New Insights into Membrane Structures: Breakthrough in SFG Microscopy
The Department of Physical Chemistry at the Fritz Haber Institute has published a remarkable study in Nature Communications. The paper, titled „Spiral Packing and Chiral Selectivity in Model Membranes Probed by Phase-Resolved Sum-Frequency Generation Microscopy," reports on a breakthrough in molecular imaging and yields unprecedented insight into the structure of biological membranes.
Biological membranes are made up of various chiral phospholipids that form complex molecular arrangements. These patterns are crucial for the membranes' functions in biological processes. However, the detailed structure of these molecular assemblies has been difficult to study due to limitations in existing imaging techniques.
The Nonlinear Interfacial Spectroscopy Group introduces a powerful new imaging method based on vibrational Sum-Frequency Generation (SFG). This tool has allowed them to observe the detailed organization of phospholipid molecules in biological membranes. The study shows that these molecules are arranged in spirals, which are governed by the molecules' chirality. Beyond this, mixtures of different enantiomers (mirror image forms) are found to generate structures which clearly break mirror symmetry. This finding highlights the role of chirality in the formation of these structures and demonstrates their strong enantioselectivity.
This research provides important insights into the molecular structure of biological membranes and sheds light on evolution’s drive towards homochirality in all forms of life. It also opens new pathways for understanding biological processes and related molecular systems by providing a significant advancement in molecular characterization at the microscopic level.
Into the Depth: Scientific Insights
At the end of last century, the lipid raft model was presented which caused significant excitement in the field of membrane research as it was recognized that phospholipid membranes could be highly heterogeneous. Since this structural heterogeneity has profound implications for many membrane-associated cellular functions, such findings spurred a substantial research effort to characterize and understand the effects of different lateral arrangements and in-plane packing structure of membrane constituents, as well as determine any role they may have in physiological processes. Nevertheless, despite decades of research, very little is known about the true nature of lipid rafts, as is clearly emphasized in the vast number of review articles spanning the last 20 years. To make a substantial step forward in our understanding of lipid rafts, a new level of experimental insight is clearly required. In particular, the elucidation of the exact molecular structures formed in such assemblies is overdue.
In this work, the team presents the first study of phospholipid monolayers using their newly developed phase-resolved vibrational sum-frequency generation (SFG) microscope. By analyzing the condensed phase domains in monolayers of mixed chiral lipids, they fully determine the details of the underlying molecular structures. They find that the seemingly symmetric mesoscopic structures (which have circular morphologies) in fact possess long-ranging structural chirality. Interestingly, the domains feature a complex hierarchal structure exhibiting spiraling molecular packing with a clear deviation from mirror symmetry between different enantiomeric mixtures. Furthermore, they show that the resulting domains possess deviating molecular purity, and orientational order. This clear breaking of mirror symmetry is an unequivocal manifestation of enantioselective interactions in the membrane and has implications in many physiological processes which rely on chiral recognition, as well as in understanding why evolution drove towards homochirality in membranes across all forms of life. While other studies employing techniques such as fluorescence and Brewster angle microscopies have characterized many growth and morphological properties of these rafts in model membranes, this is the first study that reveals their exact molecular structure including the distribution of molecular in-plane orientations – a crucial aspect in elucidating their properties.
Equally important to the result of this study is the technological aspect of the development. While there are many previous studies using SFG microscopy, the minute signals that are generated have placed significant restrictions on the types of samples that can be investigated, specifically either much thicker films, or those on metallic substrates where no in-plane information on the molecular orientations can be obtained. Here, they overcome this limitation by employing a revised microscopy geometry in combination with a newly developed imaging system. In consequence, this work represents the first successful vibrational SFG imaging of a molecular monolayer on a non-metallic substrate, which is a mandatory condition to fully exploit its intrinsic strength in the detailed elucidation of molecular structures. As shown in this work, it especially allows for direct studies of the important relation between molecular and structural chirality in molecular assemblies. This achievement therefore opens the door to a new class of structural characterization spanning a wide range of research areas where local heterogeneity can have important effects. This includes molecular self-assemblies such as membranes and surfactants, as well as other material classes e.g., polymeric composites and polycrystalline materials, and even the microscopic study of inorganic 2D materials such as phononic single crystals. The team anticipates that these results will spark a wave of advanced structural investigations in these fields.
