In addition to the peaks observed along the diagonal, off-diagonal features are present in the 2DIR spectra that increase in intensity with a longer delay time. The intersections of dashed lines along the off-diagonal direction in Figure show the approximate positions of the cross peaks. The peak shape can be qualitatively described by lineshapes that span the frequency region around (ω pump , ω probe ) = (1630, 1650) cm -1 in all samples. Off-diagonal peaks are weak at short pump-probe delays but become more pronounced at later delays. These off-diagonal features, or cross-peaks, result from the energy redistribution among the backbone vibrational modes . For example, if a cross peak is observed at the cross of two modes (ω pump and ω probe ), energy flows from one mode at ω pump to the other mode at ω probe . At an early delay time, coupling between the amide I modes of the strong and weaker bands at 1650 and 1630 cm -1 shows weak cross-peaks in all samples, whereas at longer delay times, the energy transfer further increases the cross-peak amplitudes. In addition, off-diagonal features around (ω pump , ω probe ) = (1650, 1675) cm -1 are increasingly prominent in strongly bundled networks. Following the same logic, these cross-peaks can be attributed to the coupling of amide I modes between the main band at 1650 cm -1 and the increased shoulder band in the 1675 cm -1 region of the strongly bundled networks. All findings in the lineshape of the 2D IR spectra are likely to indicate three secondary structures along the backbone of actin biopolymers that undergo structural changes under depletion-driven morphological control.