The second challenge is membrane asymmetry. Specialized lipids are often enriched in microdomains and unevenly distributed across leaflets. For example, phosphatidylserine (PS) is mainly distributed in the inner leaflet of the plasma membrane in healthy eukaryotic cells due to the activity of transporter proteins. Therefore, characterizing PS distribution is crucial for understanding the functions of lipids in cellular homeostasis and membrane trafficking. Sumfrequency generation spectroscopy could provide a direct measure of net-orientation, and thus measure enrichment across the leaflets. However, challenges would certainly arise in future experimental design of in-vivo SFG measurements of plasma membranes. Another challenge, as discussed above, is the large diversity of lipid species present in cell membranes. While simple lipid compositions have been extensively investigated, building more complex or realistic lipid membranes remains challenging. Vibrational spectroscopy provides ensemble-averaged results across all oscillators, irrespective of lipid species, and isotope-labeling, e.g., 13 C (41, 47, 143, 144), or functional-group-labeling, e.g., nitrile or thiocyanate groups (145, 146), can help identify the contribution of each species individually. MD simulations can also help interpret complex spectra.