Martin T. Zanni

Action-detected two-dimensional electronic spectroscopy (A-2DES) has emerged as a powerful alternative to coherent 2DES, offering unique advantages, including freedom from geometrical constraints imposed by directional phase-matching requirements, lower demands on sample quality, operando capability, and seamless integration with microscopy. By measuring incoherent observables, such as fluorescence, photocurrent, photoelectrons, or photoions from excited-state populations, action detection enables studies of systems that might be difficult to access using traditional coherent methods. However, this approach introduces fundamental challenges, including incoherent mixing, additional Liouville pathways, and detection nonlinearities that require careful consideration. This article provides the theoretical foundations, experimental implementations, key advantages, and critical limitations of action-detected spectroscopy, providing guidance for optimal application of this rapidly evolving technique across diverse systems ranging from single molecules to working photovoltaic devices.

Incoherent population mixing creates a large background in ultrafast, action-detected pump–probe and 2D spectroscopies. This background often obscures the desired coherent responses and leads to incorrect assignment of spectral features. In a previous study, we postulated three classes of incoherent population mixing but could only measure one. In this work, we report spectra collected using a two-quantum (2Q) pulse sequence generated by a fully phase stable spectrometer, enabling spectra containing all three classes of incoherent responses to be measured. We test the postulated formalism describing incoherent population mixing by collecting and simulating spectra measured using a 2Q pulse sequence. A 2Q pulse sequence reported here also provides a means of determining the magnitude of these backgrounds relative to the desired coherent response. Using this technique, we obtain results consistent with the existence of three incoherent population mixing responses, reveal their spectroscopic signatures, and establish a formalism for their description, enabling future methods for their identification and elimination.

Oligomeric peptides are implicated in amyloidosis, yet only a few atomic structures of amyloid oligomers have been identified. Recently, we reported an atomic structural model of human islet amylin polypeptide (hIAPP) oligomer [Shivani, S. T.; et al.Proc. Natl. Acad. Sci. U.S.A.2026, 123, e2528103123.]. The model was generated by trapping the oligomers with three alanine substitutions in a sequence termed 3A-hIAPP. These substitutions neither impact the physiologically critical β-sheet region of hIAPP nor the cytotoxicity and oligomer size measured through biochemical assays. However, their effect on the structure of the N-terminal helices could not be assessed. By modifying our 2D IR spectrometer, we interwove the acquisition of multiple pulse sequences, enabling waiting-time-dependent spectra of hIAPP oligomers to be measured during protein aggregation. Spectra acquired at a waiting time of 1.2 ps are decongested of disordered structures, revealing features originating from the oligomeric N-terminal α-helix and the adjacent kink and disulfide-loop regions. The spectra of hIAPP oligomers closely match those of 3A-hIAPP, strengthening the structural relevance of our atomic model for hIAPP oligomers. These findings also establish a workflow for testing and generating atomic structures of hIAPP oligomers associated with early-onset type 2 diabetes and for oligomers from other amyloid diseases.

Action detected two-dimensional spectroscopy interrogates molecular systems and materials via the response of the system to the stimuli of the pulse sequence rather than measuring its optical response. Recent experiments revealed that, in addition to the coherent signal, action 2D spectra contain incoherent population mixing signals, which are created when two independent linear responses are mixed during the detection time. Disentangling coherent and incoherent contributions is essential for maximizing the informational content and interpretability of action 2D spectroscopy. We use simulations to propose a set of polarizations that selectively suppress the coherent signal and expose spectral signatures of multi-exciton processes occurring during the detection time. We showed that rephasing and non-rephasing spectra contain different incoherent responses. In addition, we showed that for molecular aggregates with efficient exciton–exciton annihilation, the ⟨0°, 60°, 120°, 30°⟩ polarization isolates electronic coherences, while ⟨0°, 30°, 90°, 150°⟩ or ⟨0°, 45°, 135°, 90°⟩ polarizations can be used to study energy transfer dynamics, both of which are otherwise obscured by incoherent signals. Our simulations provide a practical strategy for experimentally isolating incoherent contributions in action-detected 2D spectroscopies.

Many conclusions about energy conversion in next-generation photovoltaic devices are gleaned indirectly from optical measurements of exciton dynamics, not directly from photocurrent itself. This method is problematic because optical measurements report on all excitons, not just productive ones. Using a new ultrafast photocurrent spectrometer, we compare exciton dynamics of semiconducting carbon nanotubes measured in films to those measured in devices using photoabsorption- and photocurrent-detected transient and two-dimensional spectroscopies. We find that photoabsorption detection greatly overestimates the importance of long-lived excitons for photovoltaic device performance. Excitons diffuse across nanotubes for picoseconds, but we find that the photocurrent is mostly created by excitons that diffuse little before dissociating at the electron transfer interface within 30 femtoseconds of being created. Thus, scientific conclusions reached from optical-only studies bear little importance to the performance of these devices, calling into question the processes thought critical for efficient photoconversion. This study points to the necessity for directly measuring photocurrent-generating exciton dynamics, not surmising them from optical spectroscopy alone.

Some mammals develop amyloid plaques and type 2 diabetes, much like humans, depending on the sequence of their islet amyloid polypeptide (IAPP). In humans, IAPP forms a toxic oligomer with a parallel β-sheet across residues ²³ FGAIL ²⁸ S. Using two-dimensional infrared spectroscopy, we monitor the structure of IAPP from seven different mammals, five of which are from species that can develop type 2 diabetes (ferret, raccoon, cat, baboon, and human) and three from those that do not (hamster, rat, and pig). G24 is isotope labeled to monitor for the presence of the oligomeric β-sheet previously found in human IAPP. For the species that develop type 2 diabetes, their IAPP is cytotoxic, and a β-sheet at G24 is observed during the lag phase prior to fibril formation. In contrast, the species that do not develop type 2 diabetes have nontoxic IAPP, and their IAPP does not form this β-sheet structure. Pig IAPP forms oligomers, but with a different structure that is nontoxic. Thus, an oligomer with a parallel β-sheet at G24 that resembles that of the known human IAPP oligomer correlates with cytotoxicity and propensity for type 2 diabetes. These results indicate that the sequence within the 20 to 29 region of human islet amyloid polypeptide (hIAPP), long known to correlate with type 2 diabetes in mammals, determines the structure and toxicity of an oligomer, supporting the oligomer hypothesis for type 2 diabetes and providing an explanation other than plaque formation for why some mammals develop insulin deficiency in late-stage type 2 diabetes, and others do not.

Amyloid oligomers of the human islet amyloid polypeptide (hIAPP) are a likely cytotoxic species driving β-cell death in type 2 diabetes, but their transient nature has precluded atomic-level structural characterization. We obtained a high-resolution structure of a physiologically relevant hIAPP oligomer. Using 2D IR spectroscopy, we identified three substitutions that slowed aggregation sufficiently for comprehensive 2D/3D NMR analysis while retaining the key wild-type structural features and cytotoxicity. The structural model reveals a dimeric assembly with N-terminal helices and a kink that facilitates an intermolecular β-sheet. The β-sheet spans the famous FGAILS portion of the sequence, helping to explain species-specific diabetes susceptibility and the origin of early-onset familial mutations. The integrated 2D IR/NMR strategy provides a unique approach to obtaining high-resolution structures of amyloid oligomers.

Little is known about structural dynamics at electrode surfaces under applied potential. Here, we report 2D-IR spectra of a 4-mercaptobenzonitrile monolayer on a gold electrode, revealing chemical exchange between two subensembles on sub- to tens-of-picoseconds time scales. Dynamics are measured in 100 mM MgCl₂, LiCl, and KCl at -200 and +300 mV vs Ag/AgCl. At +300 mV, all electrolytes exhibited ≥48 ps exchange, while at -200 mV, the slower exchange component ranged from ≥33 to ≥43 ps. The dynamics correlate with ion concentration, according to radial distribution functions calculated from molecular dynamics simulations, suggesting that local ion densities, regardless of valency or sign, slow dynamics. Results show solvation, electric double layer formation, and monolayer reorientation. This work reveals how electrolyte composition modulates molecular reorientation and hydrogen bonding at functionalized electrodes.

Heterogeneous catalysis involves the chemical transformation of molecules at an electrode surface, but experimentally probing the impact of the electric field on the structural dynamics of the molecules and any accompanying solvent dynamics remains a significant challenge. In this study, we conducted molecular dynamics simulations of a system comprising pure water in contact with gold (Au) electrodes coated with 4-mercaptobenzonitrile (4-MBN) molecules, as recently measured using 2D IR spectroscopy (Ryan, M. J. Proc. Nat. Acad. Sci. U.S.A. 2023, 120, e2314998120, 10.1073/pnas.2314998120). Our simulations revealed that the 4-MBN molecules can adopt two distinct conformations with different tilt angles and that the kinetics of switching between the two conformations depends on both the applied voltage and the packing of the monolayer. There is an accompanying change in solvation upon conformational switching. The results illustrate that the application of a voltage to an electrode alters both the solvation and molecular dynamics at the interface, which has implications for heterogeneous electrochemistry.