Vibrational spectroscopy is a powerful technique to measure conformational ensembles and local environments in complex membranes as vibrational frequencies are highly sensitive to molecular structure, molecular interactions, and local environments. The main challenge in using vibrational spectroscopy lies in the interpretation of spectra within heterogeneous biological mixtures, as overlapping peaks produce ultra-congested lineshapes that obscure many features. As a result, at present model bilayers such as small unilamellar vesicles, which topologically resemble the most fundamental structures of cell membranes, are commonly used as model systems for membrane spectroscopy . Lipid molecules contain several intrinsic vibrational probes that report on the interfacial interactions and other local environments (Figure ). The O-H stretch of water is a useful vibrational probe given its ability to probe the extended hydrogen-bond networks in water . Furthermore, both the phospholipid headgroups and alkyls can serve as sensitive vibrational probes reporting on specific regions of the bilayer. For example, the phosphate P-O stretching modes are useful for investigating the headgroup hydration, orientations, and lipid-lipid interactions . Interpreting vibrational lineshapes for phosphate groups can be challenging since the region is congested by the partial overlap among symmetric PO2 -, asymmetric PO2 -, and R-O-P-O-R' stretching modes. Additionally, the ester carbonyls are excellent reporters with wellknown characteristics for interfacial hydration, interfacial dynamics, and lipid-protein interactions . The amide groups of proteins are also strong IR probes for protein structure and local environments . Given the rich molecular information derived from vibrational spectroscopy, many IR-based techniques including time-resolved methods have been used to investigate membranes and membrane proteins. Carbon, oxygen, nitrogen, and phosphate atoms are colored as gray, red, blue, and orange, respectively. Interfacial water molecules are shown in translucent blue. The approximate frequency of each probe is shown below the corresponding chemical structure. Bottom: FTIR spectrum of a dried 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid film measured under ambient conditions. The shaded areas indicate the frequency regions of the phosphate stretching mode (green), -OH bending mode (blue), amide I stretching mode (blue), ester carbonyl stretching mode (purple), -CH2-stretching mode (yellow), and -OH stretching mode (brown). The horizontal axis is frequency in cm -1 , and the vertical axis represents the IR absorbance in arbitrary units. The absorbances across different regions are scaled for clarity.