Fluorescence-Encoded Infrared Spectroscopy: Ultrafast Vibrational

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Spectroscopy and Photochemistry; General Theory

Fluorescence Encoded Infrared Spectroscopy: Ultrafast Vibrational Spectroscopy on Small Ensembles of Molecules in Solution Lukas Whaley-Mayda, Samuel B. Penwell, and Andrei Tokmakoff J. Phys. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 03 Apr 2019 Downloaded from http://pubs.acs.org on April 3, 2019

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The Journal of Physical Chemistry Letters

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Fluorescence Encoded Infrared Spectroscopy: Ultrafast Vibrational Spectroscopy on Small Ensembles of Molecules in Solution Lukas Whaley-Mayda‡, Samuel B. Penwell‡, and Andrei Tokmakoff* Department of Chemistry, James Franck Institute, and Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois 60637, United States Corresponding Author * E-mail: [email protected]

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2 ABSTRACT

Fluorescence encoded infrared (FEIR) spectroscopy is an ultrafast technique that uses a visible pulse to up-convert information about IR-driven vibrations into a fluorescent electronic population. Here we present an updated experimental approach to FEIR that achieves high sensitivity through confocal microscopy, high repetition-rate excitation, and single photon counting. We demonstrate the sensitivity of our experiment by measuring ultrafast vibrational transients and Fourier transform spectra of increasingly dilute solutions of a coumarin dye. We collect high quality data at 40 μM (~2 orders of magnitude below the limit for conventional IR), and make measurements down to the 10-100 nM range (~5 orders of magnitude) before background signals become overwhelming. At 10 nM we measure the average number of molecules in the focal volume to be ~20 using fluorescence correlation spectroscopy. This level of sensitivity opens up the possibility of performing fluctuation correlation vibrational spectroscopy or – with further improvement – single-molecule measurements.

TOC GRAPHIC

KEYWORDS Multidimensional spectroscopy, Fourier transform spectroscopy, photon counting, high sensitivity, high repetition-rate.


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3 Time-resolved infrared (IR) vibrational spectroscopies present a structurally sensitive approach to studying molecular dynamics in the condensed phase. A large body of traditional and ultrafast methods that track vibrational dynamics across multiple dimensions in time and frequency have been developed to help resolve the molecular details of complex chemical and biophysical problems.1-7 However, IR techniques are typically limited to highly concentrated systems due to low absorption cross-sections, limitations in mid-IR detector technology, and short vibrational lifetimes that preclude luminescence. Besides restricting the variety of systems that may be studied, this limitation fundamentally constrains the manner in which dynamics experiments are performed to ensemble-averaged ‘pump-probe’ measurements that use a perturbation to trigger or synchronize the molecules and lose information as their trajectories diverge. On the other hand, single-molecule experiments have the unique ability to observe the distinct behavior of individuals rather than highly averaged information. This allows dynamics to be studied for different members within an inhomogeneous ensemble, or directly through the continuous trajectory from a given molecule at equilibrium. For example, IR experiments utilizing scattering-scanning near-field optical microscopy with femtosecond pulses have revealed the distinct character of ultrafast fluctuations in small sub-ensembles of vibrational oscillators,8 while techniques based on surface- and tip-enhanced Raman scattering have been able to track the realtime evolution of vibrational spectra from single molecules on the timescale of seconds.9-11 The time resolution of this latter class of dynamics measurements is often limited by the averaging time required to resolve the weak single-molecule signal. However, in single-molecule fluorescence spectroscopy methods based on time correlation or statistical analysis have been developed that are only limited by the shortest interval between photon arrivals in real time.12-17 While such



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4 fluorescence-based approaches have been successfully applied to studying dynamics in solution, they lack the structural sensitivity for investigations at the level of chemical structure. Strategies to enhance the sensitivity of IR techniques often operate by coupling vibrational information to more sensitive observables.18-20 Action spectroscopy using fluorescence emission as the detected observable has seen widespread use in femtosecond and multidimensional visible spectroscopies21-24, which have recently been performed as sensitive microscopy25-26 and singlemolecule27-28 experiments. Fluorescence detection was applied to time-resolved IR spectroscopy by the pioneering work of Laubereau et al29, who anticipated its sensitivity potentials despite predating the first IR picosecond transient absorption experiments. Recently our group has adapted this fluorescence detection approach, which we call fluorescence encoded IR (FEIR) spectroscopy, to experiments using modern femtosecond technology and demonstrated the information content of ultrafast and multidimensional measurements using one and two IR pulses.30-31 Here we present a new approach to FEIR aimed at exploring the sensitivity of the technique.

Figure 1. Concept of the FEIR measurement and diagram of the instrument. (a) Favorable FEIR resonance occurs when the visible encoding frequency (blue) is below the 0-0 electronic transition


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5 but resonant with transitions from IR-excited (red) vibrational states. (b) Fluorescence excitation (black) and emission (dotted gray) spectra of coumarin 6 (structure top right) in acetonitrile with the center frequencies of the visible (blue) and the sum (red) of visible and IR fields overlaid. The detected portion of the fluorescence spectrum is indicated by the dashed black box. (c) Diagram of the FEIR experiment. Visible encoding line and microscope: λ/2 = half waveplate, PBS = polarizing beamsplitter, DBS = dichroic beamsplitter, SPAD = single photon avalanche photodiode. IR line: BS1&2 are 50:50 ZnSe beamsplitters, MCT is a single element roomtemperature photovoltaic HgCdTe detector. Conceptually, the sensitivity advantages of FEIR arise from the background-free nature of fluorescence, meaning that the majority of detected light can be useful signal, coupled with the availability of sensitive detectors for the visible spectrum, allowing for weak signals to be collected on a photon-by-photon basis. Fluorescence encoding works by using an ultrashort visible pulse to convert vibrational excitations into excited electronic population. As shown schematically in Figure 1a, the visible encoding field is tuned below resonance with the transition to a fluorescent electronic excited state when the molecule is in vibrational equilibrium, but in resonance with transitions originating from IR-pumped vibrational excited states. In practice, this resonance condition is achieved when the frequency of the visible field is red-shifted beyond the molecule’s electronic absorption edge, while the sum of the visible and IR frequencies lies within the band, as shown in Figure 1b for our model system, coumarin 6. The presence of a vibrational excitation at the time the encoding pulse is incident on the molecule is therefore detected as an increase in total fluorescence emission, which requires that the IR and visible pulse durations be at least as short as the typically picosecond vibrational lifetimes. Short IR pulses with large spectral bandwidths also allow multiple vibrational bands to be studied simultaneously by Fourier



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6 transform frequency-resolved measurements. A further condition necessary for FEIR signal generation is the presence of coupling between the vibration and electronic transition. In the case of coumarin 6 this may be largely understood in terms of Franck-Condon activity.32-33 The overlap between the visible absorption profile and encoding field alone gives rise to a fluorescence background that competes with the FEIR signal. To achieve high sensitivity it is important to reduce this background, which is most practical for a steep absorption edge where the overlap with the encoding field can be made small while still maintaining some degree of resonance with the sum of IR and visible frequencies. In practice, the sensitivity potential of FEIR is facilitated by the diversity and technical maturity of fluorescence-based microscopy and single-molecule methodology that may be readily incorporated into an FEIR experiment. In particular, our approach is based on adapting a confocal epifluorescence microscope. A high numerical aperture (NA) microscope objective defines a small focal volume where the visible encoding pulse is delivered, and hence where the signal is generated, while subsequently collecting fluorescence with high efficiency. As shown in Figure 1c, the fluorescence is then sent through a pinhole before being refocused onto a single photon counting detector. Passing through the confocal pinhole reduces out of focus fluorescence, thereby restricting the observation volume of the experiment to the region where molecules are most efficiently excited by the visible encoding field.34-35 For single photon counting to be efficient in a pulsed experiment, the repetition-rate should be as high as possible while still allowing the system to fully relax before the next pulse sequence arrives. However, for nonlinear measurements using ultrashort pulses, especially with weak midIR vibrational transitions, pulse energies must also be high enough to efficiently drive population into excited states. We strike a balance between high repetition-rate and pulse energy by employing


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7 a 1 MHz Yb fiber amplified system to pump our experiment. Femtosecond IR pulses are generated by a home-built OPA36 and delivered to the sample from below with a high NA aspheric lens. The tight focusing of the IR beam guarantees sufficiently high peak field intensities at the sample. However, the correspondingly large average power densities, especially considering the high repetition-rate, can result in thermal loads that produce artifacts in the data (Supporting Information Section 7). For the present work we mitigate thermal effects by using a solvent (deuterated acetonitrile) with high transparency across the IR pulse spectrum. Due to the order of magnitude mismatch in diffraction-limited sizes of the IR and visible beams, the microscope is aligned to place the visible focal volume inside the most intense region of the much larger IR focus (Figure 1c).

Figure 2. FEIR experiments on coumarin 6 in deuterated acetonitrile at 40 μM concentration. (a) 1-pulse experiment with the amplitude of the background S0 and signal S1 at τvis = 0 indicated by blue arrows. The inset shows the background-subtracted S1 signal on a log scale. (b) 2-pulse experiment in the time domain at zero encoding delay. The τIR = 0 amplitudes of the 2-pulse signal S12, 1-pulse signals S1 and S2, and background S0 are approximately indicated by blue arrows. The maximum modulation ratio (at τIR = 0) is M = 3.3. (c) Real part of the 2-pulse signal in the frequency domain (FT FEIR spectrum). (d) Comparison of the FTIR and IR pump-normalized FT



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8 FEIR spectra. The concentration for the FTIR was at the saturation limit of ~3 mM, and the maximum OD at the 1526 cm-1 ring mode is 0.04 (50 μm pathlength). The FTIR is normalized to match the brightest FEIR feature at 1586 cm-1. The carbonyl stretch is not visible in the FT FEIR spectrum due to the lack of intensity in the IR field, shown in gray. The simplest FEIR measurement, which we term the 1-pulse experiment, uses one IR pulse followed by the visible encoding pulse after a variable delay, τvis (Figure 2a). When the encoding pulse precedes the IR (τvis < 0), there is a small baseline amplitude of background fluorescence, indicated by S0, due to weak excitation of the red wing of the molecule’s absorption profile, as well as other undesirable background contributions including solvent scattering and autofluorescence. When the IR pulse is incident first (τvis ≥ 0), there is an increase in fluorescence, indicated by S1, which relaxes back to the baseline with a fast and slow component of 1 and 10 picoseconds, respectively (fitting shown in Supporting Information Section 5). At the simplest level, this transient signal may be interpreted as tracking the relaxation of vibrational modes initially excited by the IR pulse, similar to a conventional IR pump-probe experiment.32,

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Formally, the FEIR signal S1 may be described by calculating the excited electronic population after two resonant interactions with the IR field and two with the visible.30-31 This description is supported by the linear power dependencies measured for each field (Supporting Information Figure S4). When the bandwidth of the IR pulse spans more than one vibrational resonance, coherent contributions can appear in the transient which oscillate at their respective difference frequencies.30 In our measurement the IR field excites the four highest frequency ring deformation modes of the molecule (Figure 2d), however coherent oscillations are not observed. This is likely due in combination to the dominance in overall transition strength of a single mode, fast dephasing


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9 from strong interactions with the polar solvent, and the length of the pulse cross-correlation (Supporting Information Section 1). It is convenient to quantify the relative strength of the FEIR signal by defining the modulation ratio, 𝑀 = 𝑆1/𝑆0, that is, the proportional increase in fluorescence amplitude when the IR field is present. For example, at the peak response in Figure 2a, 𝑀 = 7.4. When the modulation ratio is large it is possible to acquire high quality data by continuously and rapidly scanning the optical delay stage and correlating photon arrivals to stage positions on the fly (Supporting Information Section 6). Numbers of photons in a given delay bin are then converted to an amplitude (count rate) by dividing by the time elapsed during stage travel. A photon is either detected after a given pulse sequence or not, so the maximum possible count rate saturates at the laser repetition rate of 1 MHz. The ability to record transient signals faster than the decorrelation time of slow laser intensity fluctuations has been shown to drastically increase signal to noise ratios.38-40 We employ this fast scanning approach in all FEIR measurements shown here. Using an IR pulse-pair we may resolve the vibrational excitation frequency with a Fourier transform (FT) measurement, in analogy to resolving the pump frequency axis in pump-probe geometry 2D IR spectroscopy.41-43 Figure 2b displays the total fluorescence signal for this measurement, which we term the 2-pulse FEIR experiment, with the encoding delay fixed at τvis = 0 while scanning the IR delay (τIR > 0 when IR 1 comes before IR 2 as in Figure 1c). The desired signal contribution, S12, is due to one interaction with each IR pulse and oscillates at IR optical frequencies in τIR. It may be isolated by an FT to the frequency domain (Figure 2c), where the two 1-pulse signals, S1 and S2, manifest as the DC component (not shown). The background fluorescence, S0, may be estimated by the total amplitude at the location of the first π out-of-phase fringe of the oscillating component, as here the IR fields exhibit nearly complete destructive



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10 interference in the sample. We can therefore define the modulation ratio for the 2-pulse FEIR experiment as 𝑀 = (𝑆1 + 𝑆2 + 𝑆12)/𝑆0. Similar to 2D IR measurements, we monitor the IR intensity in the dark channel of the Mach-Zehnder interferometer (Figure 1c) for phase referencing of the FT data in order to produce absorptive spectra and discard the improperly time-ordered response (Supporting Information Section 6). The FT FEIR spectrum at zero encoding delay (Figure 2c) may be compared to a conventional linear IR absorption (FTIR) spectrum after normalizing its amplitude by the IR pulse spectrum (Figure 2d). The four features at 1483, 1526, 1586, and 1617 cm-1 in the FTIR have previously been assigned to the high frequency ring deformation modes of the dye, while the absorption at 1712 cm-1 is of predominantly carbonyl stretch character.32, 44 The frequencies and linewidths of the features in the FT FEIR spectrum appear similar to the FTIR, however their relative amplitudes are dramatically reweighted. This may be understood as reflecting the differing vibronic coupling strength of each mode to the electronic transition. It is possible to conduct ultrafast relaxation experiments by tracking the evolution of the FT FEIR spectrum in the encoding delay. The coherent contributions that appear as oscillations in the 1-pulse transients manifest as features that undergo periodic phase modulation between absorptive and dispersive lineshapes as a function of the encoding delay. These ‘phase-twisted’ coherence features sit on top of the absorptive population features and can be isolated by performing a second FT along this dimension.31


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Figure 3. Low concentration FEIR measurements and average molecule number characterization. (a) FT FEIR spectrum (red) collected at 50 nM, with the IR pulse spectrum (gray) overlaid. The FT FEIR spectrum has not been amplitude normalized by the spectrum of the IR field. The maximum count rate for this measurement was ~20 kHz. (b) 1-pulse FEIR transient at 10 nM. The acquisition time for both (a-b) of these FEIR measurements was ~30 minutes. (c) Fluorescence correlation functions 𝐺(𝑡) (open circles) of identically prepared samples of rhodamine 6G from 1 to 50 nM with fits (solid lines). (d) Inverse early time amplitude, i.e. 〈𝑁〉 (open circles), from the fits in (c) plotted versus nominal concentration, with a linear fit (black). The FEIR measurements shown in Figure 2 were performed at a concentration of 40 μM, which, while being roughly two orders of magnitude lower than that typically required for conventional IR pump-probe and 2D measurements, is a comfortably high concentration for our instrument. To demonstrate the current sensitivity limits of the FEIR experiment, we repeat the same measurements with three more orders of magnitude in sample dilution. Figures 3a and 3b show the FT FEIR spectrum at 50 nM and the 1-pulse FEIR transient at 10 nM, respectively. As the concentration is lowered, the power of the visible encoding pulse can be increased to keep the total count rate at sufficiently high levels. However, below a certain concentration (typically 0.1 – 1



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12 μM), the background count rate S0 begins to overtake the FEIR signal, leading to a loss in the modulation ratio. For example, in Figure 3a the maximum modulation ratio is 𝑀 = 0.54, and in Figure 3b 𝑀 = 0.09. The onset of this relative increase in background photons represents the current practical sensitivity floor of the measurement. Due to the small ~1 μm3 observation volume of the FEIR experiment, a more relevant metric of sensitivity is the average number of molecules being probed at a given time rather than the bulk concentration. To characterize this average molecule number we use fluorescence correlation spectroscopy (FCS), which measures the autocorrelation function of stochastic fluctuations in the fluorescence emission: 𝐺(𝑡) = 〈𝛿𝐹(0)𝛿𝐹(𝑡)〉 〈𝐹〉2. When fluorescence fluctuations are due solely to the diffusion of molecules through the observation volume (i.e. if triplet blinking and other photophysical processes are negligible), the early time amplitude of the correlation function is dictated by the Poissonian statistics of the molecule occupation number, 𝑁. Explicitly, the relation 〈𝑁〉 = 1 𝐺(𝑡→0) provides a natural definition of the average number of molecules in the observation volume.45 We use a cw visible laser with the same frequency as the FEIR encoding pulse, and perform FCS measurements (Figure 3c) on a series of rhodamine 6G solutions prepared identically to the coumarin 6 solutions and in the same sample cell as the FEIR experiments. The cw beam is passed through the same spatial filter and uses the same optical path in the microscope as the FEIR encoding beam, so the observation volumes should be effectively identical. We keep the excitation intensity low at 1.6 kW/cm2 to avoid creating appreciable triplet fractions that reshape the correlation function at early times.46 In Figure 3d we plot the inverse of the early time amplitude extracted by fitting the correlation functions (Supporting Information Section 4) against the nominal sample concentration. Using the linear trend as a calibration curve, we assign the


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13 average molecule numbers for the FEIR measurement in Figures 3a and 3b to be 〈𝑁〉 ≈ 100 and 〈𝑁〉 ≈ 20, respectively. We have demonstrated that FEIR spectroscopy performed with the standard instrumentation of confocal fluorescence microscopy can yield unprecedented sensitivity to ultrafast vibrational spectroscopy. In particular, we have shown that it is possible to conduct ultrafast time-resolved and Fourier transform vibrational measurements on countably small ensembles of molecules in solution. With this level of sensitivity it is feasible to perform fluctuation correlation experiments that employ vibrational frequencies as a way of tagging molecular structures as they evolve or interconvert at equilibrium. These small ensemble fluctuation experiments represent conceptually straightforward extensions to the conventional FCS measurements shown above, and would provide a structurally sensitive approach to studying chemical kinetics with time resolution given by the inverse of the laser repetition rate. With higher NA fluorescence collection and time-tagged detection of every photon we believe that background levels might be suppressed sufficiently to perform experiments with true single-molecule sensitivity. Possible FEIR experiments that record continuous photon streams from individual molecules could reveal details of their dynamic structural history that are fundamentally inaccessible with ensemble measurements. It is an especially interesting open question to consider if it is possible to incorporate ultrafast information from scanning optical delays with real-time trajectories. EXPERIMENTAL METHODS The mid-IR OPA is pumped by 30 W of the 1033 nm Yb fiber amplifier (Coherent Monaco 1035-40) fundamental, and produces 85 nJ pulses centered at 6.3 μm with 120 cm-1 FWHM bandwidth. The output is sent through a Mach-Zehnder interferometer (MZI) to produce a collinear pulse-pair. The IR pulses are coupled into a modified Bruker Hyperion FTIR microscope and



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14 delivered to the sample with a 0.73 NA ZnSe asphere (ISP Optics ASPH-ZC-25-12). The individual IR pulse energy at the sample is 11 nJ and the spot size is ~10.5 μm FWHM (Supporting Information Section 2), which produces peak field intensities on the order of ~50 GW/cm2. The IR pulse duration at the sample is 250 fs, with a transform limit of 130 fs (Supporting Information Section 1). For the 1-pulse FEIR measurements, the beamsplitters of the MZI were removed and a mirror was placed in the second location (BS2 in Figure 1c), which results in 34 nJ of pulse energy at the sample. The visible encoding pulse is generated by doubling (to 516 nm) a variable amount of the fiber amplifier fundamental, and after travelling through a delay line the beam is expanded with a spatial filter to fill the back-aperture of a 0.8 NA dry objective (Zeiss A-Plan 63x) mounted in the microscope. The visible spot size is ~0.4 μm FWHM (Supporting Information Section 2), and the pulse energy is typically adjusted between ~1 pJ and ~1 nJ based on the sample concentration. The visible pulse duration can be estimated by the rise time of the 1-pulse FEIR signal to be ~315 fs, with a transform limit of >200 fs (Supporting Information Section 1). The visible and IR pulses are set to have parallel polarizations at the sample. A dichroic beamsplitter (Semrock FF535-SDi01) before the objective passes the visible pulse and reflects the fluorescence, which passes through two band rejection filters (Thorlabs NF514-17), and an emission bandpass filter (575DF40). The fluorescence is then focused by a 125 mm tube lens (Thorlabs AC254-125A) through a 35 μm pinhole, and reimaged onto the SPAD (MPD PDM 50) with a 1:1 achromat pair (Thorlabs MAP051919-A). The sample solution is held in a sandwich cell with a #1.5H coverslip (Thorlabs CG15XH) on top, a 1 mm CaF2 window (Crystran) on bottom, and separated by a 50 μm Teflon spacer. All FEIR and FCS measurements were performed with the visible focus 20 μm below the coverslip. FCS measurements used a cw diode laser at 517 nm (Thorlabs CPS520) for excitation (Supporting Information Section 4), and correlation functions were computed by a


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15 custom software correlator written in LabVIEW based on the algorithm of Lee et al.47 Coumarin 6 (Arcos) and rhodamine 6G (Sigma) were used without any further purification. Solutions for FEIR and FCS were prepared by serial dilution in deuterated acetonitrile (Sigma) starting from 1 mM chloroform stock solutions. Fluorescence excitation and emission spectra were collected in a Horiba FluoroLog 3, and FTIR spectra in a Bruker Vertex. Further experimental details are provided in the Supporting Information. ASSOCIATED CONTENT Supporting Information IR pulse duration and temporal instrument response characterization, IR and visible focus characterization, IR and visible power dependencies, further details of sample preparation and FCS measurements, fitting of the 1-pulse FEIR transient, continuous scanning acquisition of FEIR data, and description of thermal artifacts in FEIR data. AUTHOR INFORMATION Corresponding Author A.T.: E-mail: [email protected] ‡ L.W.M. and S.B.P. contributed equally to this work. Notes The authors declare no competing financial interests. ACKNOWLEDGEMENTS We would like to thank Abhirup Guha for assistance in preparing samples and acquiring data, and Nicholas H.C. Lewis for careful reading of the manuscript. This work was supported by a grant from the National Science Foundation (CHE-1561888).



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16 REFERENCES (1) Nibbering, E. T. J.; Fidder, H.; Pines, E., Ultrafast Chemistry: Using Time-Resolved Vibrational Spectroscopy for Interrogation of Structural Dynamics. Annu. Rev. Phys. Chem. 2005, 56, 337-367. DOI:10.1146/annurev.physchem.56.092503.141314 (2) Kolano, C.; Helbing, J.; Kozinski, M.; Sander, W.; Hamm, P., Watching Hydrogen-Bond Dynamics in a β-Turn by Transient Two-Dimensional Infrared Spectroscopy. Nature 2006, 444, 469-472. DOI:10.1038/nature05352 (3) Butler, J. M.; George, M. W.; Schoonover, J. R.; Dattelbaum, D. M.; Meyer, T. J., Application of Transient Infrared and Near Infrared Spectroscopy to Transition Metal Complex Excited

States

and

Intermediates.

Coord.

Chem.

Rev.

2007,

251,

492-514.

DOI:10.1016/j.ccr.2006.12.002 (4) Ganim, Z.; Hoi, S. C.; Smith, A. W.; Deflores, L. P.; Jones, K. C.; Tokmakoff, A., Amide I Two-Dimensional Infrared Spectroscopy of Proteins. Acc. Chem. Res. 2008, 41, 432-441. DOI:10.1021/ar700188n (5) Fayer, M. D., Dynamics of Liquids, Molecules, and Proteins Measured with Ultrafast 2D IR Vibrational Echo Chemical Exchange Spectroscopy. Annu. Rev. Phys. Chem. 2008, 60, 21-38. DOI:10.1146/annurev-physchem-073108-112712 (6) Roberts, S. T.; Ramasesha, K.; Tokmakoff, A., Structural Rearrangements in Water Viewed through Two-Dimensional Infrared Spectroscopy. Acc. Chem. Res. 2009, 42, 1239-1249. DOI:10.1021/ar900088g


Page 17 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


17 (7) Anna, J. M.; Baiz, C. R.; Ross, M. R.; McCanne, R.; Kubarych, K. J., Ultrafast Equilibrium and Non-Equilibrium Chemical Reaction Dynamics Probed with Multidimensional Infrared Spectroscopy. Int. Rev. Phys. Chem. 2012, 31, 367-419. DOI:10.1080/0144235X.2012.716610 (8) Atkin, J. M.; Sass, P. M.; Teichen, P. E.; Eaves, J. D.; Raschke, M. B., Nanoscale Probing of Dynamics in Local Molecular Environments. J. Phys. Chem. Lett. 2015, 6, 4616-4621. DOI:10.1021/acs.jpclett.5b02093 (9) Haran, G., Single-Molecule Raman Spectroscopy: A Probe of Surface Dynamics and Plasmonic Fields. Acc. Chem. Res. 2010, 43, 1135–1143. DOI:10.1021/ar100031v (10) Park, K.-D.; Muller, E. A.; Kravtsov, V.; Sass, P. M.; Dreyer, J.; Atkin, J. M.; Raschke, M. B., Variable-Temperature Tip-Enhanced Raman Spectroscopy of Single-Molecule Fluctuations and Dynamics. Nano Lett. 2016, 16, 479-487. DOI:10.1021/acs.nanolett.5b04135 (11) Zrimsek, A. B.; Chiang, N.; Mattei, M.; Zaleski, S.; McAnally, M. O.; Chapman, C. T.; Henry, A.-I.; Schatz, G. C.; Van Duyne, R. P., Single-Molecule Chemistry with Surface- and TipEnhanced

Raman

Spectroscopy.

Chem.

Rev.

2017,

117,

7583–7613.

DOI:10.1021/acs.chemrev.6b00552 (12) Lippitz, M.; Kulzer, F.; Orrit, M., Statistical Evaluation of Single Nano-Object Fluorescence. ChemPhysChem 2005, 6, 770–789. DOI:10.1002/cphc.200400560 (13) Yang, H.; Xie, X. S., Probing Single-Molecule Dynamics Photon by Photon. J. Chem. Phys. 2002, 117, 10965–10979. DOI:10.1063/1.1521154 (14) Brokmann, X.; Bawendi, M.; Coolen, L.; Hermier, J.-P., Photon-Correlation Fourier Spectroscopy. Opt. Express 2006, 14, 6333–6333. DOI:10.1364/OE.14.006333



Page 18 of 23

18 (15) Wang, Q.; Moerner, W. E., Single-Molecule Motions Enable Direct Visualization of Biomolecular Interactions in Solution. Nat. Methods 2014, 11, 555–558. DOI:10.1038/nmeth.2882 (16) Phelps, C.; Israels, B.; Marsh, M. C.; Von Hippel, P. H.; Marcus, A. H., Using Multiorder Time-Correlation Functions (TCFs) to Elucidate Biomolecular Reaction Pathways from Microsecond Single-Molecule Fluorescence Experiments. J. Phys. Chem. B 2016, 120, 1300313016. DOI:10.1021/acs.jpcb.6b08449 (17) Otosu, T.; Ishii, K.; Tahara, T., Microsecond Protein Dynamics Observed at the SingleMolecule Level. Nat. Commun. 2015, 6, 7685. DOI:10.1038/ncomms8685 (18) Boyle, E. S.; Neff-Mallon, N. A.; Wright, J. C., Triply Resonant Sum Frequency Spectroscopy: Combining Advantages of Resonance Raman and 2D-IR. J. Phys. Chem. A 2013, 117, 12401-12408. DOI:10.1021/jp409377a (19) Burke, N. L.; Redwine, J. G.; Dean, J. C.; McLuckey, S. A.; Zwier, T. S., UV and IR Spectroscopy of Cold Protonated Leucine Enkephalin. Int. J. Mass spectrom. 2015, 378, 196–205. DOI:10.1016/j.ijms.2014.08.012 (20) Winterhalder, M. J.; Zumbusch, a.; Lippitz, M.; Orrit, M., Toward Far-Field Vibrational Spectroscopy of Single Molecules at Room Temperature. J. Phys. Chem. B 2011, 115, 5425-30. DOI:10.1021/jp109652s (21) Scherer, N. F.; Carlson, R. J.; Matro, A.; Du, M.; Ruggiero, A. J.; Romero‐Rochin, V.; Cina, J. A.; Fleming, G. R.; Rice, S. A., Fluorescence‐Detected Wave Packet Interferometry: Time Resolved Molecular Spectroscopy with Sequences of Femtosecond Phase‐Locked Pulses. J. Chem. Phys. 1991, 95, 1487–1511. DOI:10.1063/1.461064


Page 19 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


19 (22) Wagner, W.; Li, C.; Semmlow, J.; Warren, W., Rapid Phase-Cycled Two-Dimensional Optical Spectroscopy in Fluorescence and Transmission Mode. Opt. Express 2005, 13, 3697– 3706. DOI:10.1364/OPEX.13.003697 (23) Tekavec, P. F.; Lott, G. A.; Marcus, A. H., Fluorescence-Detected Two-Dimensional Electronic Coherence Spectroscopy by Acousto-Optic Phase Modulation. J. Chem. Phys. 2007, 127, 214307. DOI:10.1063/1.2800560 (24) De, A. K.; Monahan, D.; Dawlaty, J. M.; Fleming, G. R., Two-Dimensional FluorescenceDetected Coherent Spectroscopy with Absolute Phasing by Confocal Imaging of a Dynamic Grating and 27-Step Phase-Cycling. J. Chem. Phys. 2014, 140, 194201. DOI:10.1063/1.4874697 (25) Goetz, S.; Li, D.; Kolb, V.; Pflaum, J.; Brixner, T., Coherent Two-Dimensional Fluorescence

Micro-Spectroscopy.

Opt.

Express

2018,

26,

3915-3915.

DOI:10.1364/OE.26.003915 (26) Tiwari, V.; Matutes, Y. A.; Gardiner, A. T.; Jansen, T. L. C.; Cogdell, R. J.; Ogilvie, J. P., Spatially-Resolved Fluorescence-Detected Two-Dimensional Electronic Spectroscopy Probes Varying Excitonic Structure in Photosynthetic Bacteria. Nat. Commun. 2018, 9, 4219-4219. DOI:10.1038/s41467-018-06619-x (27) Weigel, A.; Sebesta, A.; Kukura, P., Shaped and Feedback-Controlled Excitation of Single Molecules in the Weak-Field Limit. J. Phys. Chem. Lett. 2015, 6, 4032-4037. DOI:10.1021/acs.jpclett.5b01748



Page 20 of 23

20 (28) Liebel, M.; Toninelli, C.; Van Hulst, N. F., Room-Temperature Ultrafast Nonlinear Spectroscopy of a Single Molecule. Nat. Photonics 2018, 12, 45-49. DOI:10.1038/s41566-0170056-5 (29) Laubereau, A.; Seilmeier, A.; Kaiser, W., A New Technique to Measure Ultrashort Vibrational Relaxation Times in Liquid Systems. Chem. Phys. Lett. 1975, 36, 232-237. (30) Mastron, J. N.; Tokmakoff, A., Two-Photon-Excited Fluorescence-Encoded Infrared Spectroscopy. J. Phys. Chem. A 2016, 120, 9178-9187. DOI:10.1021/acs.jpca.6b09158 (31) Mastron, J. N.; Tokmakoff, A., Fourier Transform Fluorescence-Encoded Infrared Spectroscopy. J. Phys. Chem. A 2018, 122, 554-562. DOI:10.1021/acs.jpca.7b10305 (32) Hübner, H. J.; Wörner, M.; Kaiser, W.; Seilmeier, A., Subpicosecond Vibrational Relaxation of Skeletal Modes in Polyatomic Molecules. Chem. Phys. Lett. 1991, 182, 315-320. DOI:10.1016/0009-2614(91)80221-I (33) Von Cosel, J.; Cerezo, J.; Kern-Michler, D.; Neumann, C.; Van Wilderen, L. J. G. W.; Bredenbeck, J.; Santoro, F.; Burghardt, I., Vibrationally Resolved Electronic Spectra Including Vibrational Pre-Excitation: Theory and Application to VIPER Spectroscopy. J. Chem. Phys. 2017, 147. DOI:10.1063/1.4999455 (34) Rigler, R.; Mets, Ü.; Widengren, J.; Kask, P., Fluorescence Correlation Spectroscopy with High Count Rate and Low Background: Analysis of Translational Diffusion. Eur. Biophys. J. 1993, 22, 169-175. DOI:10.1007/BF00185777


Page 21 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


21 (35) Hess, S. T.; Webb, W. W., Focal Volume Optics and Experimental Artifacts in Confocal Fluorescence Correlation Spectroscopy. Biophys. J. 2002, 83, 2300-2317. DOI:10.1016/S00063495(02)73990-8 (36) Penwell, S. B.; Whaley-Mayda, L.; Tokmakoff, A., Single-Stage MHz Mid-IR OPA Using LiGaS2

and

a

Fiber

Laser

Pump

Source.

Opt.

Lett.

2018,

43,

1363-1363.

DOI:10.1364/OL.43.001363 (37) Dahinten, T.; Baier, J.; Seilmeier, A., Vibrational Energy Transfer Processes in Dye Molecules after Ultrafast Excitation of Skeletal Modes. Chem. Phys. 1998, 232, 239-245. DOI:10.1016/S0301-0104(98)00072-X (38) Helbing, J.; Hamm, P., Compact Implementation of Fourier Transform Two-Dimensional IR Spectroscopy without Phase Ambiguity. J. Opt. Soc. Am. B 2011, 28, 171-171. DOI:10.1364/JOSAB.28.000171 (39) Kearns, N. M.; Mehlenbacher, R. D.; Jones, A. C.; Zanni, M. T., Broadband 2D Electronic Spectrometer Using White Light and Pulse Shaping: Noise and Signal Evaluation at 1 and 100 kHz. Opt. Express 2017, 25, 7869-7869. DOI:10.1364/OE.25.007869 (40) Fox, Z. W.; Blair, T. J.; Weakly, R. B.; Courtney, T. L.; Khalil, M., Implementation of Continuous Fast Scanning Detection in Femtosecond Fourier-Transform Two-Dimensional Vibrational-Electronic Spectroscopy to Decrease Data Acquisition Time. Rev. Sci. Instrum. 2018, 89, 113104-113104. DOI:10.1063/1.5048523



Page 22 of 23

22 (41) Gallagher Faeder, S. M.; Jonas, D. M., Two-Dimensional Electronic Correlation and Relaxation Spectra: Theory and Model Calculations. J. Phys. Chem. A 1999, 103, 10489-10505. DOI:10.1021/jp9925738 (42) DeFlores, L. P.; Nicodemus, R. A.; Tokmakoff, A., Two-Dimensional Fourier Transform Spectroscopy

in

the

Pump-Probe

Geometry.

Opt.

Lett.

2007,

32,

2966-2968.

DOI:10.1364/OL.32.002966 (43) Shim, S.-H.; Zanni, M. T., How to Turn your Pump–Probe Instrument into a Multidimensional Spectrometer: 2D IR and Vis Spectroscopies via Pulse Shaping. Phys. Chem. Chem. Phys. 2009, 11, 748-761. DOI:10.1039/B813817F (44) van Wilderen, L. J. G. W.; Messmer, A. T.; Bredenbeck, J., Mixed IR/Vis Two-Dimensional Spectroscopy: Chemical Exchange beyond the Vibrational Lifetime and Sub-Ensemble Selective Photochemistry. Angew. Chem. Int. Ed. 2014, 53, 2667-2672. DOI:10.1002/anie.201305950 (45) Widengren, J.; Mets, Ü., Conceptual Basis of Fluorescence Correlation Spectroscopy and Related Techniques as Tools in Bioscience. In Single Molecule Detection in Solution, Zander, C.; Enderlein, J.; Keller, R. A., Eds. Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, FRG, 2002; pp 69-120. (46) Widengren, J.; Mets, Ü.; Rigler, R., Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study. J. Phys. Chem. 1995, 99, 13368-13379. DOI:10.1021/j100036a009


Page 23 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


23 (47) Lee, H.-Y.; Lin, H.-Y.; White, J. D.; Fann, W., High-Speed Low-Cost Correlator for Single Molecule Fluorescence Correlation Spectroscopy. Proceedings of SPIE 7185, Single Molecule Spectroscopy and Imaging II 2009, 7185R. DOI:10.1117/12.809426


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