PhD oral, Wednesday April 15

Title:
Fluorescence interferometry applied to cell membrane model systems

Prasad Ganesan
Advisor: Steven Boxer
Department of Chemistry

2 pm, Wednesday, April 15
Chemistry Gazebo

Abstract:
Fluorescence interference contrast microscopy (FLIC) is an experimentally straightforward means for determining the position of fluorescent objects in one dimension with nanometer accuracy. It is therefore a useful method for studying properties of fluorescent objects in supported phospholipid bilayers, a common cell membrane model system. Unfortunately, in its conventional form there are limits on the kinds of systems and questions that can be probed using FLIC. To address this issue, extensions to existing interferometry approaches have been developed to be applicable to a wider range of problems than those than can be investigated with laterally homogeneous supported phospholipid bilayers.

One extension takes the form of a new imaging technique that allows the extraction of distance information for fluorescent objects that are not laterally homogeneous. In variable incidence angle fluorescence interference contrast microscopy (VIA-FLIC), a fluorescent sample is assembled above a reflective silicon interface and the incidence angle of excitation light is varied by placing annular photomasks with different radii in the aperture diaphragm plane of the microscope. Constructive and destructive interference occur near the reflective interface, and varying the incidence angle alters the interference pattern, and hence the intensity of detected fluorescence. By collecting a series of images of a single fluorescent object, an intensity profile as a function of angle of incidence can be constructed, and this profile is characteristic of a specific distance between the fluorophore and the interface.

A second extension is the development of a model membrane system that can be probed using interferometry techniques, while also positioning the phospholipid bilayer hundreds of nanometers from the substrate surface. This separation distance is sufficient that cell membrane proteins conceivably could be incorporated into this system without the surface interaction problems typically observed for proteins in supported phospholipid bilayers. Although many challenges remain to be addressed, the architecture of this system raises the possibility of studying protein conformational dynamics using fluorescence.