Amide I Infrared Spectra Show Multiple Secondary Structures. The amide I 2D IR spectra of actin samples at two selected pump-probe delay times (t 2 ) in the filament (no PEG), weakly bundled network (PEG 6 kDa, 0.1% w/v), and strongly bundled network (PEG 20 kDa, 0.1% w/v) are shown in Figure . The amide I stretch lineshapes indicate the backbone structure of the actin networks. The changes in the lineshapes as a function of the delay time (pump-probe delay) report on the local fluctuations and energy redistribution rates among the delocalized amide I modes along the backbone. The positive peaks (shown in red) correspond to the ground state bleach and involve a vibrational transition from the ground to the first excited state, while the negative peaks (shown in blue) correspond to the transition from the first to the second excited states. Two selected delay times (t 2 ) are included for clarity. Spectra measured at early delay times, such as 0.15 ps, show diagonally elongated peaks due to the high correlation between the pump (ω pump ) and probe (ω probe ) frequencies. The shape of the peaks becomes rounder at later delays due to the gradual decorrelation between the pump and probe frequencies, which results from the frequency fluctuations of individual amide groups, referred to as spectral diffusion, or energy redistribution within the backbone modes. Background noise features become increasingly prominent at longer delay times because of the overall vibrational relaxation of the amide I modes, which further degrades the already-weak 2D IR signals at the low actin concentrations used for all samples.