Studies of Membrane Biophysics Using Fluorescence Correlation Spectroscopy

Date of Award

Summer 8-15-2013

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Computational & Molecular Biophysics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

This work is dedicated to developing theories and applications of techniques related to Fluorescence Correlation Spectroscopy (FCS) to study membrane biophysics. One of the theories presented in this work is inverse FCS, which provides a way to study the diffusion rate, size and number of dark particles immersed in a high concentration of fluorescent probes. The size determination using inverse FCS can be diffusion-independent. By using inverse FCS, we found two different domain growth pathways in DLPC/DSPC membranes. In one of these two pathways, nanoscopic domains appear at first and then gradually grow to micron size. In the other pathway, the domains reach micron size quickly and their number gradually increases.

The second theory we have developed is an FCS theory for closed systems for both periodic and reflective boundaries. We demonstrate that this theory can be applied to the study the diffusion in the nanotube membranes by providing accurate diffusion coefficients. The developed methodology is also useful in the single molecule studies.

We also used FCS and fluorescence microscopy to investigate the composition heterogeneity in GUV (giant unilamellar vesicle) membranes made by electroformation. We found that there is a large composition heterogeneity among GUVs and this heterogeneity is caused by a phase separation in the depositing step in the preparation. This heterogeneity can be reduced by a pre-heating (annealing) treatment after the deposition.

Finally, by using FCS and fluorescence microscopy, we found two different gel phases in DLPC/DSPC GUVs. These two gel phases have different affinities for a variety of fluorescent lipid probes. We also found the gel domains in the GUVs are preserved after the GUVs rupture on a cover glass. Our AFM (atomic force microscopy) studies on the ruptured GUVs indicate that the two different gel phases behave differently during the rupture.

Language

English (en)

Chair and Committee

Elliot L Elson

Committee Members

Paul H Schlesinger, Guy M Genin, Carl Frieden, Jin-Yu Shao, Linda J Pike

Comments

Permanent URL: https://doi.org/10.7936/K77D2S2Q

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