Examples of 2D IR spectra and lineshape analysis for POPS:DMPC at a 1:9 molar ratio are depicted in Figure . The left panel displays the 2D IR spectra in the ester carbonyl region at early (0.15 ps) and late (4.0 ps) waiting times. The signal is centered at approximately 1740 cm -1 , consistent with the FTIR spectrum (Figure ). The broad band along the diagonal originates from the two hydrogen-bonding species (0HB and 1HB), as described previously (Figure ), with 1HB red-shifted by approximately 15 cm -1 . The evolution of the lineshapes as a function of waiting time (t2) is used to extract the dynamics of the local environments around lipid ester carbonyl groups. In Figure , we provide an example of 2D lineshape analysis using the center line slope (CLS) method . In brief, CLS is the slope of the line connecting the maxima of peaks, which are a series of cuts through the 2D spectrum parallel to the pump frequency axis.