Shani Elbaum demonstrates our single-molecule setup.

Our instrumentation is capable of one- and two-color confocal and surface-based fluorescence techniques. Some of the techniques we use to study protein dynamics include:

• FRET (Forster resonance energy transfer), in which the efficiency of energy transfer between two different dyes serves as a sensitive "molecular ruler" of the distances between them. When applied to individual protein molecules or small complexes diffusing through a confocal volume, FRET can provide very useful information on conformational changes and heterogeneity.
• FCS (fluorescence correlation spectroscopy), in which the autocorrelation decay rates of fluorescent particles diffusing through the confocal volume are used as to determine diffusion rates and hence molecular dimensions. A variant, FCCS (fluorescence cross-correlation spectroscopy) uses two different dyes to study rapid binding and dynamics.
• FFS (fluorescence fluctuation spectroscopy), a family of techniques that includes photon-counting histograms and fluorescence intensity distribution analysis, involves measuring the stoichiometry of protein-protein and protein-vesicle complexes by analyzing the intensity from multiple fluorophores in each complex.
• TIRF-M (total internal reflection fluorescence microscopy), in which an evanescent electromagnetic field generated by total internal reflection is used to excite fluorophores within a few hundred nanometers of a surface. This allows us to selectively monitor particles bound to a surface while eliminating background emission from fluors in solution. We can use TIRF-M to measure binding and dissociation of ligands to proteins or phospholipid membranes, and to monitor diffusion of membrane-associated peptides in vesicles.

Another view of our microscope, showing elements of the confocal and TIRF laser pathways.

Much of our research focuses on the process of amyloid formation in protein misfolding diseases. Amyloid plaques are insoluble, linear protein aggregates associated with many diseases including Alzheimer's, Parkinson's, type II diabetes, Huntington's and kuru. There is emerging evidence that oligomeric intermediates in the fiber formation process are more toxic than mature fibers.

We are interested in characterizing these prefibrillar intermediates, and in understanding how membranes enhance or mitigate their toxicity, using α-synuclein (Parkinson's disease) and islet amyloid polypeptide (type II diabetes) as model systems.

We also study the microtubule-associated protein tau, which forms neurofibrillary tangles in Alzheimer's disease, and γ-synuclein, a neuronal protein of unknown function which has been strongly linked to breast cancer.

Apart from protein misfolding, we are also interested in using single-molecule fluorescence to study drug-metabolizing enzymes, naturally-occuring fluorescent proteins and protein folding mechanisms.