| Cellular membranes are functional interfaces that control the flow of information between cells and their environment. It is well established that lateral (x-y) membrane fluidity allows in-plane associations and rearrangements in response to external stimuli, thus resulting in the transduction of signals across the membrane. However, it is also increasing apparent that small perturbations in out-of-plane (z) structure due to membrane bending and the topography of interacting proteins in opposing membranes play an important role in signal transduction.; The relevant size scale (nanometers) and the nature of these soft interfaces pose significant challenges towards probing these structures. The resolution of most optical microscopies is limited by diffraction to hundreds of nanometers, while these interfaces are inaccessible to high-resolution scanning probe microscopies. In the following, we describe three fluorescence microscopy methods tailored towards of the goal being able to detect these nanometer-scale changes at membrane interfaces in real time: variable incidence angle total internal reflection fluorescence microscopy (VIA-TIRFM), an evanescent wave microscopy, and fluorescence interference contrast microscopy (FLIC), and variable incidence angle fluorescence interference contrast microscopy (VIA-FLIC), both interferometric techniques. These methods utilize structured illumination, which is illumination that varies in z, to encode z-position in the observed fluorescence intensity. Modulation of the structured illumination allows the z-position of fluorescent objects to be quantitatively determined. Improvements to the existing techniques allow us to demonstrate resolution on the order of tens of nanometers using VIA-TIRFM and nanometer resolution using FLIC. Both of these techniques have their own advantages and disadvantages. VIA-TIRFM can probe z-structure of localized structures, however alignment issues make it difficult to routinely implement. In contrast, FLIC is a very simple method that is well suited for studying z-position of uniformly labeled fluorescent structures that extend over tens of microns, but typically cannot probe localized structures. To capture the advantages of both techniques, we present a novel imaging technique, VIA-FLIC, which preserves the experimental simplicity of FLIC, but adds the ability to z-image isolated objects like VIA-TIRFM. The developments in VIA-TIRFM, FLIC , and VIA-FLIC presented provide improved methods of examining z-structure at cellular and model membrane interfaces with nanometer resolution. |
