Seed - Advanced Imaging
Jay Groves, Chemistry, University of California, Berkeley
Phase-separated domains on cell membranes, sometimes called lipid rafts, are important in many biological contexts. Groves and Longo are beginning a collaboration to interrogate the shape and mobility of these domains using a combination of techniques. Domains in lipid compositions such as those developed by Longo can be imaged without the need for fluorescent probes using an electrostatic imaging technique recently developed by Groves. This approach relies on measuring the 3D positions of many sedimented colloidal microspheres as they passively scan over a planar substrate (Figure 5). Averaging the vertical positions in the field of view reveals a force map that, when interpreted in terms of an electrostatic model, yields an independent image of local surface charge density. Although the readout is entirely optical, the lateral resolution of this imaging technique is not diffraction limited. The theoretical imaging resolution limits of this inherently statistical technique predict lateral resolutions of 100 nm or better are achievable. Because the readout is electrostatic, protein binding or other compositional changes in phase-separated domain can be studied. This is expected to be complementary to the domain imaging developed by Longo and Boxer.
Figure 5. Surface electrostatic imaging using passively scanning probes. (A) Many colloidal microspheres passively scan across a patterned surface. (B) Microsphere equilibrium height is set by a balance between gravity and local electrostatic forces. The patterned surface has regions of (C) negatively charged silica and (D) 3-aminopropyltriethoxysilane (APTES) which locally increases the surface charge. Covalently attached Alexa Fluor 680 (-R, negatively charged) is used to independently visualize the pattern. (E) 2 µm resolution surface charge density map. (F) Fluorescence micrograph. (G) Overlay.
Additionally, Groves has applied a combination of infrared and fluorescence correlation spectroscopies to characterize lipid membrane phase behavior as a function of protein binding. Surprisingly, cholera toxin, which binds specifically to the headgroup of the liposaccharide GH1, was found to alter the long-range lateral diffusion of fluorescently labeled probe lipids not directly involved in the binding interaction. This effect is amplified near the bulk lipid gel-fluid transition temperature, where changes in long-range diffusion were observed at protein coverage densities as low as 0.02 area fraction. These results, together with spectral shifts of stretching modes in the lipid acyl chain, confirm that protein binding alters the fraction of lipid in the gel-phase.
