Excitation spectra recorded under such conditionsare found to differ significantly from previously reported spectra.Pump–probe studies find that intersystem crossing is the dominantdecay pathway of the excited singlet manifold, in agreement with previoussolution phase studies and quantum chemical predictions for the isolatedmolecule. Microsolvation studies on flavone–water clustersreveal that the addition of one and two water molecules leads to considerableshifts in excitation energies but that further complexation does notresult in further noticeable shifts. The relaxation pathways of theelectronically excited states, on the other hand, do not appear tobe influenced by interactions with the solvent molecules. Finally,photoionization spectra have enabled the accurate determination ofthe adiabatic ionization energy to the ground state of the molecularion—key to the antioxidant properties of flavone—as65,415 cm As yet, more than 8000 flavonoids have been identifiedin nature that differ by the substituents on these rings.17 Because of this strong absorption, flavones have attracted considerableinterest for applications such as UV filters in commercial sunscreenformulations.19 In particular, the 3- and 5-hydroxy-substituted derivatives havefrom this point of view been of interest as it has been shown thatin these compounds the photon energy can be dissipated into harmlessheat efficiently and on an ultrafast timescale by excited-state intramolecularproton transfer.25 In order to tailor the photoprotective properties of flavone-basedcompounds, significant effort is presently dedicated to obtain a fundamentalunderstanding of the influence of substituents and environment onthe spectroscopic and dynamic properties of the electronically excitedstates involved in absorption and dissipation of the photon energy.The photoprotective properties of flavonesderive from their strongabsorption of light in the UV-B region.1nπ* state with negligible oscillator strength,while at higher excitation energies a strong UV absorption is observedthat has been assigned to two close-lying 1ππ*states.30 Photoexcitation of these states has been shown to be followed bya very efficient intersystem crossing (ISC) process that populateson a picosecond timescale the triplet manifold with a nearly unitquantum yield.32 Further detailed insight intothese processes has been obtained from high-level density functionaltheory and multireference configuration interaction (DFT/MRCI) calculationsthat determined vertical and adiabatic excitation energies of electronicallyexcited singlet and triplet states, as well as spin–orbit couplingmatrix elements between singlet and triplet states.29The starting point for such considerations is the electronic manifoldof flavone, the core chromophore of such compounds 1. Previo2(ππ*) state. Nevertheless,when these features are considered in more detail, one rapidly comesto the conclusion that their assignment is still far from clear. Inorder to provide further insight into the spectroscopic and dynamicproperties of flavone, we have therefore performed similar R2PI studiesof the excitation spectrum of flavone in combination with ns pump–probemeasurements. In contrast to the previous studies, however, we havenot used the same laser for excitation and ionization but have adopteda two-color scheme that employs an ArF excimer laser (193 nm) forionization to ensure that ionization indeed takes place via a (1 +1′) ionization process. For practical applications, it is importantto determine how the properties of the compound under isolated conditionsare modified by its interactions with the environment. Analogous studieshave therefore been performed on complexes of flavone with a varyingnumber of water molecules. Finally, flavone is also well-known andused for its antioxidant properties as has been mentioned above. Akey parameter determining these properties is the adiabatic ionizationenergy of the compound. As yet, only low-resolution He(I) photoelectronspectra have been reported.34 In order to improveon these energies, photoionization spectra have therefore been recordedthat indeed have allowed for a significantly more accurate value ofthe adiabatic ionization energy.Although flavone hasbeen studied extensively under solvated conditions,studies of the compound under isolated conditions are notoriouslylacking. In fact, the only report of an excitation spectrum recordedunder such conditions has been provided in the transient absorptionstudies of ref  in which35 .Briefly, flavone was heated up to 150 °C within a glass containerin order to obtain sufficient vapor pressure and expanded using neonat 1.5 bar as a carrier gas through a General Valve pulsed nozzlewith a 0.5 mm orifice diameter, which was kept 5 °C higher thanthe main body in order to avoid clogging. After being skimmed by a2 mm skimmer, the molecular beam entered an ionization chamber whereions or electrons were detected using either a reflection time-of-flight(R.M. Jordan Co.) setup for mass-resolved ion detection or a custom-builtsetup (R.M. Jordan Co.) for electron detection.Flavone was purchased from Sigma-Aldrich chemicalsand used withoutany purification. R2PI experiments have been performed employing amolecular beam setup described previously.(1 + 1′)R2PI excitation spectra were recorded using a frequency-doubledSirah Cobra-Stretch dye laser operating on DCM/Pyrromethene 597 pumpedby a Spectra-Physics Lab 190 Nd/YAG laser for excitation and a NeweksPSX-501 ArF excimer laser  for ionization. Typically,pulse energies of 10–50 μJ and 1 mJ were used for excitationand ionization, respectively. For recording ionization threshold spectra,the same pulsed dye laser system has been used in combination withanother pulsed dye laser system consisting of a frequency-doubledSirah Precision Scan dye laser operating on DCM or Pyrromethene 597and pumped by a Spectra Physics Lab 190 Nd/YAG laser. In these experiments,typical pulse energies were employed of 1 mJ for the pump laser and2–4 mJ for the probe laser.0) and cation (D0), while time-dependent DFT(TD-DFT) was employed to optimize the geometry of the molecule inthe first three electronically excited singlet states of the neutraland to determine the associated harmonic force fields. Such calculationshave been performed at both the B3LYP/TVZP and wB97XD/cc-pVDZ level.39 For comparison with the experimental results, the obtained equilibriumgeometries and force fields were used to obtain Franck–Condonspectra at wB97XD/cc-pVDZ level for Sn ← S0 transitions for which vibrational frequencieswere scaled using a scaling factor of 0.953.41 All calculations have been performed with the Gaussian16, Rev.C.01suite of programs.42In order to analyze the observedelectronic transitions and todetermine the ionization energy of flavone and its complexes, DFThas been used to determine the equilibrium geometries and harmonicforce fields of flavone in the electronic ground state of the neutral(S–1 (327.9–289.9 nm). Importantly, no furtherbands could be detected below 30,500 cm–1 or athigher excitation energies up to 35,000 cm–1. Thisspectrum is dominated by a broadband with a maximum at about 32,560cm–1 (∼307 nm) and width on the order of1000 cm–1. As the band does not have a Lorentzianprofile, it has to be concluded that it consists of overlapping vibronicbands that have become unresolved because of extensive Franck–Condonactivity of low-frequency modes and/or lifetime broadening as is indeedconfirmed by our quantum chemical calculations (vide infra). The onsetof the band suggests that the vibrationless transition to the pertainingelectronic state is located between 31,200 and 31,600 cm–1. Although the band does not display a well-resolved substructure,the low-energy side of the band appears to display a vibrational progressionof a mode with a frequency of about 400 cm–1. Athigher excitation energies, the spectrum displays a weak activityin the region around 34,000 cm–1 where the activityof C=C and C=O stretch vibrations might be expected.The black curve in –1 (321.5 nm) and 31,397 cm–1 (318.5 nm).29 These two bands are notobserved in the present experiments. Ionization threshold spectrathat will be discussed below show that the adiabatic ionization energyto the ground state D0 of the ion is 65,415 cm–1. This implies that in the one-color REMPI experiments, ionizationcan only take place at the previously reported energies by a three-photonionization process, in contrast to the present experiments in whichthe energy of the ionizing 193 nm photon is large enough to allowfor (1 + 1′) ionization. The differences between the two spectrathus lead to the conclusion that the bands that are observed in theone-color spectra should be attributed to resonance enhancement atthe two-photon level, that is, the ionization process should be describedas a (2 + 1) ionization process. In view of the associated two-photonexcitation energies the states that are then responsible for the resonanceenhancement are most probably Rydberg states.The spectrum shown in 0 →S1 transition, while S2 is a ππ*state with a significant oscillator strength in agreement with absorptionspectra observed in solution.29 Interestingly,these calculations also indicate that at slightly higher excitationenergies the transition to the second ππ* state shouldbe located with an oscillator strength that is even larger than thatof the transition to S2. We have performed additional TD-DFTcalculations of vertical and adiabatic excitation energies of thelower-lying electronically excited singlet states using the dispersion-correctedwB97XD functional and the cc-PVDZ basis set. Comparison with TD-DFTcalculations at the B3LYP/TZVP level and the results of the previouslyreported DFT/MRCI calculations  state to the lower-lying S1(nπ*)state is expected. In this respect, the rate of (170 fs)−1 leading to linewidths of 32 cm–1 as measured inthe gas phase for S1(nπ*) ← S2(ππ*)internal conversion in trans-azobenzene, a similarly-sized compoundwith a similar S1-S2 energy gap, is indicative.45Inthe DFT/MRCI study, it was found that the molecule adopts ageometry in the ground state in which the benzopyrone and phenyl planesare twisted by ca. 28°. Upon excitation to the electronicallyexcited S2(ππ*) ←S0 and/or S3(ππ*) ← S0 transitions we have calculated such spectra for these twotransitions. These wB97XD/cc-PVDZ calculations confirm the previouslyreported geometry characteristics as a twisting angle of 21°is found for S0 while in the two electronically excitedstates the molecule becomes nearly planar, and give rise to S2(ππ*) ← S0 and S3(ππ*) ← S0 Franck–Condon spectradepicted in –1 and anout-of-plane torsional mode with a frequency of 55 cm–1. Importantly, in the absence of lifetime broadening one would haveexpected to observe well-resolved transitions to the various levelsof such modes.35 The observation that suchis not the case indicates that the observed spectrum is indeed subjectto extensive lifetime broadening.In order to determine whether such progressionsmight indeed accountfor the experimentally observed band profile and to investigate towhat extent predicted Franck–Condon spectra can resolve whetherthis band is associated with the S–1 is larger (roughly a factor of 2–3)than predicted theoretically for the two transitions. One could thereforespeculate that this band actually consists of overlapping S2(ππ*) ← S0 and S3(ππ*)← S0 transitions, but it is clear that with thepresent data, it is not possible to go beyond the level of speculation.The Franck–Condon spectrum predicted for the S2(ππ*)← S0 transition is further characterized by vibrationalactivity in the ∼700 and ∼1650 cm–1 regions, while for the S3(ππ*) ← S0 transition such activity is dispersed over a much largerregion. In this respect, the experimentally observed spectrum wouldappear to favor an assignment to the S2(ππ*)excitation spectrum, although we also directly notice that the relativeintensities of the pertaining vibrational regions are not as largeas predicted by the calculations. We, therefore, conclude that theexperimentally observed excitation spectrum is in agreement with whatis to be expected on account of the planarization of the benzopyroneand phenyl moieties upon excitation but that further theoretical studiesare needed to come to a quantitative understanding of the furtherdetails of this spectrum.Focusing for the moment onthe onset of the two transitions, weobserve that the width of the experimentally observed band at 32,560cm–1—the maximum of the band observedin SI2). The trace thus actuallyindicates that after excitation molecules are ionized from a statethat does not decay on the timescale during which they are “visible”to the ionization laser. Such a conclusion is in line with solutionexperiments in which an almost near-unit triplet formation is observedupon UV radiation.29 We, therefore, concludethat in our experiments we observe ions that have been generated byionization from triplet states. A further observation that can bemade is that the trace does not show a fast decay for near-zero-timedelays between excitation and ionization lasers followed by an apparentslower decay caused by the removal of excited molecules from the ionizationspot. On the timescale of the present experiments fast internal conversionof the initially excited ππ* state to the nπ* statewill have taken place. The absence of a fast decay thus indicatesthat for time-overlapping excitation and ionization lasers, the contributionto the signal of ions associated with ionization from the excitednπ* singlet state is negligible. We, therefore, also concludethat intersystem crossing from the excited singlet manifold to thetriplet manifold occurs at rates that are at least one or two ordersof magnitude larger than the inverse of the pulse durations of theexcitation and ionization lasers. Such a conclusion nicely supportsthe results of calculations on intersystem crossing rates, which predictrates on the order of 1011 s–1 for flavoneunder isolated conditions.46Pump–probe traces of the ionyield obtained after excitationat 32,563 cm2(ππ*) ← S0 band. However, suchexperiments were not successful, most probably due to rapid electronicand vibrational autoionization processes.47 We, therefore, reverted to measuring photoionization spectra todetermine the adiabatic ionization threshold. One-color photoionizationwas in this case not possible since the wavelengths that would needto be used are in the region of –1—which is in the very tail ofthe band observed in the excitation spectrum of –1 is obtained,which yields an adiabatic ionization energy of 65,424 ± 30 cm–1 (8.112 ± 0.004 eV) when extrapolated to zeroelectric field.48 A further determination and confirmationof this energy have been obtained using electron detection for whichmuch lower electric fields are used. The ionization threshold spectrumobtained in this way is shown as the black trace in –1 (8.109 ± 0.004 eV), which is in good agreementwith the value obtained with ion detection. Photoionization spectrarecorded at several other excitation wavelengths (see Supporting Information SI3) lead to the same threshold and show thatthis threshold can indeed be associated with the adiabatic ionizationenergy and not with a threshold to a vibrationally excited cation.Previous He(I) photoelectron studies have reported a vertical ionizationenergy of 8.52 ± 0.2 eV33 implyingan internal reorganization energy upon ionization of 0.41 eV, whichis in line with what is expected. These numbers also compare favorablywith our calculations, which predict adiabatic and vertical ionizationenergies to the ground ionic state of 8.18 and 8.40 eV, respectively.The excitation spectrum of flavone is of importancefor assessingits properties as a UV absorber and, related to that, its potentialapplications in sunscreen formulations. As discussed in the introduction,flavonoids are also well known for their antioxidant properties forwhich detailed knowledge of their ionization energies is important.In the first instance, we have tried to perform zero-kinetic-energypulsed-field-ionization (ZEKE-PFI) experiments via the observed S2O)n complexes with up to eight water molecules (see SupportingInformation SI4 for a further discussionof the possible role of dissociative ionization). These spectra showthat the addition of one water molecule leads to a redshift of thespectrum of ∼300 cm–1. A similar shift (∼300cm–1) results from the addition of a second watermolecule, but it has to be noticed that the spectrum at the same timesignificantly broadens. Interestingly, further addition of water moleculesleads to significantly smaller redshifts of the spectrum and no furtherbroadening of the spectrum. Apparently, conformational heterogeneitystarts to be of influence for two water molecules and more, but itseffects on the excitation spectrum are minor once two water moleculeshave been added. The observed redshifts are in line with the generalnotion that ππ* states shift to lower energies in polarsolvents. Similarly, one would expect the lower-lying nπ* stateto be shifted to higher excitation energies upon increasing microsolvation,but in the spectra that have been recorded here, there is no indicationof the presence of the S1(nπ*) state within the recordedexcitation energy range.Under biological conditions flavone is not isolatedbut subjectto interactions with solvent molecules. From this point of view, itis of interest to determine how the spectroscopic properties of flavoneas determined above are modified by a solvent. To this purpose, wehave performed microsolvation studies in which we study flavone complexedwith an increasing number of water molecules. 2O complex arises fromcomplexation at the carbonyl site. In addition, and in agreement withour expectations, these calculations find that further addition ofwater molecules preferably occurs by binding to the already complexedwater molecules as this affords strong hydrogen bonds. In this respect,it is interesting that the addition of a second water molecule leadsto a similar redshift of the flavone–H2O spectrumas the redshift observed in going from flavone to flavone–H2O since the second water molecule is not directly attachedto the aromatic system of flavone. Apparently, delocalization of theπ orbitals does not extend beyond the second water moleculeor is considered from another point of view, the charge reorganizationoccurring upon forming a hydrogen bond between water and the carbonyloxygen is influenced by the hydrogen bond formed between the two watermolecules in flavone–(H2O)2 but not bya third or more water molecule.Quantum chemical calculations show as expectedthat the lowest-energystructure of the flavone–H1(nπ*) andT1(ππ*) states in polar solvents, the presentpump–probe experiments do not give evidence for such a decreasenor for a change in intersystem crossing yield. We thus conclude thatunder the intrinsic excited state decay dynamics of flavone are retainedunder the microsolvated conditions explored here.Pump–probe experimentson microsolvated flavone show thatcomplexation with water does not lead to a discernible influence ondecay dynamics. In all cases, long-lived species are observed whoseR2PI signal disappears because the excited complexes travel away fromthe excitation spot. Although one would expect that under solvatedconditions the intersystem crossing rate will decrease because ofthe increasing energy gap between the S2(ππ*) state but involve valence or Rydberg states atthe two-photon level. The one-photon excitation spectrum recordedhere shows in contrast primarily a very broadband with minor discerniblevibrational resolution. Quantum chemical calculations confirm thatthe absence of resolved vibrational bands can very well be attributedto extensive Franck–Condon activity of low-frequency normalmodes activated because of planarization of the phenyl and pyroneplanes upon excitation, in combination with lifetime broadening dueto internal conversion from the initially excited bright S2(ππ*) state to the dark S1(nπ*) state.Pump–probe studies of the R2PI signal find that the observedion signal is associated with ionization from an electronically excitedstate that is instantaneously populated on the timescale of our experimentsand that has a lifetime longer than can be probed in our experiments.Such conclusions are in agreement with the generally accepted decaypath of the S2(ππ*) state, which involves ISCto the triplet manifold with near-unity quantum yield.The present study has provided detailedinsight into the spectroscopicand dynamic properties of flavone, a key chromophore in providingprotection from harmful UV radiation as well as from harmful radicals.Our molecular beam studies have shown that previously obtained excitationspectra are not associated with the strongly absorbing SMicrosolvationstudies in which flavone is increasingly complexedwith water molecules demonstrate that the addition of one and twomolecules results in significant redshifts of the excitation spectrumas would indeed be expected for a ππ* state but that furthercomplexation has limited influence on both the maximum and width ofthe absorption band. On the accessible timescales of the present experiments,the observed decay dynamics, on the other hand, do not appear to beinfluenced by microsolvation.–1 (8.110 ± 0.004 eV)].Finally, our studies have alsocontributed to the further knowledgeof the antioxidant properties of flavone. These properties are closelyrelated to its ionization energy. The present study has enabled amuch more accurate determination of the adiabatic ionization thresholdthan possible up till now [65,415 ± 30 cm


Around these singularities, reflection spectra demonstrate rapid phase changes while the structure behaves as a perfect absorber. Furthermore, we demonstrate that such topological phase singularities are ubiquitous for the entire class of atomically thin TMDCs and other high-refractive-index materials, making it a powerful tool for phase engineering in flat optics. As a practical demonstration, we employ PdSe2 topological phase singularities for a refractive index sensor and demonstrate its superior phase sensitivity compared to typical surface plasmon resonance sensors.Atomically thin transition metal dichalcogenides (TMDCs) present a promising platform for numerous photonic applications due to excitonic spectral features, possibility to tune their constants by external gating, doping, or light, and mechanical stability. Utilization of such materials for sensing or optical modulation purposes would require a clever optical design, as by itself the 2D materials can offer only a small optical phase delay – consequence of the atomic thickness. To address this issue, we combine films of 2D semiconductors which exhibit excitonic lines with the Fabry-Perot resonators of the standard commercial SiO 2/Si Fabry-Perot resonators in order to realize topological phase singularities in reflection. Around these singularities, the reflection spectra demonstrate rapid phase changes while the structure behaves as a perfect absorber.The authors combine films of two-dimensional semiconductors, which exhibit excitonic spectral features, with SiO Generally, the phase acquired by light upon reflection, scattering, or transmission varies rapidly when the amplitude of the light  goes to zero7. Reflection zeros can be encountered in the effect of plasmonic blackbody8, perfect absorption10, coherent perfect absorption11, Brewster angle13 or more sophisticated examples of zero-reflection modes14. Such zeros of response function always reveal phase singularities16 which are accompanied by a non-trivial topological charge 16.Optical waves carry energy and information encoded in their electric field amplitude, phase, and polarization. While light field amplitude is still used the most in various applications, optical phase manipulation could lie in the core of next-generation information technologies2/Si substrate. For a given thickness of HRIM film, an angle of incidence (E), and polarization, reflection from the structure can be made exactly zero by calculating appropriate values for the dielectric permittivity of the HRIM film (Re(ε) and Im(ε)), which can be always found by the nature of Fresnel coefficients for the structure17. When we change an angle of incidence and a photon energy in some range, a zero-reflection surface will appear, see the blue surface in Fig. E, Re(ε) and Im(ε)) and hence will inevitably cross the surface of zero-reflection as shown in Fig. 17, there will be two intersection points between the material dispersion curve and the zero-reflection point resulting in two zeros of reflection from the structure in Fig. 6. Indeed, minor variations of the material dispersion curve caused by material imperfections will not change the relative alignment of the curve and the zero-reflection surface and cannot lead to the disappearance of the zero-reflection points which lead to the idea of topological darkness6. Our structure relies on the atomic flatness of the interfaces and on the sharp changes of the refractive indexes, which offer a non-trivial possibility to realize zero-reflection and topological singularities for atomically thin layers in this structure at visible light, which was never achieved before.To demonstrate how phase singularities associated with zero-reflection can be topologically protected, let us consider light reflection from a planar structure shown in Fig. E and Zero-reflection implies perfect absorption of the light that falls onto the discussed structure (as transmission through the silicon substrate should be zero). Zero-reflection entails phase singularity due to the singular nature of light phase at zero light amplitude (where the phase of light is not defined). Figure 20. Such “flat optics” paradigm enables miniaturized metalenses23 and meta-holograms26, and two-dimensional (2D) materials27 such as graphene28, transition metal dichalcogenides29, and organic semiconductors30 provide an excellent platform for the implementation of these components. Although these works constitute an important step towards truly flat optics with electrically controlled properties, the efficiency of current devices is limited by the fundamental constraints: the majority of optical devices require a sufficient phase accumulation, while for atomically thin layers, this value is naturally low31. Indeed, the typical wavefront manipulation applications require that the phase accumulated by a light wave upon interaction with such a device can be tuned at least within the range of t ∼ 0.65 nm and n ∼ 4) for visible light (λ ∼ 60  nm), the resulting phase delay, which is approximately determined by the optical thickness of the 2D material layer, is only about 0.01The most exciting consequence of such optical phase control is the realization of “flat optics” paradigm—flexible manipulation of the optical wavefront by an arrangement of subwavelength planar objects to shape the desired phase pattern2, graphene, MoS2, and WS2 films placed on SiO2/Si substrate. Combined theoretical and experimental analysis indicates that the zero-reflection points are accompanied by a non-trivial topological charge. We derive an analytical condition for such points to occur in layered structures containing optically thin films and predict the occurrence of these points in structures containing a broader family of atomically thin HRIMs and substrates. The observed effect is highly robust and does not require complicated fabrication steps guaranteeing its reproducibility and reliability. In contrast to optical darkness observed for dielectric materials and multilayers which disappear with layer irregularities  the effect is topologically protected. It can be used in numerous applications, including label-free bio- and chemical sensing, photo-detection and photo-harvesting, perfect light absorption in 2D monolayers, quantum communication, and security. As a practical application of this platform, we demonstrate a refractive index sensor that can rival modern plasmon resonance-based counterparts32. Therefore, our phase engineering approach as a whole offers an advanced tool for current and next-generation 2D flat optics.Here, we demonstrate a platform for efficient optical phase manipulation presented by atomically thin high-refractive-index materials (HRIMs) that often possess excitonic resonances. We experimentally observe zero-reflection, phase singularities, and rapid phase variation of reflected light in extremely thin layers (down to single monolayer!) of PdSe2/Si substrates, we utilized spectroscopic ellipsometry (Methods). The unique advantage of this technique is the simultaneous determination of reflection amplitude and phase in terms of the ellipsometric parameters ρ:33To examine the topological properties of the light reflectance from thin HRIM films placed on SiO2 as well as for monolayers of graphene, MoS2, and WS2 on SiO2/Si substrate showed a number of zero-reflection points as explained in Fig. 34. However, the spectra of C. Therefore, for the first time we experimentally proved that these points are phase singularities, which can occur if and only if the response function (reflection in our case) takes zero magnitude at that point. Figure 2, two for graphene, and one for MoS2 and WS2 monolayers. Interestingly, PdSe2 has the largest number of topological phase singularities . It is worth mention that these phase singularities do not originate from Brewster or interference effect38. Indeed, the Brewster effect requires a lossless dielectric while the interference effect needs a substantial phase accumulation, which is negligible for atomically thin PdSe2. Hence, our concept of topological phase singularities  and dielectric were used to generate zero reflection and phase singularities. Figure 2/Si. The reason for this counterintuitive result is a rapid dielectric function variation  in atomically thin HRIM. This rapid variation guarantees intersection with the zero-reflection surface  define zero-reflection points for the particular material of the film, as shown in Fig. 40.Equations  and 3)  3) defin2, which demonstrates rich 2 film, and then switch to unique applications and features of topological phase gradient.Spectral positions of topological phase singularities can be controlled by either the thickness or the dielectric permittivity of the material. The derived analytical expressions allow us to generalize the effect of phase singularities to other high-refractive-index materials 41 resulted in a uniform sample as confirmed by representative optical and scanning electron microscopy (SEM) images in Fig. 42. Next, we validated the material’s purity by X-ray photoemission spectroscopy (XPS) in Fig. 2 with puckered pentagonal crystal structure presented in Fig. 3a43. This crystal configuration naturally has high geometrical and, therefore, high optical anisotropy  in Fig. 2 has pronounced excitonic peaks46 and a metallic Drude response caused by p-doping revealed by XPS /Si substrate, making system PdSe2/SiO2(280 nm)/Si promising for enhancement of the PdSe2 optical response.To investigate anisotropic optical response, we measured Mueller matrices (Methods), which nonzero off-diagonal elements relate to sample anisotropy  and 7.1 nm PdSe2 thin film to have topological zero in the operation range of our device since liquid changes zero’s spectral and angle position according to Eqs. , we injected isopropanol into the solution and recorded 4 degrees per refractive index unit (deg/RIU) exceeds that of the cutting-edge sensor based on plasmonic surface lattice resonance with 5.7 × 104 deg/RIU  since it measures amplitude and phase of complex reflection ratio simultaneously. Measurements were done over a wide wavelength range from 248 to 1240 nm (1 – 5 eV) in steps of 1 nm and multiple angles of incidence in the range 15°–80° with 0.5° step. Ellipsometer has a single chamber monochromator with two gratings: 1200 g/mm for visible light (248 – 1040 nm) with 4.6 nm bandwidth and 600 g/mm for near-infrared interval (1040–1240 nm) with 9.2 nm bandwidth.2 in water.For accurate refractive index sensing, we performed the nulling ellipsometry with Accurion nanofilm_ep4 ellipsometer. During the measurement light was initially directed through the polarizer then through the compensator, whose settings were adjusted until the reflection from the sample became linearly polarized. Afterward, the analyzer was set to achieve the minimum in the signal at the photodetector. The final positions of polarizer and analyzer at signal’s minimum uniquely define ellipsometric parameters m12, m13, m21, m22, m23, m24, m31, m32, m33, m34) on Accurion nanofilm_ep4 ellipsometer over 400–1000 nm wavelength range in 5 nm step with 4 nm bandwidth at 50° incident angle in a rotation compensator mode to get access to Stokes parameters in the input branch of ellipsometer.To probe anisotropic response, we also measured 11 elements of Mueller Matrix  with a digital camera DS-Fi3.The surface images (2400 × 2400 pixels) of PdSe2 film were accurately characterized by an atomic force microscope  using AFM in a peak-force mode at ambient conditions. AFM measurements were carried out using cantilever tips from NT-MDT  with a spring constant of 3.5 N/m, a tip radius < 10 nm and a resonant frequency of 140 kHz. Images of the PdSe2 surface were taken with 512 × 512 pixels and scan rate of 0.5 Hz, after that data were analyzed by Gwyddion software.The thickness and surface morphology of the PdSe2 film surface within different areas using 1960 × 1280 pixel scan.Scanning electron microscopy  with a Schottky emitter in secondary electron imaging mode with a voltage of 30 kV and current of 67 µA, and a working distance of ~6.3 mm was applied to study in detail surface features and homogeneity of the PdSe−9 mBar) with a monochromatic Al-Kα X-ray source (1486.6 eV). Photoelectron spectra were acquired using fixed analyzer transmission (FAT) mode with 50 eV pass energy. The spectrometer energy scale was calibrated on the Au 4f7/2 line (84.0 eV).The chemical states of the elements in the film, as well as the valence band were analyzed by X-ray photoelectron spectroscopy (XPS) in Theta Probe tool (Thermo Scientific) under ultrahigh vacuum conditions  equipped with Cu Kα radiation −1, as the reference was used the reflection from the gold mirror.Fourier-transform spectrometer Bruker Vertex 80 v has been used to measure the reflection coefficient at the normal incident at room temperature in the frequency range from 1000 to 24,000 cm−1, for each of the 45 × 45 points in the scan, with an integration time of 500 ms at each point and incident power P = 1.7 mW.The experimental setup used for Raman measurements was a confocal scanning Raman microscope Horiba LabRAM HR Evolution . All measurements were carried out using linearly polarized excitation at wavelengths 532 and 632.8 nm,1800 lines/mm diffraction grating, and ×100 objective (N.A. = 0.90), whereas we used un-polarized detection to have a significant signal-to-noise ratio. The spot size was approximately 0.43 μm. The Raman images were recorded by mapping the spatial dependence of Raman intensity integrated at the main Raman peaks within the shift range 136–156 cmSupplementary Information


Raman maps capture sub-cellular responses with the progression of tumor cells into metastasis. Raman feature extraction via cluster analysis allows for the identification of specific cellular constituents in the images. For the first time, this work demonstrates a promising potential of Raman imaging, PCA, and cluster analysis to discriminate between cancer cells at different stages of metastatic tumorigenesis.Metastatic prostate cancer colonizes the bone to pave the way for bone metastasis, leading to skeletal complications associated with poor prognosis and morbidity. This study demonstrates the feasibility of Raman imaging to differentiate between cancer cells at different stages of tumorigenesis using a nanoclay-based three-dimensional (3D) bone mimetic in vitro model that mimics prostate cancer bone metastasis. A comprehensive study comparing the classification of as received prostate cancer cells in a two-dimensional (2D) model and cancer cells in a 3D bone mimetic environment was performed over various time intervals using principal component analysis (PCA). Our results showed distinctive spectral differences in Raman imaging between prostate cancer cells and the cells cultured in 3D bone mimetic scaffolds, particularly at 1002, 1261, 1444, and 1654 cm Metastatic prostate cancer colonizes in the bone marrow to pave the way for bone metastasis, leading to cytoskeletal complications and poor prognosis and morbidity. There has been an increasing demand for translational models that recapitulate bone metastasis of prostate cancer to understand the mechanisms that underlie bone metastasis, mainly due to the lack of availability of human prostate cancer metastasized bone samples and in vivo mouse models for spontaneous prostate cancer bone metastasis. Moreover, the complexity of this disease itself poses a challenge for the diagnosis and assessment of populations with therapeutic resistance. The basic histopathological examination is the current gold standard for determining the prognosis of prostate cancer in patients. Immunohistopathology has been used extensively for the analysis of biopsy samples in the last decade. Nevertheless, immunohistochemistry has limitations due to the difficulty of analyzing large volumes of tissue sections by staining and the inability to detect multiple signals simultaneously. Also, the reproducibility and robustness of genomic data remain a concern due to the heterogeneity of tumors. It is, therefore, necessary to develop robust diagnostic and classification tools that have reproducibility and translational application with clinical samples.Prostate cancer is the most frequently diagnosed cancer amongst men. According to recent statistics, the 5-year survival rate of patients with primary prostate cancer is 100% when diagnosed early, while it declines to 30% in the case of metastases4. A recent study demonstrated the feasibility of the complementary vibrational spectroscopy technique for accurate and precise chemical analysis of organic liquids by combining IR and Raman spectroscopy5. Raman spectroscopy has been widely used to differentiate between normal, nonmalignant, and malignant cell lines, from breast7, colorectal8, gastrointestinal9, lung10, and prostate cancer cells/tissues13. Recent studies demonstrate the use of Raman spectroscopy to distinguish between the osteoblastic and osteolytic bone metastases14. A few studies have addressed stage-based classifications16. A recent study demonstrates the use of Raman mapping to evaluate sub-cellular responses in prostate cancer cells (PC-3) to X-ray exposure while also elucidating the advantage of Raman mapping techniques over single-point measurement17. Also, recent studies demonstrate the use of Raman spectroscopy and artificial intelligence to evaluate the probability of breast cancer in a clinical environment18. A recent study describes the use of optical diffraction tomographic imaging and Raman spectroscopy to combine morphological and molecular data for phenotyping breast cancer cells19. Furthermore, most previous studies used Raman spectroscopy in prostate cancer research based on two-dimensional (2D) culture models20. It has been reported that in the 2D system, cells lose their in vivo characteristics, resulting in poor cell–cell and cell–matrix interactions21. In contrast, three-dimensional (3D) in vitro models recapitulate a physiological environment that mimics in vivo conditions, leading to improved predictions22. The primary goal of this study is to evaluate Raman spectroscopy as a potential tool for monitoring cancer progression at bone metastasis using a nanoclay-based 3D bone mimetic in vitro model of prostate cancer bone metastasis. For this purpose, we developed a 3D bone mimetic in vitro model23 using nanoclay-based scaffolds24 along with osteogenically differentiated human mesenchymal stem cells (MSCs) and human prostate cancer cells. Our group has investigated nanoclays extensively to prepare polymer clay nanocomposites26 using both experimental and simulation-based studies, with applications towards bone tissue engineering scaffolds27. Our previous studies have shown osteogenic differentiation of human MSCs into osteoblasts and MSCs mediated mineralization through the biomimetic process of vesicular delivery on nanoclay based scaffolds without using osteogenic supplements28. Recently, we have reported the late stage of prostate and breast cancer pathogenesis to the bone using the sequential culture of MSCs with human prostate and breast cancer cells on nanoclay based scaffolds31. In another study, Wnt/β-catenin signaling has been shown to play a key role in osteogenesis in the 3D bone mimetic in vitro model of breast cancer bone metastasis32. We have also shown the influence of prostate cancer phenotype on bone mineralization at metastases33. In a related study from our laboratory, we have evaluated the feasibility of FT-IR spectroscopy for monitoring breast cancer progression on the 3D bone mimetic in vitro model of breast cancer bone metastasis34. A recent study showed how bone microenvironment secreted cytokines confer chemoresistance in breast cancer cells at bone metastases35. We have also described the mechanobiology of breast cancer cells36 and prostate cancer cells37 with time at the bone metastasis site using the 3D bone mimetic in vitro model.Raman spectroscopy is an inelastic scattering phenomenon used to probe molecular vibrations, thus providing a molecular fingerprint that is especially suited for biological macromolecules. This technique is based on a change in polarizability of a molecule on the absorption of photons. In contrast, complementary infrared (IR) spectroscopy depends on a change in the dipole moment. Raman spectroscopy is an information-rich spectroscopic method, like FT-IR, capable of detecting specific groups and the environment-sensitive cell surface composition of biological macromolecules consisting of carbohydrates, proteins, and lipids, among others39 have only used cells/tissues targeted using antibodies or ligands labelled using SERS particle for signal enhancement.In the present study, we describe the feasibility of Raman spectroscopy as a powerful imaging method for differentiating prostate cancer cells from prostate cancer bone metastasis tumor cells and discerning cancer progression at the bone site. We are using 2D and 3D bone mimetic cell culture models in conjunction with the application of principal component analysis (PCA) and cluster analysis. The present work addresses the: (1) potential of Raman imaging to discriminate between cancer cells at different stages of tumorigenesis; and (2) determination of key biochemical components that account for the differences observed in the cells at different stages of cancer progression as biomarkers for future prognostic application of prostate cancer bone metastasis. This is the first study that uses label-free Raman spectroscopy to differentiate tumor tissues of various stages by imaging a large tumor tissue. Previous studies−1 (ring breathing of cytosine and thymine), 850 cm−1 (v(C–C) ring breathing of tyrosine and proline), 935 cm−1 (v(C–C) of α-helix conformation for proteins), 1002 cm−1 (v(C–C) ring breathing of phenylalanine), 1080 cm−1 (v(C–C) of lipids), 1124 cm−1 (v(C–O) in carbohydrates), 1261 cm−1 (amide III δ(N–H) of proteins), 1296 cm−1 (δ(CH2) deformations of lipids and proteins), 1336 cm−1 (CH3CH2 twisting of nucleic acids), 1444 cm−1 (δ(CH2) of proteins and lipids), and 1654 cm−1 (amide I v(C=O) of proteins, α- helical conformation)45  MSCs + PCa SC, while 2D cancer cell culture is referred to as 2D PCa, where X = 23 days of MSCs culture and Y = 5, 10, and 15 days of cancer cell culture after 23 days of MSCs culture. The mean Raman spectra of 2D PCa, 3D d (23 + 5) MSCs + PCa SC, 3D d (23 + 10) MSCs + PCa SC, and 3D d (23 + 15) MSCs + PCa SC are shown in Fig. −1, 1261 cm−1, 1444 cm−1, and 1654 cm−1 in the Raman spectra. A comparison of the Raman spectra intensities ± SD at each of the four identified spectral sub-regions for all sample types studied is illustrated in the bar chart in Fig. −1, 1261 cm−1, 1444 cm−1, and 1654 cm−1) between 2D PCa and 3D d(23 + 5) MSCs + PCa SC; however, we observed a significant reduction in intensities at all four bands for 3D d(23 + 10) MSCs + PCa SC and 3D d(23 + 15) MSCs + PCa SC, as compared to both 2D PCa and 3D d(23 + 5) MSCs + PCa SC as shown in Fig. −1 signal in 3D d(23 + 10) MSCs + PCa SC and 3D d(23 + 15) MSCs + PCa SC, as opposed to 3D d(23 + 5) MSCs + PCa SC or 2D PCa, indicating a decrease in the amount of phenylalanine relative to the total Raman-active constituents in cancer cells during cancer progression  MSCs + PCa SC and 3D d(23 + 15) MSCs + PCa SC as opposed to 3D d(23 + 5) MSCs + PCa SC  analysis, whose score and loading plots showed subtle differences, as shown in Fig. 52 to cluster data into groups. If data in the specimen image is in the same cluster as the pure component stripe (actin or myosin), those regions within the map were considered similar to the component of the strip. Figure 53. We observed changes to the spatial distribution of both actin and myosin from 2D to the 15-day 3D tumor samples. The uniform actin and myosin distribution within cells, as observed in 2D samples, are disturbed as the tumor grows  ring breathing of phenylalanine), 1261 cm−1 (amide III δ(N–H) of proteins), 1444 cm−1 (δ(CH2) of proteins), and 1654 cm−1 (amide I v(C=O) of proteins, α-helical conformation)54  ring breathing of phenylalanine), 1444 cm−1 (δ(CH2) of proteins), 1654 cm−1 (amide I v(C=O) of proteins, α-helical conformation) (shifted from 1670 cm−1 in reference myosin spectra) (not shown), and 1336 cm−1 (CH3CH2 twist) (shifted from 1320 cm−1 in reference myosin spectra) (not shown)55  is used to inform the likelihood of a suspicious area being a clinically significant cancer. Based on future more extensive studies, we see the potential for using Raman imaging results to calculate PI-RADS scores or a similar quantitative reporting system for clinically relevant diagnosis.Collectively, the observations from this study suggest that there are significant changes in the intensities of specific biomolecules relative to the total Raman-active constituents in 2D and 3D bone mimetic cultures. The 3D bone mimetic cultures mimic prostate cancer metastasis at the bone site37 are valuable for development of predictive tools of cancer. Also, the use of Raman based tools enables a non destructive and in situ evaluation that is of great clinical importance. Our methods suggest that incorporating Raman spectroscopy and statistical techniques, such as PCA and cluster analysis, makes the classification of cancer progression straightforward.The present study marks the first application of Raman spectroscopy to classify 3D in vitro prostate cancer bone metastasis and to discriminate between cancer cells at different stages of tumorigenesis. Our results show that lipid, aromatic amino acid, and extracellular matrix components are involved in staging prostate cancer at the secondary bone site. Raman spectroscopic properties of prostate cancer metastasized at bone tissue can be effectively translated into a great wealth of diagnostic information providing new insights into biochemical and architectural changes of prostate cancer cells undergoing different stages of tumorigenesis. Also, from the point of clinical applications, it is possible to use hand-held Raman imaging of tissues ex vivo or in vivo for diagnostics at the patient level and would be very valuable for accessible cancers. The clinical application of Raman spectroscopy based endoscopy techniques for colon and prostate cancer diagnosis, and also the future application of the Raman signatures developed from this study for cancer staging and treatment response to therapy from biopsy samples address unmet needs towards new therapeutics for cancer. In addition,besides bone stabilizing drugs, clinicians use bone replacement materials and pins to stabilize damaged bone. It is of great interest to know the progression of metastasis in a patient to identify suitable therapies or combination thereof. Also from a scientific perspective, eventual in silico oncology approaches will need a dependable predictive capability of metastasis progression that is reliable for individual patients. The data collected with Raman imaging tools presented here in combination with nanomechanical evaluation of metastasis progression58. In brief, the 5-aminovaleric acid solution was added to preheated (60 °C) MMT suspension, and the mixture solution was kept for stirring. After one hour, the obtained slurry was centrifuged and washed to remove chloride ions, then dried at 70 °C, ground, and sieved to obtain a fine powder. Na-MMT clay was procured from Clay Minerals Respiratory at the University of Missouri, Columbia, and 5-aminovaleric acid was obtained from Sigma-Aldrich.The detailed procedure for the modification of Na-MMT clay is described elsewhere27. Briefly, the organically modified MMT clay powder was dissolved into Na2HPO4 solution (23.8 mM) by stirring at room temperature for two hours. Further, 39.8 mM of CaCl2 solution was added, and this suspension was stirred vigorously for eight hours (pH 7.4). The obtained precipitate was centrifuged, dried (70 °C), and subsequently ground and sieved to obtain a fine powder. Na2HPO4 and CaCl2 were purchased from JT Baker.We have followed the procedure described in our previous studies to prepare in situ HAPclay24. 3D PCL scaffolds were prepared with ten wt. % in situ HAP clay. In a typical procedure, the PCL solution was prepared by dissolving 3.6 g of polymer in 40 ml of 1,4-dioxane. Another solution was prepared by dissolving 0.4 g of prepared in situ HAPclay in 20 ml of 1,4-dioxane, followed by sonicating for 18 min. Freshly prepared in situ HAPclay solution was added to the polymer solution and stirred for two hours. We used the freeze extraction method to obtain 3D scaffolds. The chemicals PCL and 1, 4-dioxane were purchased from Sigma Aldrich.3D PCL/in situ HAPclay scaffolds were prepared according to the procedure described in earlier studies4 cells per scaffold and cultured for 23 days to allow bone tissue formation. Further, prostate cancer cells PCa were seeded on newly formed bone tissue in the 3D bone mimetic scaffolds at a density of 5 × 104 cells and maintained in 1:1 MSCs and PCa medium  (Lonza) were maintained in MSCGM™ Bulletkit™ medium . Human prostate cancer cell line MDA-PCa-2b (PCa) is purchased from ATCC and kept in 80% BRFF-HPC-1 (AthenaES) and 20% FBS (ATCC). For 2D cultures, cells were seeded on tissue culture-treated Petri dishes. For 3D sequential culture, MSCs were seeded at a density of 5 × 10−1 fingerprint region was used for the subsequent analysis . Specially developed codes in MATLAB™ were used for simultaneous baseline correction of all Raman spectra in each image. The open-source Raman Tool Set was used for mapping the spectra. Subsequently, additional MATLAB™ codes were utilized for contour map development to allow uniform scaling across all maps generated for a given band. MATLAB™ codes were also developed to enable data to be transferred between Raman Tool Set and MATLAB™. Cosmic ray removal was also implemented before the spectra were used to create maps for predefined Raman bands in MATLAB (Mathworks Inc.). Further, 20 cell spectra from each sample were selected for intensity calculation of chosen Raman bands and principal component analysis. The mean ± standard deviation for the average spectrum obtained for each sample shown in Fig. −1 before analysis. Cancer cells, because of their rapid proliferation, possess multiple copies of DNA per cell in a given time compared to normal cells within the tumor. In addition, cancer cells are the predominant population compared to normal cells  in cancer tissues. Hence, we used Raman spectra respective to 780 cm−1 which represents the ring breathing of cytosine and thymine, the parts of DNA as internal control for normalization.Raman spectra were acquired using a custom-built NIR confocal Raman microscopy system. The excitation source comprises a 785 nm wavelength from Ti: Sapphire laser . The laser beam is focused on the sample, and backscattered light is collected using the same objective lens . Raman signal is collected after a series of Rayleigh rejection filters by a collection fiber connected to a high-throughput imaging spectrograph  with a thermoelectric-cooled, back-illuminated, and deep depleted CCD . The 785 nm wavelength is ideal for cell studies as it is just outside the visible range. Visible wavelengths cause degradation and photodamage to biological samples even at low power and short acquisition times. The primary goal of the present study is to investigate biochemical nuances at the cellular level, and thus a collection of cells in pellets from both 2D and 3D cultures was analyzed using point spectroscopic measurements. Briefly, after replacing the culture medium with PBS, cell pellets were formed by centrifugation and placed on top of the quartz coverslip followed by fixation with 4% paraformaldehyde for 10 min at room temperature, washing with phosphate-buffered saline (PBS) for 2–3 times before performing Raman experiments  and 80 columns . PCA was performed in the spectral region of 700–1800 cm−1 using a covariance matrix .Principal Component Analysis (PCA) is an unsupervised statistical method that reduces variability by generating principal components (PCs). PCs are originated from a linear combination of original variables and are arranged based on the variance in the original dataset to explain correlation to one another. The first two PCs, i.e., PC1 and PC2, generally describe most of the data variance and generate a score plot. In a score plot, samples are positioned according to PC scores. For instance, samples with similar scores will take up a similar position while those with different scores will be placed in distance, thus making it easier to classify among samples60. It is extensively used in data mining, machine learning, pattern recognition, image analysis, bioinformatics, and computer graphics in fields as diverse as astronomy, ecology, social sciences, and biology. Although there are many ways of conducting cluster analysis, we have combined cluster analysis with PCA to analyze spectral data from Raman mapping. Unsupervised algorithms such as principal component analysis and cluster analysis have been recently investigated for disease identification, including epithelial tissue tumors, brain tumors, skin tumors, bone diseases, atherosclerosis, kidney stones, gallstones, diabetes, and osteoarthritis61. A digital stripe of 5 μm width and 76 μm length is created outside of the Raman image for cluster analysis. The digital stripe comprises uniformly distributed pixels containing the reference spectra obtained separately for the pure constituents (actin or myosin). The reference spectra obtained from the Raman instrument are baseline corrected and scaled to the average cellular spectra using the 1444 cm−1 band for actin and 1002 cm−1 band for myosin. PCA was used to identify the different principal components. These principal components were then used to build clusters of the Raman data, followed by mapping. Specifically, we chose principle components PC1 to PC6 for actin and PC1 to PC9 for myosin. Next, the clusters were constructed using the CDP Rodriguez method52 in the Raman Toolset software. We used pure component spectra that were obtained from commercial sources of actin and myosin.Cluster analysis is the method of grouping objects into clusters of similar items to distinguish and segregate a large volume of dissimilar datap value) was verified by one-way ANOVA followed by post hoc Tukey test for multiple comparisons .All the data were expressed as mean ± standard deviation (n = 20), and the statistical significance (Supplementary Information.


Modeling water’s dynamic bond network and accurately predicting physical properties is challenging. As a result, small water clusters are crucial templates to understand hydrogen bonding at a simpler level. Unfortunately, such clusters can only be studied under cryogenic conditions and often with uncontrolled charge or size. Here, we introduce a technique to trap and analyze neutral water clusters in ambient conditions, by encapsulating them in barrel-shaped molecules (cucurbiturils) and measuring them with Raman spectroscopy. We observe a confined water dimer and demonstrate facile tuning of its environment and even isotopic composition, paving the way for probing ambient water cluster dynamics in situations difficult to access in previous experiments. Nanoconfined few-molecule water clusters are invaluable systems to study fundamental aspects of hydrogen bonding. Unfortunately, most experiments on water clusters must be performed at cryogenic temperatures. Probing water clusters in noncryogenic systems is however crucial to understand the behavior of confined water in atmospheric or biological settings, but such systems usually require either complex synthesis and/or introduce many confounding external bonds to the clusters. Here, we show that combining Raman spectroscopy with the molecular nanocapsule cucurbituril is a powerful technique to sequester and analyze water clusters in ambient conditions. We observe sharp peaks in vibrational spectra arising from a single rigid confined water dimer. The high resolution and rich information in these vibrational spectra allow us to track specific isotopic exchanges inside the water dimer, verified with density-functional theory and kinetic population modeling. We showcase the versatility of such molecular nanocapsules by tracking water cluster vibrations through systematic changes in confinement size, in temperatures up to 120° C, and in their chemical environment. Whilng bonds , 19, 21.rocesses –25. Meano theory –28.−1 (OD at −1) but can downshift by as much as 600 cm−1 upon hydrogen bonding, when electron densities are pulled away from the OH (OD) bond , allowing us to probe an isolated hydrogen bond in ambient conditions. We connect our findings to density functional theory (DFT) and rate equation models that track H–D isotope exchange in these dimers. Finally, we highlight the versatility of this system and how it enables new experiments on ambient water clusters, demonstrating control of the degree of confinement, the effects of temperature, and how bonding interactions of such confined water clusters can be modified.Raman spectroscopy is a powerful tool for probing hydrogen bonding in water . Gas-pha1 OD at ∼,700 cm−12O or D2O dries  and 830 cm−1  (−1 (orange shaded) (−1 (blue shaded)  , and OH  shaded) . The CB4contents , while O2O experiment shows broad OD peaks of bulk D2O and comparatively smaller OH peaks from HDO , the integrated signals of both bands strongly decrease as the bulk water evaporates  characteristic of discrete water clusters . These broad bands are similar to those observed in previous reports on water complexation with CB[n]  with CB[5] expected to only incorporate two water molecules. This means that stage III water@CB[5] signals likely arise from discrete water dimers: (H2O)2 or isotopologues thereof. We propose (below) that this sequestered water dimer is in a rigid state as a result of the highly restrictive space in the CB[5] cavity  2@CB[5] is the first to analyze the vibrational spectra of the confined dimer in depth. We note that few works discuss Raman spectra of water clusters rather than infrared spectra (Previous experiments and theory agree that different sizes of CB electronegative carbonyls while also acting as a hydrogen-bond acceptor from a ‘cavity water’ sequestered inside the CB[5].DFT is used extensively to model free water clusters, and inclusion of water clusters in CB has been shown to only slightly perturb their geometry . Therefo2O)2@CB[5] are calculated , yielding 12 unique isotopologues. This produces a wide fan of modes across the OD and OH bands. A labeling scheme is employed where underlining labels the cavity water, e.g., DOH-ODD represents a DOH cavity water hydrogen bound (through its H atom) to a D2O portal water. The relative peak heights and orderings from these frequency calculations can be simply explained as a perturbation of the carbonyl binding onto free water dimer modes  using a mass-spring model and DFT . Further supporting the validity of this model, the DFT calculations produce water-to-CB[n] peak ratios of the same order as observed in experiment .To explain the D→H isotopic exchange dynamics observed, the expected vibrational frequencies for all deuterium isotopologue variants of , no deuterium exists in the system, so only HOH-OHH can be observed. The observed experimental spectrum of a strong central peak with two weak side peaks matches well with the DFT frequency calculation. Therefore, we assign the strong observed peak to the OHβ vibration and the low-intensity side peaks to the OHɑ and OHδ vibrations. These bands are fit  to OH spectra from five different locations on the sample , resulting in peak centers and FWHMs reported in SI Appendix, Table S2. The OH vibrations of (H2O)2@CB[5] observed are at lower frequencies  than in gas-phase molecular beam experiments , indicating that in general, hydrogen bonding is stronger for the CB-confined dimer than for the free dimer. This strong redshift is expected because the calculated O–O distances in the CB-confined dimer are much closer to those of ice than those of free water dimers . The extra hydrogen bonds between portal water and CB carbonyls mean that the vibrational frequencies of the CB-confined dimer overlap more closely with previously observed spectra for the water trimer (H2O)3 . The bands measured here are also broader, which could arise from inhomogeneities or the increased temperature relative to cryogenic experiments .To match with experimental data, the DFT modes are split into four frequency bands: ɑ–δ for both OD and OH  Throughout the D2O experiment, 4 narrow peaks are seen in the OD region but only 3 narrow peaks in the OH region. This suggests that observed isotopologues have more deuterium than hydrogen. Hence, we assign peaks ɑ, β, ɣ, and δ inside the OD band, but only assign peaks ɑ, β, and ɣ inside the OH band. These peaks are fit  throughout the time of the experiment ; fitting results are reported in SI Appendix, Table S3. 2) The ODβ peak disappears faster relative to the other OD peaks ODɑ, ODɣ, and ODδ. 3) Initially, the two OH peaks OHβ and OHɣ have roughly equal intensity, but after isotope exchange, the OHβ peak has higher intensity.Three key observations help match the DFT bands to the D2O experiment is that throughout the isotopic exchange, the states with DOD cavity water dominate the system and exchange occurs at the portal, i.e., the dominant states are DOD-ODD , DOD-OHD, and DOD-OHH . This hypothesis explains 1) because these states have more deuterium than hydrogen. It also explains 2) because ODβ only appears for DOD-ODD, which is quickly removed from the system during the transition DOD-ODD DOD-ODH. It explains 3) because OHɣ only appears for DOD-OHD, while OHβ only appears for DOD-OHH. Thus, under this model, the replacement of DOD-OHD by DOD-OHH will result in increasing OHβ peak intensity relative to that of OHɣ.The simplest hypothesis explaining all three observations in the D2O experiment reveals that the CB[5] cavity protects the cavity water from isotopic exchange, while the portal water is free to exchange isotopes with its surroundings. The CB[5] thus acts as a proton guard, gated by the portal water, and allows no access to the cavity water. This model both explains why a month of immersion in D2O is needed to deuterate the cavity with initial H2O and also explains experiments where D2O was incubated in CB[5] solution for shorter times. Incubating CB[5] in D2O for only 1 week or less yields spectra showing features of nonfully deuterated cavity water . The alignment of this model to experiments suggests that the dimer stays attached to one set of carbonyl portals without migrating to the other carbonyl portal because otherwise the portal water and cavity water would exchange roles. This is reasonable given the strong portal water-cavity water hydrogen bond  and the potential barrier required for the portal water to leave the electronegative environment near its carbonyl portals. The small accumulation of the OHɑ peak in experimental data arises from small fractions of cavity water becoming deuterated, representing a higher-order correction to our simple hypothesis. The modes from the isotopologue (D2O)2@CB[5] are also redshifted relative to previous spectra from free (D2O)2 . For reference, the calculated vibrational frequencies for CB-confined dimers from DFT are compared directly with the experimental spectra in SI Appendix, Fig. S11.The ability of this model to explain the D2O experiment, a simple rate equation model is used  to fit population dynamics to the experimental fitted peak heights of ODɑ, β, ɣ, δ and OHɑ, β, ɣ . For all locations, SI Appendix, Fig. S15B). While −2 min−1, 450 peak can also be fitted using the kinetic model in a consistent way across all four sample locations . This verifies that the isotopic water exchange and movement of the CB450 peak are correlated processes.Fitted rate constants differ at different locations of the sample (which correspond to slightly different solid-state structures) but all display qualitatively similar behavior and support the same kinetic model for isotopic exchange . Moreover, there are nonmonotonic changes in the spectral components representing networked vs. free water as n increases from 6 to 8 . Overall, the general agreement between experiment and prediction indicates that water clusters within the four major CB[n] homologues can be isolated and analyzed using Raman spectroscopy, enabling further studies on cluster dynamics for different numbers of water molecules.A diverse range of fundamental studies on discrete water clusters, many of them difficult to access in existing systems, are enabled using cucurbituril nano-encapsulants. Three examples are provided here. First, the cluster size dependence: Various sizes of the CB[2O)2@CB[5]  shift and broaden with heating, ultimately disappearing completely at 120° C. This shows that superheated solid water dimers can be studied using our method, though further experiments and simulation will be required to fully elucidate the dynamics and energy barriers of these water dimers in the CB environment. The OHɑ, OHβ, and OHδ peaks are fit over the temperature sweep. The strong central OHβ peak shows the most consistent trends across all measured sample locations, while OHɑ and OHδ peak fits are noisy due to weaker signal 2@CB[5] C shows tr signal D.β to shift to higher frequencies. In this regime, the linewidth is unchanged. Hence, watching the temperature-dependent vibrational shift up to 80° C is a clear way to controllably decouple water molecules from their immediate environment and slowly weaken hydrogen bonds. It would clearly be interesting to tune the dimer temperature from 80° C to below room temperature to observe the sharp transition from weak hydrogen bonding to moderate/strong hydrogen bonding, where the potential becomes rapidly anharmonic and vibrational frequency rapidly decreases  . Moreover, the number of observed peaks decreases, suggesting that the original dimer becomes a monomer in the presence of Fe3+. While the D2O@CB[5] without Fe3+ shows D 2O@CB[5] capped with Fe3+ exhibits narrower OD bands that persist over several hours in ambient atmospheres . No isotope exchange was observed over >10 h showing that the confined dimer isotopic labeling can be protected even in hydrogen-rich environments. In addition, using larger CB[n] homologues also allows dynamics between host–guest chemistry and confined water clusters to be studied, as shown for the inclusion of ferrocene, which binds strongly into the CB[7] cavity interior and displaces cavity water . By engineering the CB[n] environment, it is thus possible to tune water cluster chemical interactions with their surroundings, which opens many possibilities and suggests that switchable systems can also be envisaged. Future work will explore the kinetics and thermodynamics of such capping with different sizes of CB[n] and with different additional guests in the cavity.Thirdly, chemical manipulation: CB from aqueous solutions in a low-humidity environment allows water clusters to be studied using Raman spectroscopy, with narrow OD/OH bands observed for CB[5]. For these water@CB[5] clusters, analysis through DFT and kinetic modeling of isotopic exchange strongly suggests that the bands originate from a cavity-confined water dimer. This dimer consists of a ‘portal water’ which is hydrogen-bound to one CB[5] portal, while the ‘cavity water’ lies inside the CB[5] cavity. CB[5] cavity encapsulation slightly perturbs the free water dimer geometry, resulting in a compressed donor-acceptor hydrogen bond length in calculations and redshifted OH stretches in experiments. Our results suggest that the CB[5] cavity walls shield the inner cavity water, while the portal water readily interacts with its environment. The readily observed single-hydrogen bond connecting them is thus available for study. This system hence isolates a single hydrogen-bonded cavity water molecule protected by the CB[5] walls, while allowing for vibrational measurements at room temperature. Our observation of a CB[5]-confined water dimer in a ‘dry’ system provides an intriguing contrast to NMR results  are prepared and allowed to rest for 1 mo to ensure full isotope exchange of the cavity water in CB[5] . For the CB[7]-ferrocene inclusion experiments, CB[7] solution is mixed with ferrocene  in a 1:1 molar ratio and sonicated for several hours. For the Fe3+ experiments, CB[5] is mixed with FeCl3  in a 1:2 CB:Fe molar ratio in deionized water and sonicated.For Dn] were modeled with the 6-31+G basis set that can well-describe geometries and energies of simple water clusters with well-defined hydrogen bonds .The Gaussian09 computational chemistry program was used to model water clusters through initial geometry optimization and then frequency calculations. Free water clusters were modeled with the B3LYP functional with 6-311++G basis set, while water dimers inside CB[en bonds  (optimizAppendix 01 (PDF)Click here for additional data file.