Binary phospholipid bilayers composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-palmitoyl-sn-glycero-3-phosphocholine (DPPC) were studied by Raman spectroscopy and differential scanning calorimetry (DSC). We examined features in Raman scattering spectra that are sensitive to the lipid phase and, therefore, could indicate the phase coexistence. It was found that the low-frequency half-width of half-maximum (LHWHM) of the 2850 cm−1 Raman line, corresponding to the symmetric CH2 stretching vibrations, unequivocally reveals the coexisting phospholipids in ordered and disordered conformational states, which correspond to ordered and disordered phases coexistence, in the DPPC mole concentration range from 0.4 to 0.9. The phase coexistence in this concentration range was supported by the particular concentration behavior of the ratio between the intensities of the 2880 cm−1 antisymmetric CH2 vibration line and the 2850 cm−1 symmetric one. It was also shown that the spectral shape of the 1300 cm−1 Raman line, corresponding to the CH2 twisting vibrations, is a good indicator for the phase state and phase coexistence in the phospholipid bilayers. Comparison with the DSC curves confirmed that in the DPPC mole concentration range from 0.4 to 0.9, the two phase transition peaks are observed in DSC curve, those positions are independent of the DPPC concentration. The outcome of the study is the robust label-free contactless approach for the detection of the lipid phase separation, which can be realized with the micrometer resolution.
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