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Translational Diffusion of Fluorescent Proteins by Molecular Fourier Imaging Correlation Spectroscopy

Michael C. Fink , Kenneth V. Adair , Marina G. Guenza ^*  and Andrew H. Marcus  

^* Institute of Theoretical Sciences, Department of Chemistry, University of Oregon, Eugene, Oregon; and Oregon Center for Optics, Eugene, Oregon

Correspondence: Address reprint requests to A. H. Marcus, E-mail: ahmarcus{at}uoregon.edu.

The ability to noninvasively observe translational diffusion of proteins and protein complexes is important to many biophysical problems. We report high signal/noise (250) measurements of the translational diffusion in viscous solution of the fluorescent protein, DsRed. This is carried out using a new technique: molecular Fourier imaging correlation spectroscopy (M-FICS). M-FICS is an interferometric method that detects a collective Fourier component of the fluctuating density of a small population of fluorescent molecules, and provides information about the distribution of molecular diffusivities. A theoretical analysis is presented that expresses the detected signal fluctuations in terms of the relevant time-correlation functions for molecular translational diffusion. Furthermore, the role played by optical orientational degrees of freedom is established. We report Fickian self-diffusion of the DsRed tetramer at short timescales. The long-time deviation of our data from Fickian behavior is used to determine the variance of the distribution of the protein self-diffusion coefficient. We compare our results to the expected outcomes for 1), a bi-disperse distribution of protein species, and 2), dynamic disorder of the host solvent.