Molecular assemblies at interfaces are fundamental to natural and industrial applications, with prominent examples including biological membranes and functionalized sensors, where they play key roles in processes like selective transport and signal detection. Understanding their molecular structure, arrangement, and orientation is crucial in biophysics, along with applications in materials science and nanophotonics. While techniques like atomic force microscopy, Brewster angle microscopy, or fluorescence microscopy have provided valuable insights, elucidating molecular conformations and order remains a formidable challenge. Vibrational sum-frequency generation (vSFG) microscopy complements these methods, offering features such as label-free detection and unique sensitivity to molecular order and orientation under ambient conditions. However, existing implementations face limitations, including low signal-to-noise ratios, complex geometries, and challenges in characterizing thin films on dielectric substrates. In this thesis, a newly designed phase-resolved vSFG microscope is presented, which ad- dresses challenges in characterizing molecular assemblies. The system integrates a collinear beam geometry and a reflective objective with a custom-drilled hole for distortion-free imaging at the sub-micron scale. To enhance signal quality, a paired pixel balanced imag- ing technique was developed, achieving a „10-fold improvement in signal-to-noise ratio. Interferometric time-domain scans with femtosecond laser pulses provide phase-resolved vibrational spectra for each pixel, ensuring high spectral resolution. Proof-of-principle measurements on a TEM grid validated its performance, confirming high sensitivity, im- proved signal quality, and the system’s capability for detailed molecular characterization. The microscope was applied to chiral phospholipid monolayers as model systems for biolog- ical membranes. At specific surface pressures, mixed lipid monolayers coexist in condensed and expanded phases, with the condensed lipids forming micron scale domains with nearly circular shape. The SFG microscopy shows that these domains exhibit curved molecular directionality and spiraling mesoscopic organization, providing new insights into their hier- archical structural features. Enantiomeric substitution experiments uncovered deviations from mirror symmetry, providing insights into enantioselective interactions and offering new perspectives on the evolution of homochirality. Furthermore, the visualization and investigation of phase-separated lipid domains enabled the extraction of molecular com- positions and out-of-plane orientational order in both condensed and expanded phases. In conclusion, this thesis work overcomes key limitations of SFG microscopy by various technical achievements mentioned above. The performance of the newly designed phase- resolved vSFG microscope was demonstrated by the study of the molecular packing struc- ture in lipid monolayers, uncovering its potential to study diverse molecular assemblies, paving the way for advancements in interfacial science.
