Two-dimensional bond-selective fluorescence spectroscopy: violations of the resonance condition, vibrational cooling rate dispersion, and super-multiplex imaging

Abstract

Multidimensional spectroscopies have shaped our understanding of molecular phenomena, but they are often limited in sensitivity. In this work, we describe two-dimensional bond-selective fluorescence-detected infrared-excited (2D-BonFIRE) spectro-microscopy: an ultrasensitive two-dimensional spectroscopy and hyperspectral imaging technique. 2D-BonFIRE spectra are richly detailed, allowing for direct measurement of vibronic coupling and strong evidence of combination modes in congested spectral regions. Additionally, 2D-BonFIRE provides new insights into the nature of vibrational relaxation, including direct experimental observation of vibrational cooling rate dispersion, illuminating the inherent heterogeneity of vibrational decays in large molecules. Finally, we demonstrate that the high specificity of 2D-BonFIRE allows for single-shot 16-colour chemical imaging, with high promise for further palette expansion. 2D-BonFIRE holds significant potential as a tool for fundamental photophysics and a basis for super-multiplex bioimaging.

Fig. 1. Overview of 2D-BonFIRE. (a) Principle of BonFIRE, comprising two narrowband excitations. Vibronic states are labelled |S^v_n〉, where n describes the electronic state and v describes the vibrational state. Vibrational states are labelled with an apostrophe (') in the S₁ manifold. (b) Principle of 2D-BonFIRE, where narrowband pulses are tuned across broad frequency ranges. (c) Picosecond-scanning methodology of 2D-BonFIRE. 2D spectra are constructed from a series of 1D sweeps, where BonFIRE intensity is measured by the peak height in a sweep of the time delay (t_D) between the IR and probe pulses.

Fig. 2. 2D-BonFIRE frequency-domain spectroscopy. (a) 2D-BonFIRE fingerprint spectrum of Rh800 at 100 μM in DMSO-d₆. (b) 2D-BonFIRE intensity as a function of ω_IR at ω_probe = 12 820 cm⁻¹ (Rh800 structure inset). 2D-BonFIRE aligns well with the FTIR spectrum (grey, 100 mM in DMSO) and the predicted IR absorption by DFT (purple; scaled by 0.975). We note that the absence of certain peaks in BonFIRE compared to FTIR is due to solvent interference from DMSO and dye aggregation due to the high concentrations needed for FTIR, as we have discussed previously. (c) Relative 2D-BonFIRE intensity as a function of ω_probe for Rh800 at ω_IR = 1300 (pink), 1440 (red), 1500 (orange), 1550 (yellow) and 1590 (green) cm⁻¹. Because these signals vary over orders of magnitude, the spectra have been scaled to facilitate a visual comparison. Vertical dashed line positions are defined relative to the UV-vis absorption maximum. (d) 2D-BonFIRE of Rh800 in the nitrile and CH-stretching regions at ω_probe = 12 120 cm⁻¹. Due to strong anharmonicity, the frequency of the nitrile-stretch is over-estimated in harmonic DFT. The CH-stretching absorptions predicted by DFT (purple; scaled by 0.975) disagree with the FTIR (grey) and 2D-BonFIRE (blue) spectra. (e) Scaled 2D-BonFIRE ω_probe-dependence of Rh800 for the high-frequency modes of Rh800. Vertical dashed line positions are defined relative to the UV-vis absorption maximum. The spectra with ω_IR > 2600 cm⁻¹ are displaced by a different energy than ω_IR, in violation of the resonance condition. (f) Summary of 2D-BonFIRE frequency-domain data for all measured modes in this work (also see Table S2). The resonance condition is well-maintained for the fingerprint modes and nitriles, but the peaks in the CH-stretching region consistently violate the resonance condition. As detailed below, these modes appear to be combination modes involving the simultaneous excitation of two fingerprint modes, where one mode is FC-coupled and the other is not. In such a case, only the FC-coupled mode should contribute to double-resonance.

Fig. 3. 2D-BonFIRE time-domain spectroscopy. (a) Representative normalized time-domain spectra of major modes of Rh800. (b) Normalized time-domain spectra of the nitrile-stretch of ATTO725 at ω_IR = 2230 cm⁻¹ and varying ω_probe. (c) ω_probe-dependence of the nitrile lifetimes of Rh800 (blue) and ATTO725 (green) at ω_IR = 2230 cm⁻¹. The nitrile lifetimes are constant, characteristic of a local oscillator mode. (d) Normalized time-domain spectra of ATTO725 at ω_IR = 1590 cm⁻¹ and varying ω_probe. (e–g) ω_probe-dependence of (e) τ₂ (dashed line at 4.5 ps), (f) τ₁ (dashed line at 1.2 ps), and (g) A₁/A₂ (trendline is a sigmoidal global fit) for ATTO665, ATTO680, and ATTO725 at ω_IR = 1590 cm⁻¹. (h) Rh800 time-domain BonFIRE at ω_IR = 1500 cm⁻¹ and ω_probe = 12 950 cm⁻¹. Judging by the oscillating residuals of the fit (obs.–calc.; bottom panel), the observed decay is not described by a pure biexponential (red solid line; residual oscillations highlighted by black arrows). Modelling VC rate dispersion with a KWW function (blue dashed line) yields improved fitting. (i) β as a function of probing energy for ω_IR = 1500 cm⁻¹ (trendline is a sigmoidal fit). β transitions from its initial value of ∼0.5 to ∼1 as ω_probe increases.

Fig. 4. 16-colour chemical imaging by 2D-BonFIRE. (a) Structures of BonFIRE nitrile dyes for super-multiplex imaging (R = (CH₂)₃CO₂⁻). Dye scaffolds are colour-coded by electronic absorption and labelled with their absorption maxima, and individual dyes are labelled by their nitrile IR frequency in cm⁻¹. (b) Lifetime-weighted IR reference spectra for BF dyes. (c) Probe reference spectra for ¹³C¹⁵N BF dyes (other isotopologues shown in Fig. S16). (d) Stitched BonFIRE images of labelled PS films acquired with varying ω_IR and ω_probe (contrast scaled for each ω_probe for visual comparison). (e) Unmixed 16-colour BonFIRE image of labelled PS films by adding conditional logic to the least absolute shrinkage and selection operator (CL + LASSO). Unmixed component images are provided for reference in Fig. S18.

Fig. 5. Vibrational lifetime multiplex imaging. (a) BonFIRE image of overlapping BF2227 and BF2231 films at ω_IR = 2229 cm⁻¹ and ω_probe = 11 930 cm⁻¹ (white dashed lines mark the approximate edges of the films). (b) Vibrational lifetime image of overlapping BF2227 and BF2231 films (low-intensity regions are masked). Film 2 exhibits systematically larger vibrational lifetimes. (c) Overlay of reference temporal profiles of BF2227 (dark grey solid curve) and BF2231 (light grey dashed curve) and temporal profiles of films 1 (turquoise dots) and 2 (purple circles) in the regions of interest marked in panel (a). The temporal profiles of films 1 and 2 align with the solution spectra of BF2231 and BF2227, respectively.