Single Molecule Fluorescence Imaging

Single Molecule Orientation Measurements to Probe Molecular Level Structure in Membranes

The view of biological membranes and their functional role in cellular processes continues to evolve as new approaches are developed to probe these intricate structures.  Biomembranes are composed of a complex matrix of lipids, proteins, sterols, and other species which combine to create highly heterogeneous and dynamic systems.  This often makes it difficult to directly link structural changes with membrane constituents, which has motivated the long historical development of model systems that mimic the natural cellular barrier.  These simplified systems offer a high degree of control over important thermodynamic and compositional parameters and have been essential for both understanding natural membranes and developing new tools for examining biological systems in a controlled manner. 

Fluorescence microscopy is one of the most widely used approaches for probing structural and dynamic attributes of both model and natural membranes.  A wide variety of fluorescent lipid analogs have been developed that readily insert into the macroscopic lipid assembly and often partition into particular domains, thus enabling heterogeneous structural features to be delineated.  This approach has been used extensively to probe specific environments within lipid monolayers and bilayers, characterize phase structure, probe models of lipid rafts, and study the dynamics and fluidity of lipid membranes.  While fluorescence-based analysis of membranes has been extensively developed and utilized, interpretation of the results and comparisons between studies is often complicated.  For example, measured diffusion constants can vary by orders of magnitude, the assignment of dye partitioning within localized domains is often contradictory, and even the same fluorescent probe can alter its domain partitioning preference as a function of the lipid system composition.  This has renewed interest in understanding and controlling how probes insert into their target system.

There has been a considerable effort to design fluorescent probes capable of sensing the deep regions of membranes by positioning the fluorophore within the lipid acyl tails.  For example, 1,6-diphenyl-1,3,5-hexatriene (DPH) has an elongated structure which is expected to insert along lipid acyl tails and has been widely used to probe order in the lipid tail region. However, using anisotropy measurements, Levine et al. has shown that DPH does not consistently insert as expected along lipid acyl tails but also inserts perpendicular to the membrane normal. 

Similar efforts have led to the development of lipid analogs incorporating the BODIPY fluorophore.  BODIPY probes are conceptually attractive for investigating the hydrophobic region of lipid membranes since they are less hydrophilic than other probes and have no net charge.  BODIPY probes also exhibit excellent fluorescent properties with high extinction coefficients, near unity quantum yields, and favorable photostability properties.  In order to examine the incorporation of this fluorophore within the structure of lipid membranes, several studies have examined insertion properties of BODIPY lipid analogs located at incrementally longer regions of the acyl tail.