Two-Photon-Excited Fluorescence-Encoded Infrared Spectroscopy

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Two-Photon-Excited Fluorescence-Encoded Infrared Spectroscopy Joseph Nathanael Mastron, and Andrei Tokmakoff J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.6b09158 • Publication Date (Web): 01 Nov 2016 Downloaded from http://pubs.acs.org on November 2, 2016

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The Journal of Physical Chemistry

Two-Photon-Excited Fluorescence-Encoded Infrared Spectroscopy

Joseph N. Mastron and Andrei Tokmakoff*

Department of Chemistry, the James Franck Institute, and the Institute for Biophysical Dynamics University of Chicago, Chicago, IL 60637 *Corresponding Author: [email protected], (773) 834-7696

Abstract: We report on a method for performing ultrafast infrared (IR) vibrational spectroscopy using fluorescence detection. Vibrational dynamics on the ground electronic state driven by femtosecond mid-infrared pulses are detected by changes in fluorescence amplitude resulting from modulation of a two-photon visible transition by nuclear motion. We examine a series of coumarin dyes and study the signals as a function of solvent and excitation pulse parameters. The measured signal characterizes the relaxation of vibrational populations and coherences, but yields different information than conventional IR transient absorption measurements. These differences result from the manner in which the ground- state dynamics are projected by the two-photon detection step. Extensions of this method can be adapted for a variety of increased-sensitivity IR measurements.

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I. Introduction: Although ultrafast infrared (IR) vibrational spectroscopy has the time resolution and structural sensitivity to explore the ultrafast structural dynamics of complex molecular systems, it is also limited by its low sensitivity when studying solutions and biophysical systems. IR transitions, often with an extinction coefficient on the order of 100 M-1 cm-1 or less, are commonly investigated at 0.1–5.0 molar concentrations in solution, and rarely under 10-4 M, for biological macromolecules whose physiological concentrations are μM–pM. Besides weak absorption cross sections and lack of radiative emission channels, experimental sensitivity is limited by low band gap IR detectors that are noisier and less sensitive than those in the visible range, and require liquid nitrogen cooling. This sensitivity limitation has driven interest in developing new types of high-sensitivity IR spectroscopy.

In gas-phase experiments, several strategies have been implemented to perform highsensitivity IR spectroscopy by measuring the influence of IR light fields on more sensitive experimental observables, for instance through photoacoustic and ion dip detection techniques1–5. However, application of these techniques to solution-phase measurements is difficult, if not fundamentally infeasible. One technique that yields promise in solution encodes IR vibrational excitations on the ground electronic state of a system onto an excited electronic population through a nuclear-dependent electronic transition, and then measures the resulting visible fluorescence as the experimental observable. Herein, we refer to the process of upconverting a ground-state vibrational excitation into an electronic fluorescence observable as “fluorescence encoding”.


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Fluorescence-encoded IR (FEIR) vibrational spectroscopy was originally investigated by Kaiser and co-workers in the 1970s as part of the emerging field of picosecond IR spectroscopy6–9, but few investigations have followed up on this concept using modern ultrafast laser technology10–14. More recently, fluorescence encoding has also been used as a strategy for super-resolution IR vibrational imaging15,16. Although a growing number of coherent mixed electronic–vibrational spectroscopies are currently being used to investigate coupled electronic–nuclear dynamics17,18, the motivation for our studies is vibrational spectroscopy on the ground electronic state19. As an analogue to fluorescence-detected coherent electronic spectroscopies20–25, this technique also presents a similar possibility of detecting Fourier transform coherent multidimensional spectroscopy through fluorescence.

Figure 1. TPE-FEIR experimental principle. Left: In absence of nuclear excitation, the NIR twophoton absorption pulse (A) is off resonance with the electronic transition, resulting in the emission of little or no fluorescence (B). Right: With the addition of a mid-IR vibrational excitation pulse (C), the two-photon absorption pulse (D) is on resonance with the electronic transition, resulting in an increase of the fluorescence intensity (E).

This work explores fluorescence-encoding through electronic two-photon absorption (TPA)26. Two-photon-excited fluorescence with a femtosecond near-IR (NIR) pulse was chosen to increase detection contrast, since the excitation wavelength can be easily filtered from the



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fluorescence emission27. The experimental principle of two-photon-excited fluorescenceencoded infrared (TPE-FEIR) spectroscopy is shown in Fig. 1. The NIR encoding pulse is chosen to be at a frequency less than half of the electronic transition frequency, such that the twophoton transition between the ground and first electronic state is off resonance at the nuclear ground state equilibrium (Fig. 1A), and on resonance upon nuclear excitation (Fig. 1D). Short mid-IR pulses vibrationally excite the transitions that are resonant within the pulse bandwidth (Fig. 1C). Following a time delay, the NIR light excites a two-photon absorption ending in an excited electronic state (Fig. 1D). The electronic excitation then relaxes and radiatively decays incoherently as fluorescence (Fig. 1B,E). The experimental observable is the increase in fluorescence intensity from the sample that results from the increase of the TPA transition resonance due to the ground-state vibrational excitation of vibronically-coupled modes.

In order to lay the groundwork for the expected incoherently-detected fluorescence signal, the steady-state vibrational and electronic spectra for the samples of interest will be characterized. Then, to test the experimental predictions, the simplest case will be presented: a single IR excitation pulse followed by a single TPA pulse. The resulting delay-time-dependent fluorescence signal is found to be sensitive to dynamics of the vibrational excitation, and shows contributions from both vibrational populations and coherences. The signal is characterized as a function of experimental variables such as pulse intensity and polarization, electronic transition resonance, and solvent. These observations allow us to describe several physical properties governing the experimental observable and discuss future extensions to more complex pulse sequences.


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II. Materials and Methods: A. TPE-FEIR NIR ultrafast pulses (93 fs FWHM by autocorrelation, 800 nm, horizontally polarized, 1 kHz) from a regenerative amplifier system (Libra, Coherent Inc) were used (Fig. 2) to pump a twostage optical parametric amplifier (OPA) with difference frequency generation (DFG) mixer attachment (TOPAS-DFG, Light Conversion). Signal and idler from the first stage at 1430 and 1900 nm were mixed to generate the mid-IR pulses at 1735 cm-1 with full-width at halfmaximum (FWHM) of 200 cm-1. The IR was then resized and collimated with a reflective telescope (Tel1) in the Z geometry, and the residual signal and idler were removed with a coated Ge bandpass filter (F1). The IR was chopped (Ch) at 500 Hz and sent through a polarizer pair (Pol) to set and control the IR intensity at the sample. A small amount of the NIR was picked off reflectively before the TOPAS, sent through a fixed delay line to match the IR path length and attenuated with a gradated filter (Thorlabs NDL-25C-4). The NIR was then sent into a precision delay line (ANT95-L-25 stage with A3200 motion composer, Aerotech) with a retroreflecting cube mirror to control the IR–NIR detection time delay (τd). After telescoping the NIR with a CaF2 lens pair to match the IR focus (Tel2), the beams were overlapped on a 2 mm Ge Brewster plate (Ge), to transmit the p-polarized IR and reflect the NIR. IR (5 μJ) and NIR (1 μJ) were focused into the sample with a 90° silver parabolic mirror (Par1) to spot sizes of

Two-Photon-Excited Fluorescence-Encoded Infrared Spectroscopy

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