The 2D IR spectra offer insights into the backbone structure and conformational changes in F-actin across three different morphologies. One-dimensional FT IR spectra of these samples are particularly difficult to measure because of the low absorbance and strong background resulting from the dilute concentrations used. As an alternative, we perform a pump slice amplitude (PSA) analysis using 2D IR spectra to obtain features that are comparable to FT IR . In short, PSA analysis projects a 1D spectrum from the 2D spectrum by computing the difference between the maxima and minima of a series of pump-probe slices that are cut through the 2D IR spectrum at each pump frequency. Here, 2D IR spectra at a delay time of 0.15 ps are chosen because, as described above, energy transfer does not significantly contribute to the measured signals at this early delay time. The PSA spectra are used for the direct comparison of amide I bands in different states of actin (Figure ). The amide I spectra can indicate different secondary structures, where α-helix gives rise to a band around 1650 cm -1 , with β -sheet on the lower frequency side and a loop structure on the higher frequency side . Similar peak features can be observed in the spectra of the actin networks (Figure ), where the strongest band at 1660 cm -1 indicates the major structure of α-helix. There is a side band at 1640 cm -1 , indicating the presence of β -sheet structure in the actin networks. Furthermore, a minor feature likely arise from the loop structure is observed, especially for the strongly bundled network sample, as indicated by the rising shoulder around 1680 cm -1 . It is worth noting that the weak peak at 1590 cm -1 corresponds to the asymmetric carboxylate stretches of glutamate and aspartate residues . The analysis of these bands is outside the scope of this study. Further interpretation of the amide I band of the secondary structures of actin networks can provide valuable information about the conformational differences between bundles and filaments.