a LQ-EDB that does not require complex Mie fittings, as shown in Arnold et al.’s works.28, 29 In a way, our detection method is more similar to that described by Lin30 in that we are using the droplet’s displacement from its original position in our measurement. However, we want to stress that we are not measuring the photophoretic force acting on the droplet nor the fractional voltage to bring the droplet back to its original position. Instead, we are mapping the magnitude of the droplet’s displacement upon IR-induced evaporation as a function or IR frequency from a tunable quantum cascade laser (QCL). We have coined this technique Single Droplet Displacement Infrared Action Spectroscopy (SiDDIRAS). SiDDIRAS does not require complex optics or post-acquisition analyses. This technique is easily reproducible, highly accurate, and, depending on scan speed and needs, can provide up to sub-wavenumber (≤ 1 cm-1) resolution.

In this work, we reported the SiDDIRA spectrum of an ~8 µm droplet containing NaCl and NaN3. Leveraging the sensitivity of the azide anion’s (N3−) asymmetric stretch to the ionic environment, we showed that this droplet remained aqueous while supersaturated with salt through SiDDIRAS and other established methods. The SiDDIRA spectrum confirmed that the N3− anions formed ion pairs with Na+ cations, and that the water hydrogen bonding network of the microdroplet is altered due to the water molecules’ interactions with ions. This work showed the robustness and sensitivity of SiDDIRAS, which will contribute to the arsenal of analytical techniques to unveil many unanswered questions in microdroplet chemistry.

Results
Since the ionic environment of microdroplets is of interest, we chose the N3− anion as a molecular vibrational probe. N3− is a popular vibrational probe because its asymmetric fundamental vibration (νas) in water at 2048 cm-1 (black trace in Figure 1A) does not overlap with other common functional groups such as -OH, -NH, carbonyls, carbonates, and especially water in the mid-IR region. Additionally, νas is sensitive to its environment. Stronger interaction with solvent molecules causes dramatic blueshifts (1987 cm-1 in vacuum31 to 2000 cm-1 in DMSO32 to 2048 cm-1 in water33). Blueshifts are also observed when ion pairs (IPs) are formed. In DMSO, a ~25 cm-1 blueshift is observed when N3− forms contact ion pairs (CIPs) with Na+ cation.32 In aqueous nanoconfined reverse micelles (RMs), a more moderate ~9 cm-1 blueshift for formation of IPs with Na+ ions is observed, presumably due to shielding effect from water-ion interactions.33 The authors in that study assigned the blueshift to formation of IPs in small, nanometer-sized RMs, due to an increase in ion concentration and decrease in water concentration. This phenomenon in nanoconfined water in RMs is known,34, 35, 36, 37, 38, 39, 40 and has been reported to be the reason for the breaking of the hydrogen bonding network,41 which leads to a decrease in proton transfer rate and a shutdown of the Grotthuss mechanism.

We emulated the results from aqueous RMs studies by taking an FTIR spectrum of a saturated NaCl bulk solution at 5.4 M, shown in red in Figure 1A. Indeed, we observed a modest (~4 cm-1) blueshift at 2052 cm-1 in the νas for Na+ ion even at saturation limit at room temperature (Figure 1A). Additionally, the FWHM also increases by ~7 cm-1 as [Na+] increases. This blue shift and peak broadening trend are consistent with previous studies for N3− forming IPs with Na+,32, 33 although those studies did not provide adequate explanations for this broadening behavior. We have previously reported similar line broadening for the nitrile (-CN) group as it forms Lewis bonds with transition metals.42, 43

With the complete knowledge of the spectral behavior of the νas when coupled to Na+, we first isolated a droplet containing NaCl and NaN3 with r = 7.944 µm in the LQ-EDB (see sizing information in Figure 2). The droplet was generated from a bulk solution containing 2.5 g/L (~43 mM) NaCl and 2.5 g/L (~39 mM) NaN3. However, as the droplets are generated in the LQ-EDB, water evaporates, and the composition of the droplet equilibrates with the chamber environment. For that reason, one cannot assume the concentrations in the droplet to be the same as the bulk solution. Determining the concentration of species in microdroplets that are generated in this way will be discussed later. We obtained the SiDDIRA spectrum for the 7.944 µm droplet, with the raw droplet z displacement shown as open black circles in Figure 1B. Details of the data processing are discussed in Section 1.3 in the SI with the raw data shown in Figure S2. The corresponding video of the droplet motion as resonant IR light is turned on and off is also attached. Additionally, for the action spectrum to be meaningful, the droplet displacement must be linear with the laser power, which is discussed in Figure S4. As the droplet displaced upwards, the irradiation efficiency may decrease if the droplet is no longer in the IR laser’s path. The IR beam size in our setup is measured to be ~0.724 mm (details in Section S1.4, Table S2 and Figure S5), which is much bigger than the droplet’s displacement (0.13 mm). Hence, we believe the irradiation efficiency change to be negligible.

The raw droplet displacement data fits well to two Gaussian peaks, shown in red and blue in Figure 1B, which combined to form the black trace in Figure 1B (fitting details discussed in Section S1.3 and the complete fitting parameters shown in Table S1). The red Gaussian centers at 2053 cm-1 with FWHM of 40 cm-1 while the broad blue Gaussian centers at 2101 cm-1 with a FWHM of 80 cm-1. We assigned the red peak as the νas. Compared to FTIR spectra in Figure 2A, the νas from the droplet shows a similar blueshift to the bulk FTIR solution at 5.4 M NaCl but is slightly broader (40 vs. 33 cm-1). Ideally, a calibration between Δz and absorption cross section is desired. However, establishing such a connection in an absolute sense is difficult because of differences in the droplet’s mass-to-charge ratio from experiment to experiment. Nevertheless, this blue shift indicates that the droplet has high ionic content, while the modest increase in spectral width hints at a slight supersaturation, similar to bulk behavior.

The peak at 2101 cm-1 is likely the combination band of the water bend fundamental and the lower frequency libration motions,44, 45, 46 sometimes known as the “association band” (hereafter denoted νcombH2O). Previous bulk FTIR study if this νcombH2O band has shown that it redshifts from ~2135 cm-1 to ~2100 cm-1 as [Na+] increases from 1 to 5 M.46 The reason for this redshift is due to ionic interactions of the ion with the water molecules, which weakens the hydrogen bonding network. The more rigid hydrogen bonding network will thus have a lower average librational frequency, which causes redshift. This feature is often ignored in many studies because of its weak signal. In fact, it can be observed in the bulk FTIR as well if we reduce the NaN3 concentration, which we discuss further in Section S2, Figure S6-S7. The fact that SiDDIRAS can detect the νcombH2O band points to its sensitivity and robustness. Additionally, the νcombH2O redshifting to 2101 cm-1 is further evidence that the droplet has high ionic content.

The deliquescence point for NaCl is at 75%,47, 48 meaning that the droplets will be supersaturated only if the RH in the chamber is below this RH value. To determine the true RH in the chamber and the salt concentrations in the droplets, we applied a dual-droplet hygroscopicity measurement originally developed in our lab.47 First, we measure the RI and radius of the droplet as a function of RH, and the bulk water activity of a high concentration solution. From these measurements, we obtain the radial growth factor (rGF), which can then be used to deduce the salt concentration in the droplets. More details are provided in Section S3 of the SI. To decompose the composition of the multicomponent salt droplet, rGF curves for pure salt and mixed salt droplets were obtained (Figure S8A). Then, using the Zdanovskii-Stokes-Robinson (ZSR) relationship,49 which relates the water content of a mixture to that of the individual solutes at the same water activity, one can estimate the mixed salt content. We assumed an equal mass ratio of the solutes NaCl and NaN3 in the mixture and the predicted rGF is compared against the measured data in Figures S8A, showing excellent agreement. Based on the measured rGF and the approximations described in Section S3, we also estimate the molarity of the solutes in the droplet as a function of RH, shown in Figure S8B. From these measurements, we also obtain the refractive index (RI) as a function of RH, reported for 589 nm in Figure S8C.

To obtain an improved estimate of the in-situ RH during SiDDIRA measurements, we compare the measured RI of the droplet to the dependence reported in Figure S8C. The size and RI of these droplets were determined from the Mie resonance spectra, shown in Figure 2A along with the best-fit simulation. From these data, the RI of the droplet was measured to be 1.384, which corresponds to an RH of ~72% according to Figure S8C. At this RH, [Na+] = 6.1 M and [N3−] = 2.9 M in the droplet, consistent with our earlier interpretation that the SiDDIRA spectrum of NaCl and NaN3, showing that the 5 cm-1 blueshift and peak broadening, is due to supersaturation of salt and formation of IPs.

Other additional factors may contribute to the observed 5 cm-1 blueshift, such as electrostatic field effects from the charged surface or microheterogenous solvation environments at the air-water interface. The electrostatic field effects from the droplet surface are expected to be negligible due to the high salt content of the droplet, resulting in short screening lengths. Furthermore, the majority of the azide ions would have to be aligned with the electric field in order to induce such a large blueshift. Additionally, the charge density on the droplet is quite small. For example, our previous studies of organic azide at the air-water interface showed that a charge density of ~1 e–/nm2 induces a shift in the νasN3 by only 1-2 cm-1.50 Previous measurements of microdroplets trapped in EDBs showed that there are 105 to 106 e– in the droplet.11 Assuming that all of the charge density is at the surface, for a r = 10 µm droplet, the surface charge density will be 10-2 to 10-3 e–/nm2. This surface charge density is several orders of magnitude smaller than the previous study at the air-water interface, and is likely too small to induce measurable shifts. Therefore, we expect the contribution from electrostatic field effects to the blue shift to be negligible. Regarding microheterogenous solvation environments at the air-water interface, we cannot comment on such effect with our current data because this method is not necessarily surface-sensitive. Future studies may use a different vibrational probe that is surface active to deconvolute surface from bulk effects.

To verify that the IR laser does not cause significant evaporation, which could influence [Na+], hence the frequency of the νasN3, we also obtain the Mie spectrum of the NaCl and NaN3 droplet under constant IR illumination at 2053 cm-1, shown in Figure 2B. This measurement shows a ~200 nm change in radius and a small increase in RI. This change in radius corresponds to a ~7.6% reduction in volume at peak absorption, leading to an increase in the concentration from 6.1 M to ~6.5 M. Given the limited sensitivity of the spectrum to [Na+], we do not expect this change to be apparent in our data. Figure 2 indicates that IR absorption by the particle can also be deduced from the change in Mie resonance, as first demonstrated by Arnold et al.28, 29 While this detection method can be more sensitive to small changes in droplet’s response, it is also more laborious, as accurately simulating the Mie spectra require additional post-acquisition analyses. Furthermore, the displacement can cause misalignment of the broadband source for detection of Mie resonances. Hence, we do not see the advantage of using this detection method over monitoring the displacement, which allows the spectral data to be acquired much faster without compromising accuracy.

To get further molecular-level insights on the Na+ −N3− IPs in the droplet, we turn to quantum computational modeling using the Q-Chem program suite at the ωB97M-V/def2-TZVPD level of theory. To verify the robustness of the method, we benchmarked the experimental νasN3 bands obtained in this study against calculated frequencies shown in Table S4. Computational details are discussed further in Section S4. To understand the effect of the hydration shell on the geometry and charge distribution of the IPs, we compared the computational results with and without water, shown in Figure 3.

Figures 3A-B show Na+ −N3− with and without water, respectively. A stark contrast between the gas-phase and hydrated geometries is the Na+ −N1 −N2 bond angle. Without water, this bond angle is 172.9°, which is almost linear, but then bends to 135.2° when hydrated by only six water molecules. Interestingly, the reorientation of the N3− anion geometry as water is added agrees with previous experimental and theoretical gas-phase study by Jungwirth and coworkers51 on microhydrated clusters of N3− ∙(H2O)n. In that study, the authors show that hydrogen bonding from water molecules can stabilize the N3− anion. Their MD simulation also shows that the preferred geometry for the N3− anion is parallel to the water clusters at the vacuum-water interface. In this case, specific hydrogen bond interactions likely cause azide reorientation.

Additionally, charge distribution on Na+ −N3− complex changes significantly upon hydration. We performed natural bond orbital (NBO) analysis for both cases, reported in Tables S6 and S7, respectively. In the bare complex (Table S6), charge distribution is localized between the Na+ −N1 bond with the Na+ carrying most of the positive charge (+0.97) and N1 carrying most of the negative charge (-0.87). The central nitrogen, N2, has some negative character (-0.31) and the terminal nitrogen is slightly positive N3 (0.22). When water molecules are present (Table S7), charges on N1 and N2 are distributed slightly more evenly (-0.62 and -0.51, respectively). This charge redistribution upon hydration can be thought of more clearly in terms of polarizability. Without water, the Na+ cation is much more polarizing to the azide anion and causes electron density to build on N1. Upon hydration, the solvent compensates for this effect and redistributes electron density throughout the complex. In our NBO analysis, we find that charge redistribution, and not Lewis-type interaction, causes the frequency shift.

We have described a new and simple method, called SiDDIRAS, to obtain an IR spectrum of a single micron-size, charged microdroplet. The IR spectrum of an ~8 µm aqueous droplet containing NaCl and NaN3 show significant blue shift of the νasN3. Using this shift in frequency and hygroscopicity measurements, we provided evidence to attribute the spectral shift and broadening from the azide asymmetric stretch to supersaturation of salt and formation of ion pairs in the ~8 µm aqueous droplet. The concentrations of Na+ and N3− were determined to be 6.1 M and 2.9 M, respectively.

Many recent reports have attributed novel behaviors of microdroplets to surface effects such as strong electric fields52 and microsolvation environment at the air-water interface.53 SiDDIRAS is a promising method to investigate these effects. The spectral resolution of this method is limited by the laser, which is 0.1 cm-1 in this case. The signal-to-noise ratio is limited by the detection of the mechanical displacement of the droplet. We note that the spectrum reported in this study is from a single scan at reduced laser power. To detect weaker vibrational modes, one can average multiple scans at higher laser power. Hence, SiDDIRAS is a promising and sensitive technique to uncover some of the unanswered questions in microdroplet chemistry. For example, electrostatic field effects can be probed by changing the droplet’s charge (both magnitude and polarity) and the droplet’s size. The latter requires better control of RH without introducing mechanical displacement of the droplet through humidified gas. Understanding the droplet’s microhetergenous solvation at the surface is also possible by using a surface-active vibrational probe,50 which will require calibrating these probes first on the bulk surface. While this report serves to introduce SiDDIRAS, we foresee performing more quantitative studies as the method matures to clarify some of the unanswered questions in droplet chemistry.

The fact that action spectroscopy is possible for microdroplets by mapping its displacement opens the door for methods and studies that were not possible. For example, droplets that contain UV-Vis chromophore species can be studied using UV-Vis action spectroscopy.54 We suspect that, because UV-Vis chromophores generally have higher absorption cross sections, hence higher energy per absorbed photon (provided there is no fluorescence), the displacement caused by light-induced evaporation might be even more sensitive to UV-Vis absorption. The rich literature of IR spectroscopy on biological systems can now be applied to study in single, substrate-free, levitated microdroplets. Experiments are ongoing to highlight the rich experimental landscape as SiDDIRA continues to mature and evolve.

Methods
The experimental method is discussed in more detail in Section S1 but will be briefly described here. The instrument diagram is shown in Figure 4A with the LQ-EDB inside an environmentally controlled chamber. On either side of the LQ-EDB are the optical setups. On the left, is a broadband LED (620-680 nm, Thorlabs M660L4) focused by a 50 mm biconvex lens which enters the LQ-EDB at an angle through a quartz window. The backscattered light, with the characteristic WGMs, is collected for sizing the droplets (See Figure S1 in Section S1.2 for details on optical setup). On the right is a QCL (MIRcat-QT, Daylight Solutions, 1950-2300 cm-1), which enters the chamber through a CaF2 window. The QCL operates in the continuous wave (CW) mode, scanning in increments of 1 cm-1 with a mechanical shutter attached to a solenoid at its entrance. The solenoid is controlled by a delay generator (SRS DG535), which alternates between allowing the laser to irradiate the droplet for 2 seconds, followed by blocking it for 2 seconds. The cycle repeats for each frequency until the frequency scan is complete. The droplet is illuminated by a 532 nm laser from the bottom of the LQ-EDB. Note that the scattered light from this 532 nm laser is used to illuminate the droplet only and is not collected for optical analysis. A color CMOS camera (Thorlabs CS165CU) mounted in the orthogonal position to the optical paths (not depicted) is used to record the droplet’s position over time as the IR laser scans the frequency range. The recorded centroid position of the droplet is then extracted and processed with a MATLAB code (see Figure S2 in Section 1.3 for details).

Figures 4B-D describe the sequence of events that take place when the IR light interacts with the levitated microdroplet in the LQ-EDB. Initially, when the IR laser is off, the droplet z position is balanced by gravitational pull and the electrical force from the DC electrode (Figure 4B):

mg = qVDC C / z, (1)

where m is the mass of the droplet, g is the gravitational acceleration, q is the droplet’s charge, VDC is the DC voltage on the electrode, z is the distance from the electrode to the droplet, and C is a constant that relates the applied potential on the balancing electrode to the potential felt by the droplet at the equilibrium position. Note that to control the relative humidity (RH), humidified gas is continuously introduced to the chamber, which would add an additional drag force term in Equation 1. However, during IR acquisition, we turn the gas off to prevent this drag force from introducing noise, hence this term is excluded. Additionally, Equation 1 does not account for the inhomogeneity of the electric field as the droplet’s z position increases and is only approximately valid when the droplet is near the balancing electrode. To establish the z distance at which Equation 1 is valid, we performed a measurement of the droplet’s displacement (Δz) as a function of applied voltage (ΔV) on the balancing electrode, shown in Figure S3. Our measurement shows that the droplet’s displacement is linear with the applied voltage up to ~1.5 mm. Because the droplet’s displacement in this study is within this range (vide infra), Equation 1 remains valid.

When the IR laser irradiates the droplet and the frequency is in resonance with IR active absorption features of molecules in the droplet (Figure 4C), the light will be absorbed and converted into heat. This heat will raise the temperature of the droplet, leading to a shift in the water vapor equilibrium and causing water to evaporate from the droplet. The droplet will lose mass and displace upwards:

(m−∆m)g = qVDC C / (z+∆z), (2)

When the laser is turned off, the surrounding moisture condenses back onto the droplet, restoring the droplet to its original position (Figure 4D).

While the radiation pressure and electrostatic coupling between the laser field and the charged droplet can potentially contribute to Δz, we rule out these interactions in our work for the following reasons: 1) Experimental evidence from this work shows that the displacement is in the wrong direction for Δz to be due to the scattering force (which is in the same direction of laser propagation) and the beam is not focused enough to cause a strong gradient force (see Section S1.4, Figure S5). 2) Electrostatic coupling between the laser field and the charged droplet is unlikely because the displacement is composition and excitation frequency dependent. Another concern is whether the droplet remains homogenous during the evaporation/condensation process. Previous studies47, 55 have shown that NaCl microdroplets can remain aqueous until 40% RH before crystallizing, hence we do not foresee the existence of salt crashing out of the droplet, causing inhomogeneity in the solvation environment. Additionally, salt cannot evaporate from the droplet, hence as moisture condenses back onto the droplet after IR irradiation, we expect the salt composition of the droplet to remain the same.

An RH probe (Elitech) was used to ensure the RH reached stable condition but, as mentioned earlier, humidified gas is turned off to eliminate drag force, which interfere with the accuracy of the RH readout. When the humidified gas is turned off, the RH and temperature of the chamber varied by 1-2% and ~0.1°C throughout the IR scan according to the probe. The manufacturer reported the precision of this particular probe to be 3% RH and 0.3°C.To eliminate uncertainty in RH measurements during collection of SiDARRA spectra, we used an in situ dual-droplet technique to infer the RH inside the chamber (vide infra, discussed in more details in Section S3), showing the internal RH to be ~72%. This evaporation/condensation process occurs on the millisecond timescale.56, 57, 58 At each frequency, the droplet interacts with the IR light on the seconds timescale, thus there is enough time for the evaporation/condensation processes to reach steady state. By mapping the displacement of the droplet (∆z) as a function of IR frequency, an IR spectrum of a single charged droplet isolated at ambient conditions in the LQ-EDB can be obtained, illustrated in Figure 4E.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Single Droplet Displacement Infrared Action Spectroscopy\nThe authors present a new method of measuring the chemical environment inside individual water droplets. The procedure\nallows access to a previously poorly understood but important environment, and combines standard tools in a novel and\nclever way. The research is understandable and approachable.\nThe paper is clearly written and convincingly argues that the authors did what they say. The writing is clear, albeit a little\nverbose.\nA few aspects of the technique could be explained more clearly. One example is the imaging system. The reader is\nchallenged to understand exactly what light is collected where. In particular, figure 1A contains no imaging optics;\npresumably both scattered 532 nm light and scattered LED light (?) are collected, but this is not clear. Obviously this\ndescription can be corrected.\nMost readers of the paper (including myself!) are not experts in droplet chemistry. It would be helpful if a summary of the\ndroplet were included. How many N3- ions are in this small, but macroscopic, droplet?\nThe technique is innovative, convincingly demonstrated, and potentially transformative in the somewhat niche field of droplet\nchemistry. In addition, the technique appears to be tractable enough that it will be reproducible by other groups.\nThe authors make a considerable effort to propose extensions of the technique. This is the crux of acceptance to Nature\nCommunications. My own judgement, from outside the field, is that there is. I am far from an expert in this field, but this is\nrelevant to electrospray ionization (certainly a widely used tool), as well as atmospheric chemistry, where water droplets are\nof course very relevant. There is a small but non-zero chance to become a widely used technique, of which this would be the\nseminal paper.\nThe paper is unusual, and innovative. It is the type of paper I believe broadens the scientific conversation, and leads to new\nscientific ideas. As such, I support its publication in nature communications.", "reviewer_2": "Comments:\nThis paper presents a novel method for observing the IR spectrum of a single droplet levitated in an electrodynamic trap.\nThe IR spectrum is measured by the displacement of the trapped droplet due to evaporation and mass loss induced by the\nabsorption of IR light by the droplet. This paper is well-written and provides a novel methodology to measure the IR\nspectrum of a single droplet. Therefore, this paper is considered worthy of publication. However, the following points are\nrequired to be considered before publication.\n1. Some papers have reported spectral measurements of a single particle in the electrodynamic trap. [1,2] The author should\nproperly cite these papers and clearly state the differences from the previous reports.\n[1] S. Arnold, M. Neuman, A. B. Pluchino, “Molecular spectroscopy of a single aerosol particle.” Opt. Lett. 1984, 9, 4–6.\n[2] S. Arnold, E. K. Murphy, G. Sageev, “Aerosol particle molecular spectroscopy” Appl. Opt. 1985, 24, 1048.\n2. Photophoretic force on a single particle in the electrodynamic trap has been measured by the displacement of the particle\nin the trap. [3,4] The author should properly cite these papers and state that they applied similar phenomena to spectral\nmeasurements.\n[3] H.-B. Lin, “Infrared absorption spectroscopy of single particles using photophoresis” Opt. Lett. 1985, 10, 68.\n[4] O. Krauß, G. Wurm, “Radiation pressure forces on individual micron-size dust particles: A new experimental approach” J.\nQuant. Spectrosc. Radiat. Transf. 2004, 89, 179–189.\n3. Equation (1) assumes that the electric field is identical from the bottom to the top of the linear trap. However, the electric\nfield is not homogeneous with respect to z due to the disturbance caused by the linear electrodes. Equation (1) is only\napproximately valid in the vicinity of the trapped particle.\n4. Figure S3 shows that the displacement of the particle reaches 0.13 mm. The authors should specify the beam size of the\nIR laser, because the irradiation efficiency may decrease due to the particle displacement.\n5. Figure 3 indicates that the IR absorption by the particle can also be detected from the spectral shifts of the Mie spectra.\nDiscussion on the applicability of the spectral shift will help the understanding of the advantage of the present method.\n6. Chemical formulas (N3-, Na+, etc) should be written in upright type.", "reviewer_3": "In this study, Prof. Dawalty and Coworkers report a novel approach, termed Single Droplet Displacement Infrared Action Spectroscopy (SiDDIRAS), which enables acquisition of IR spectra from single, levitated microdroplets confined within a linear quadrupole electrodynamic balance (LQ-EDB). By tracking the droplet’s displacement as a function of IR frequency, they convert mass-loss-driven motion into an “action spectrum.” The study represents an exciting methodological advance, addressing a long-standing challenge in probing the molecular environments of individual microdroplet systems central to both analytical and atmospheric chemistry. The work is technically sophisticated and conceptually impactful; however, several key aspects require clarification and control to ensure robustness of these techniques. On this, I have several queries and suggestions, which are outlined below.\n1. The central claim of the work, that IR absorption induces droplet motion through resonant heating and evaporation,\nrequires more stringent control. Could radiation pressure or electrostatic coupling between the laser field and the charged\ndroplet also contribute to Δz? Quantitative analysis or modeling of the force balance would strengthen confidence in the\ninterpretation. The author should address this.\n2. Is there a calibration curve linking Δz to a known absorption cross-section or optical density, perhaps using droplets of\nother composition (e.g., D2O)? Without such calibration, the technique risks being limited to qualitative shifts rather than\nquantitative intensity analysis. Authors are suggested to clarify this.\n3. What are the error margins in droplet radius, RI, and the ZSR-based solute estimation?\n4. Please comment on whether the droplets were homogeneous throughout the optical measurement period.\n5. The observed 5 cm⁻¹ blueshift and significant broadening of ν(N₃⁻) are attributed to Na⁺–N₃⁻ ion-pairing and\nsupersaturation, but additional factors may contribute. Could electrostatic field effects from the charged droplet surface or\nmicroheterogeneous solvation play roles? Please comment.\n6. The authors may consider comparing droplets of different charge states or sizes to distinguish between electrostatic vs.\nconcentration-driven shifts.\n7. The authors should specify the minimum detectable frequency shift (sensitivity) or absorbance achievable by this method.\nWhat is the signal-to-noise ratio for small absorptions, and could SiDDIRAS detect weakly absorbing vibrational modes? A\nquantitative limit of detection would make the method more broadly comparable to Raman or fluorescence action\nspectroscopy.\n8. Clarify the temperature and RH control precision during IR scans, given the high sensitivity of evaporation to these\nparameters.\n9. A brief paragraph comparing SiDDIRAS to existing optical-tweezers-IR or substrate-free aerosol IR techniques would\ncontextualize its novelty and advantages (maybe as an SI note).\n10. The authors mention the potential for UV–Vis action spectroscopy. It would be valuable to discuss practical limitations\n(e.g., optical trapping stability, droplet heating) and expected signal magnitude.\n11. “More recently, using ESI and pneumatic sprays, studies spearheaded by the Zare and\nCooks groups, have reported enhanced chemical reactivity in microdroplets” – authors should also acknowledge and\nappropriately cite other research groups that have been actively contributing to the advancement of microdroplet chemistry\nsuch as Prof. Shibdas Banerjee (IISER Tirupati), Prof. Xinxing Zhang (Nankai University), Prof. Agustin J. Colussi (Caltech)."}, "author_response": "Author Response: We thank the reviewer for the kind and positive comments on our\nwork. We have addressed the Reviewer’s comments and suggestion below.\nA few aspects of the technique could be explained more clearly. One example is the\nimaging system. The reader is challenged to understand exactly what light is collected\nwhere. In particular, figure 1A contains no imaging optics; presumably both scattered\n532 nm light and scattered LED light (?) are collected, but this is not clear. Obviously\nthis description can be corrected.\nAuthor Response: We thank the reviewer for pointing out this potentially confusing\nexperimental diagram. The green 532 nm light is only for illumination purposes so that we\ncan record the position of the droplet with a camera. The scattered green light is not\ncollected by the spectrometer. To make this point clearer, we have included the following\nsentences:\nNote that the scattered light from this 532 nm laser is used to illuminate the droplet only and is not\ncollected for optical analysis. A color CMOS camera (Thorlabs CS165CU) mounted in the\northogonal position to the optical paths (not depicted) is used to record the droplet’s position over\ntime as the IR laser scans the frequency range. The recorded centroid position of the droplet is then\nextracted and processed with a MATLAB code (see Figure S2 in Section 1.3 for details).\nMost readers of the paper (including myself!) are not experts in droplet chemistry. It\nwould be helpful if a summary of the droplet were included. How many N3- ions are in\nthis small, but macroscopic, droplet?\nAuthor Response: We thank the reviewer for the suggestion. We have included the\nfollowing paragraph to summarize our findings:\nThe IR spectrum of an ~8 µm aqueous droplet containing NaCl and NaN show significant blue\n3\nshift of the 𝜈𝑎𝑠. Using this shift in frequency and hygroscopicity measurements, we provided\n𝑁3\n1\nevidence to attribute the spectral shift and broadening from the azide asymmetric stretch to\nsupersaturation of salt and formation of contact ion pairs in the ~8 µm aqueous droplet.\nThe concentrations of Na+ and N− were determined to be 6.1 M and 2.9 M, respectively.\nin the somewhat niche field of droplet chemistry. In addition, the technique appears to\nbe tractable enough that it will be reproducible by other groups.\nThe authors make a considerable effort to propose extensions of the technique. This is\nthe crux of acceptance to Nature Communications. My own judgement, from outside the\nfield, is that there is. I am far from an expert in this field, but this is relevant to\nelectrospray ionization (certainly a widely used tool), as well as atmospheric chemistry,\nwhere water droplets are of course very relevant. There is a small but non-zero chance\nto become a widely used technique, of which this would be the seminal paper.\nThe paper is unusual, and innovative. It is the type of paper I believe broadens the\nscientific conversation, and leads to new scientific ideas. As such, I support its\npublication in nature communications.\nAuthor Response: We thank the reviewer for the positive comments on our work and\nfor recommending the manuscript for publication.\nAuthor Response: We thank the reviewer for the positive comments on our work and\nfor recommending the manuscript for publication.\n1. Some papers have reported spectral measurements of a single particle in the\nelectrodynamic trap. [1,2] The author should properly cite these papers and clearly state\nthe differences from the previous reports.\n[1] S. Arnold, M. Neuman, A. B. Pluchino, “Molecular spectroscopy of a single aerosol\nparticle.” Opt. Lett. 1984, 9, 4–6.\n[2] S. Arnold, E. K. Murphy, G. Sageev, “Aerosol particle molecular spectroscopy” Appl.\n2. Photophoretic force on a single particle in the electrodynamic trap has been\nmeasured by the displacement of the particle in the trap. [3,4] The author should\nproperly cite these papers and state that they applied similar phenomena to spectral\nmeasurements.\n[3] H.-B. Lin, “Infrared absorption spectroscopy of single particles using photophoresis”\nOpt. Lett. 1985, 10, 68.\n[4] O. Krauß, G. Wurm, “Radiation pressure forces on individual micron-size dust\nparticles: A new experimental approach” J. Quant. Spectrosc. Radiat. Transf. 2004, 89,\n179–189.\nAuthor Response: We thank the reviewer for directing us toward these important and\nrelevant papers! Indeed, the reviewer is correct that they also use a form of action\nspectroscopy, albeit detecting the action using other methods. To clarify the differences\nbetween our method and the methods described by Arnold et al and Lin, we have cited\nthese works and discussed them in the following paragraphs:\nAction IR spectroscopy on aerosol particles trapped in an EDB were first introduced by several\ngroups in the 1980s. Arnold et al.28,29 described a technique called Structure Resonance Molecular\nSpectroscopy (SRMS), where the authors induced an ammonium sulfate droplet’s evaporation at\na certain IR frequency using an SiC Glo-Bar. The fractional change in a droplet’s scattering pattern\nupon resonant IR-induced evaporation is then fitted with Mie theory and mapped as a function of\nIR frequency to produce an IR spectrum. Lin30 followed with a method for measuring an action IR\nspectrum of solid particles, where the photophoretic force acting on a trapped solid ammonium\nsulfate particle upon absorbing IR photons from a CO laser displaced the particle from its original\n2\nposition. Thus, by measuring the fractional change in balancing voltage required to bring the\nparticle back to its original position (ΔV/V) and mapping it to the IR frequency, an action spectrum\nwas obtained.\nBased on these seminal works, we described below an action IR technique for aqueous\nmicrodroplets in a LQ-EDB that does not require complex Mie fittings, as shown in Arnold et\nal.’s works.28,29 In a way, our detection method is more similar to that described by Lin30 in that\nwe are using the droplet’s displacement from its original position in our measurement. However,\nwe want to stress that we are not measuring the photophoretic force acting on the droplet nor the\nfractional voltage to bring the droplet back to its original position. Instead, we are mapping the\nmagnitude of the droplet’s displacement upon IR-induced evaporation as a function or IR\nfrequency from a tunable quantum cascade laser (QCL). We have coined this technique Single\nDroplet Displacement Infrared Action Spectroscopy (SiDDIRAS). SiDDIRAS does not require\ncomplex optics or post-acquisition analyses. This technique is easily reproducible, highly\naccurate, and, depending on scan speed and needs, can provide up to sub-wavenumber (≤ 1 cm-1)\nresolution.\nseem to be of relevance here, since the authors in that paper were interested in\nmeasuring the radiation pressure of micron-size particles in a force-free environment, so\nwe have opted to not include this reference.\n3. Equation (1) assumes that the electric field is identical from the bottom to the top of\nthe linear trap. However, the electric field is not homogeneous with respect to z due to\nthe disturbance caused by the linear electrodes. Equation (1) is only approximately valid\nin the vicinity of the trapped particle.\nAuthor Response: The reviewer is correct that the electric field is not homogenous wrt\nto z as z increases. The reviewer also correctly pointed out that Eq. 1 is approximately\nvalid in the vicinity of the trapped particle. Indeed, the response of the droplet is\napproximately linear in the displacement range that we reported here. To clarify this\npoint, we have added Figure S3 and discussed its results in the Supporting Information:\nAs the droplet displaces upwards, however,\n1.8\nthe electric field from the balancing electrode\n1.6 R2=0.994\nmight no longer be homogenous, potentially\n1.4\ncausing non-linear response to the 1.2\ndisplacement. To establish the displacement )m1.0\nm\nrange at which the droplet’s response to the (\nz\n0.8\nΔ\nelectric field from the balancing electrode is 0.6\nlinear, we performed a control experiment 0.4\nmeasuring the droplet displacement (Δz) as a 0.2\n0.0\nfunction of change in applied voltage on the\n-0.2\nelectrode (ΔV). The result, displayed in\n0 20 40 60 80\nFigure S3, shows that the droplet’s response ΔV (V)\nto the applied voltage is linear up to ~1.5 mm.\nFigure S3. NaCl droplet Δz displacement as a\nFigure S2 shows that the droplet’s max\nfunction of change in balancing electrode applied\ndisplacement at ~2052 cm-1 is much smaller\nvoltage (ΔV).\n(~0.13 mm), hence within the range of linear\nresponse. This control experiment means that Equation 1 in the main text is valid in our instrument\nat least up to Δz = 1.5 mm.\nWe have also included the following sentences in the manuscript to point out the linear\nresponse of the droplet’s displacement (Δz) as a function of DC voltage applied on the\nlevitating electrode:\nAdditionally, Equation 1 does not account for the inhomogeneity of the electric field as the\ndroplet’s z position increases and is only approximately valid when the droplet is near the\nbalancing electrode. To establish the z distance at which Equation 1 is valid, we performed a\n4\nmeasurement of the droplet’s displacement (Δz) as a function of applied voltage (ΔV) on the\nbalancing electrode, shown in Figure S3. Our measurement shows that the droplet’s displacement\nis linear with the applied voltage up to ~1.5 mm. Because the droplet’s displacement in this study\nis within this range (vide infra), Equation 1 remains valid.\n4. Figure S3 shows that the displacement of the particle reaches 0.13 mm. The authors\nshould specify the beam size of the IR laser, because the irradiation efficiency may\ndecrease due to the particle displacement.\nAuthor Response: We thank the reviewer for this suggestion. We have added Section\nS1.4 in the supporting information detailing the measurement for the IR beam size. The\nresults from this measurement are summarized in Table S2 and Figure S5:\n1.4.IR laser beam size\nTo ensure that the droplet is\nilluminated by the IR laser while it is\nFit\ndisplacing upwards, we measured the\n250\nlaser spot size. The beam profile of\n)\nW\nthe IR laser was measured using the m200\nknife-edge method.4 The setup\n(\nr e\nw150\nconsists of an IR beam followed by a o\nP\nR\nCaF 2 focusing lens, a translation I100\nstage with a razor blade and a power\n50\nmeter (Coherent). The razor blade\nwas attached to the translation stage\n0\nalong the x-axis and placed at the 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2\nfocal point of a f ~8.4 cm (3.3”) CaF Distance (mm)\n2\nfocusing lens. This distance Figure S5. IR power measured at 2053 cm-1 as a function\ncorresponds to the distance between of distance from the peak power using a knife-edge method.\nthe focusing lens and the droplet\nposition in the EDB chamber. A micrometer attached to the translation stage was used to increment\nthe position of the blade by 0.05 mm across the laser spot beginning at a position of maximum IR\npower. The laser power was measured using a thermopile sensor at each position of the blade. The\nIR power profile as a function of the distance moved is shown in Figure S5. Assuming a Gaussian\nbeam profile for the laser intensity results in the following function for power versus distance:\n𝑥 −𝑥\n0\n𝐼 = 𝐴(1+erf( ))+𝑦 𝑆2\n0\n√2\n𝐹𝑊𝐻𝑀\n2.355\n5\nwhere 𝐹𝑊𝐻𝑀 is the full width half maximum, 𝑥 is the point of the peak intensity of the beam,\n0\n𝑦 is the total power of the beam and 𝐴 is a constant. The 𝐹𝑊𝐻𝑀 of the beam from the fit is\n0\n~0.724 mm.\nTable S2: Summary of results obtained from fitting the power profile of the IR beam as per\nEquation S2 using Matlab R2024b.\nCoefficients Value 95% Confidence Bounds\n𝑭𝑾𝑯𝑴 0.724 (0.714,0.734)\n𝑨 -150.69 (-151.44,-149.94)\n𝒙 0.899 (0.895,0.901)\n𝟎\n𝒚 305.49 (304.41,306.58)\nAdditionally, we discussed this results in the following sentence in the manuscript:\nAs the droplet displaced upwards, the irradiation efficiency may decrease if the droplet is no\nlonger in the IR laser’s path. The IR beam size in our setup is measured to be ~0.724 mm (details\nin Section S1.4, Table S2 and Figure S5), which is much bigger than the droplet’s displacement\n(0.13 mm). Hence, we believe the irradiation efficiency change to be negligible.\n5. Figure 3 indicates that the IR absorption by the particle can also be detected from the\nspectral shifts of the Mie spectra. Discussion on the applicability of the spectral shift will\nhelp the understanding of the advantage of the present method.\nAuthor Response: The reviewer is correct that the action spectrum can also be\nobtained from Mie spectral shifts, as shown by Arnold et al. While this detection method\ncan potentially be more sensitive to changes, the analysis is a bit more complex than\njust detecting the displacement. We have included the following to clarify this point:\nFigure 3 indicates that IR absorption by the particle can also be deduced from the change in Mie\nresonance, as first demonstrated by Arnold et al.28,29 While this detection method can be more\nsensitive to small changes in droplet’s response, it is also more laborious, as accurately\nsimulating the Mie spectra require additional post-acquisition analyses. Furthermore, the\ndisplacement can cause misalignment of the broadband source for detection of Mie resonances.\nHence, we do not see the advantage of using this detection method over monitoring the\ndisplacement, which allows the spectral data to be acquired much faster without compromising\naccuracy.\n6. Chemical formulas (N3-, Na+, etc) should be written in upright type.\nAuthor Response: We have changed the font as suggested.\nAuthor Response: We thank the reviewer for the kind and positive comments on our\nwork. We have addressed the reviewer’s comments and suggestions below.\n1. The central claim of the work, that IR absorption induces droplet motion through\nresonant heating and evaporation, requires more stringent control. Could radiation\npressure or electrostatic coupling between the laser field and the charged droplet also\ncontribute to Δz? Quantitative analysis or modeling of the force balance would\nstrengthen confidence in the interpretation. The author should address this.\nAuthor Response: We thank the reviewer for pointing out additional possibilities to\nconsider. Experimental evidence from this work shows that the displacement is in the\nwrong direction for it to be due to the scattering force (which is in the same direction of\nlaser propagation) and the beam is not focused enough to cause a strong gradient force\n(see Figure S5). We can also rule out electrostatic coupling between the laser field and\nthe charged droplet because the displacement is composition and excitation frequency\ndependent. To make this point clearer, we have included the following in the manuscript:\nWhile the radiation pressure and electrostatic coupling between the laser field and the charged\ndroplet can potentially contribute to Δz, we rule out these interactions in our work for the\nfollowing reasons: 1) Experimental evidence from this work shows that the displacement is in\nthe wrong direction for Δz to be due to the scattering force (which is in the same direction of\nlaser propagation) and the beam is not focused enough to cause a strong gradient force ((see\nSection S1.4, Figure S5). 2) Electrostatic coupling between the laser field and the charged\ndroplet is unlikely because the displacement is composition and excitation frequency dependent.\n2. Is there a calibration curve linking Δz to a known absorption cross-section or optical\ndensity, perhaps using droplets of other composition (e.g., D2O)? Without such\ncalibration, the technique risks being limited to qualitative shifts rather than quantitative\nintensity analysis. Authors are suggested to clarify this.\nAuthor Response: We thank the reviewer for raising this concern. One strategy to\nmeasure absolute absorption cross section could be to measure the ΔV necessary to\n7\nkeep the droplet in the same position upon irradiation, which will allow the change in\nrelative mass (Δm) to be measured. Once the Δm values are established, the change in\ntemperature can be estimated because this corresponds to a specific shift in water\nvapor pressure (assuming accurate hygroscopic growth information was measured for a\nspecific sample). Thus, the heat loss from the droplet is in equilibrium with the heat\nbeing absorbed. Then, with a model of thermal transport and good knowledge of the\nillumination intensity, the absolute absorption cross section can be calculated. However,\nthis is a long analysis which requires a lot of assumptions, so it isn’t feasible for this\nwork. Additionally, because droplets generated in our method vary physically (charge\nand mass) from droplet to droplet, hence the force of gravity acting on the droplet will\nvary. Therefore, even if there is a calibration curve linking Δz to a known absorption\ncross-section or optical density, one cannot extrapolate this information from experiment\nto experiment. We have added the following to the manuscript:\nIdeally, a calibration between Δz and absorption cross section is desired. However, establishing\nsuch a connection in an absolute sense is difficult because of differences in the droplet’s mass-to-\ncharge ratio from experiment to experiment.\n3. What are the error margins in droplet radius, RI, and the ZSR-based solute\nestimation?\nAuthor Response: We thank the reviewer for pointing out the lack of discussion on the\nuncertainty of our measurements. We have added the following paragraphs to Section\nS3 in the SI section on the discussion of uncertainty on radius, RI, and ZSR analyses:\nThe uncertainty in the radius is typically less than ±5nm (around 0.1%), and this arises from the precision\nto which the peak positions are measured. The uncertainty in the RI is less than ±0.005, with measurements\nusually closer to ±0.001.\nThe uncertainty in that size propagates to the uncertainty in the solute concentration estimates, via the radial\ngrowth factor (rGF), although the exact uncertainty depends on the specifics of the measurement.\nIn general, the uncertainty in the rGF is the error additive sum of the uncertainty in the radius and the\nuncertainty in the dry size. The uncertainty in the latter comes from the accuracy of the bulk solution\nconcentration measurement, which we estimate to be better than ±0.1% based on mass balance precision\nand volumetric flask precision, the accuracy of the bulk water activity measurement, which is ~0.3%, and\nthe accuracy of the water activity measured in the LQ-EDB using the dual-droplet approach, on the order\nof ~0.1%. In our method, the droplet radius at a water activity equal to the bulk water activity is used to\ndetermine the dry size. The uncertainty in the water activity meter dominates the uncertainty in this process,\nleading to approximately 0.5% uncertainty in the droplet size at the bulk activity measurement water\nactivity. Thus, the dry size has an error of approximately the same size. The error in the rGF is thus 0.6%\nwhich corresponds to an error in the molarity of 1.8%.\nThe ZSR estimate was shown to match closely with experiments; however, these values were not used in\nthe analysis and we report them merely to demonstrate that the solution is well-behaved and follows\n8\nstandard mixing rules. The measurement of the individual and mixed solutions were used directly to\ndetermine molarity, with the uncertainty as reported above.\n4. Please comment on whether the droplets were homogeneous throughout the optical\nmeasurement period.\nAuthor Response: We interpret the reviewer’s question regarding homogeneity in two\nways and will address both. If the reviewer meant whether the droplet contains salt\nnanocrystals upon reaching supersaturation, hence making the solvation environment\ninhomogeneous, then we can confidently say that this does not occur. If such salt\ncrystals exist, they would serve as nucleation sites, causing the salt in the droplet to\ncrystalize and the droplet will no longer be aqueous. Because we were able to measure\nthe Mie resonances coming from the droplet under IR irradiation (Figure 3B), the\ndroplet remains aqueous, hence the ions are under a homogenous solvation\nenvironment.\nIf the reviewer meant whether the droplet contains the same salt content as it\nundergoes light-induced evaporation/condensation, we can also confidently say that this\nis true because salt crystals cannot evaporate from the droplet. While the droplet is\nundergoing further evaporation upon IR irradiation at peak absorbance (2053 cm-1),\nhence becoming even more supersaturated, the droplet remained aqueous (Figure 3B).\nTherefore, we can conclude that the droplet retained the same salt composition\nthroughout the optical measurement period. To clarify these points, we have included\nthe following in the manuscript:\nAnother concern is whether the droplet remains homogenous during the evaporation/condensation\nprocess. Previous studies31,32 have shown that NaCl microdroplets can remain aqueous until 40%\nRH before crystallizing, hence we do not foresee the existence of salt crashing out of the droplet,\ncausing inhomogeneity in the solvation environment. Additionally, salt cannot evaporate from the\ndroplet, hence as moisture condenses back onto the droplet after IR irradiation, we expect the salt\ncomposition of the droplet to remain the same.\n5. The observed 5 cm⁻¹ blueshift and significant broadening of ν(N₃⁻) are attributed to\nNa⁺–N₃⁻ ion-pairing and supersaturation, but additional factors may contribute. Could\nelectrostatic field effects from the charged droplet surface or microheterogeneous\nsolvation play roles? Please comment.\nAuthor Response: We thank the reviewer for bringing up these factors. Electrostatic\nfield effects from the droplet surface are expected to be quite small due to the high salt\ncontent of the droplet, which results in short screening lengths. The majority of the azide\nions would have to be aligned with the electric field in order to induce such a large\nblueshift. Additionally, the charge density on the droplet is quite small. For example, we\nhave measured the field required to shift to the organic azide stretch by 1-2 cm-1 on the\n9\nair-water interface,54 which is ~1 e–/nm2. For a r = 10 µm droplet generated and trapped\nin the EDB, the charge has been measured to be ~105 to ~106 e–.11 Assuming that all of\nthis charge is at the surface, the charge density will be ~10-2 to 10-3 e–/nm2, several of\nmagnitude smaller than that measured at the air-water interface, which could only\nproduce a small shift in the organic azide stretch. Therefore, we do not believe that\nelectrostatic field plays a major role here.\nRegarding microhetergenous solvation, we have discussed above (bullet point 4) that\nwe do not believe the droplet is inhomogeneous during our measurement. If the\nreviewer meant microheterogeneity at the droplet surface, we cannot make definite\nassignments on such effect with our current data. In order to probe the effects of the\nsurface, it would be required to bring the probe to the surface of the droplet. Then, one\ncould potentially test the role of the electrostatic field on the azide shift of the droplet by\nincreasing the charge as well which we will pursue in future studies (see our answer in\nbullet point 6).\nTo address the reviewer’s point, we have added the following in the manuscript:\nOther additional factors may contribute to the observed 5 cm-1 blueshift, such as electrostatic field\neffects from the charged surface or microheterogenous solvation environments at the air-water\ninterface. The electrostatic field effects from the droplet surface are expected to be negligible due\nto the high salt content of the droplet, resulting in short screening lengths. Furthermore, the\nmajority of the azide ions would have to be aligned with the electric field in order to induce such\na large blueshift. Additionally, the charge density on the droplet is quite small. For example, our\nprevious studies of organic azide at the air-water interface showed that a charge density of\n~1 e–/nm2 induces a shift in the 𝜈𝑎𝑠 by only 1-2 cm-1.54 Previous measurements of microdroplets\n𝑁3\ntrapped in EDBs showed that there are 105 to 106 e– in the droplet.11 Assuming that all of the charge\ndensity is at the surface, for a r = 10 µm droplet, the surface charge density will be 10-2 to 10-3\ne–/nm2. This surface charge density is several orders of magnitude smaller than the previous study\nat the air-water interface, and is likely too small to induce measurable shifts. Therefore, we expect\nthe contribution from electrostatic field effects to the blue shift to be negligible. Regarding\nmicroheterogenous solvation environments at the air-water interface, we cannot comment on such\neffect with our current data because this method is not necessarily surface-sensitive. Future studies\nmay use a different vibrational probe that is surface active to deconvolute surface from bulk effects.\n6. The authors may consider comparing droplets of different charge states or sizes to\ndistinguish between electrostatic vs. concentration-driven shifts.\nAuthor Response: This is a great suggestion and is currently being pursued in our lab!\nThe main subject of this paper is to introduce the technique. As the instrument stands,\nthere are several modifications needed (such as gentler introduction of humidified air to\nreduce noise in droplet’s displacement) before we can perform more quantitative\nmeasurements. We foresee that as the method matures, these measurements will be\n10\ntaken and, hopefully, be able to clarify many unanswered questions in droplet chemistry!\nWe have included this discussion to the main text of the manuscript:\nMany recent reports have attributed novel behaviors of microdroplets to surface effects such as\nstrong electric fields56 and microsolvation environment at the air-water interface.57 SiDDIRAS is\na promising method to investigate these effects. The spectral resolution of this method is limited\nby the laser, which is 0.1 cm-1 in this case. The signal-to-noise ratio is limited by the detection of\nthe mechanical displacement of the droplet. We note that the spectrum reported in this study is\nfrom a single scan at reduced laser power. To detect weaker vibrational modes, one can average\nmultiple scans at higher laser power. Hence, SiDDIRAS is a promising and sensitive technique to\nuncover some of the unanswered questions in microdroplet chemistry. For example, electrostatic\nfield effects can be probed by changing the droplet’s charge (both magnitude and polarity) and the\ndroplet’s size. The latter requires better control of RH without introducing mechanical\ndisplacement of the droplet through humidified gas. Understanding the droplet’s microhetergenous\nsolvation at the surface is also possible by using a surface-active vibrational probe,54 which will\nrequire calibrating these probes first on the bulk surface. While this report serves to introduce\nSiDDIRAS, we foresee performing more quantitative studies as the method matures to clarify\nsome of the unanswered questions in microdroplet chemistry.\n7. The authors should specify the minimum detectable frequency shift (sensitivity) or\nabsorbance achievable by this method. What is the signal-to-noise ratio for small\nabsorptions, and could SiDDIRAS detect weakly absorbing vibrational modes? A\nquantitative limit of detection would make the method more broadly comparable to\nRaman or fluorescence action spectroscopy.\nAuthor Response: Regarding frequency shifts, our method is only limited by the laser\nresolution, which is 0.1 cm-1 in this case, and the intrinsic spectral width of the absorber\nin sample. Regarding signal-to-noise, because we are detecting the mechanical action\nof the droplet, small movement from the droplet in the z direction will contribute to this\nnoise. If one looked at the non-absorbing region of Figure S2 (~2000 cm-1), one can see\nthat the noise level is ~0.01 mm, hence anything above this noise level is theoretically\ndetectable. We also want to highlight that our spectrum came from one single scan at\nlower laser power. For weaker vibrational modes, we can always increase the laser\npower and average multiple scans together. To clarify this point, we have added the\nfollowing:\nThe spectral resolution of this method is limited by the laser, which is 0.1 cm-1 in this case. The\nsignal-to-noise ratio is limited by the detection of the mechanical displacement of the droplet. We\nnote that the spectrum reported in this study is from a single scan at reduced laser power. To\ndetect weaker vibrational modes, one can average multiple scans at higher laser power. Hence,\nSiDDIRAS is a promising and sensitive technique to uncover some of the unanswered questions\nin microdroplet chemistry.\n11\n8. Clarify the temperature and RH control precision during IR scans, given the high\nsensitivity of evaporation to these parameters.\nAuthor Response: We used a commercial temperature and RH probe from Elitech.\nAccording to the manufacturer, the precision for temperature for this particular probe is\n0.3 °C and for RH is 3%. We note that in order to obtain the scan without interference\nfrom the humidified air, we turned off the bubbler during the optical scan. Because the\nchamber holding the EDB is vacuum tight, the RH does not change drastically over the\ncourse of the scan. The RH and temperature remain relatively stable, with the RH\ndropping by a few percent over hours. We have included the following discussion in the\ntext:\nWhen the humidified gas is turned off, the RH and temperature of the chamber varied by 1-2%\nand ~0.1°C throughout the IR scan according to the probe. The manufacturer reported the\nprecision of this particular probe to be 3% RH and 0.3°C.\n9. A brief paragraph comparing SiDDIRAS to existing optical-tweezers-IR or substrate-\nfree aerosol IR techniques would contextualize its novelty and advantages (maybe as\nan SI note).\nAuthor Response: Please see our response to bullet point 1 and 2 to Reviewer 2\nabove.\n10. The authors mention the potential for UV–Vis action spectroscopy. It would be\nvaluable to discuss practical limitations (e.g., optical trapping stability, droplet heating)\nand expected signal magnitude.\nAuthor Response: For UV-Vis action spectroscopy, we would expect molecules to\nhave higher absorption cross sections, hence higher energy per absorbed photon\n(provided there is no fluorescence). Hence, we suspect the displacement caused by\nlight-induced evaporation might be even more sensitive to UV-Vis absorption. We briefly\ncommented on this point in the following sentence:\nWe suspect that, because UV-Vis chromophores generally have higher absorption cross sections,\nhence higher energy per absorbed photon (provided there is no fluorescence), the displacement\ncaused by light-induced evaporation might be even more sensitive to UV-Vis absorption.\n11. “More recently, using ESI and pneumatic sprays, studies spearheaded by the Zare\nand\nCooks groups, have reported enhanced chemical reactivity in microdroplets” – authors\nshould also acknowledge and appropriately cite other research groups that have been\nactively contributing to the advancement of microdroplet chemistry such as Prof.\nShibdas Banerjee (IISER Tirupati), Prof. Xinxing Zhang (Nankai University), Prof.\nAgustin J. Colussi (Caltech).\nWe have cited their works as suggested:\n“…studies by several groups have reported enhanced chemical reactivity in microdroplets.4-8”"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have answered my concerns, and I recommend for publication. The work is weird, bold, and innovative.", "reviewer_2": "The manuscript has been appropriately revised by incorporating the reviewers’ comments. The reviewer recommends\npublishing this paper in Nature Communications.", "reviewer_3": "The authors have addressed my concerns adequately in the revised draft. Therefore, I support its publication."}, "author_response": ""}]

Decision letter