Research Overview

Our group develops and applies ultrahigh-resolution, ultrahigh-sensitivity fluorescence imaging and spectroscopy tools to solving outstanding problems in biology.

The ability to watch one molecule at a time helps us obtain unique information on distribution functions of relevant observables, resolve subpopulations in heterogeneous samples, and record asynchronous time trajectories of observables that would otherwise be hidden. Single molecule techniques resolve important phenomena that are otherwise “averaged out” by ensemble measurements.

Of particular relevance is our application of single molecule techniques to studying conformational changes of macromolecules. Several of our techniques make use of small, inert labels which are attached to molecules of interest to detect changes in their conformations. For example, dynamic distance changes between two sites on a macromolecule (or between two different molecules) can be measured via single-pair fluorescence resonance energy transfer (spFRET) by following spectral changes in the emission of a single donor-acceptor pair. Orientational changes can be detected via single-molecule fluorescence polarization anisotropy (smFPA) by following changes in the dipole orientation of a rigidly-attached or tethered probe. Having spent years developing and improving these tools, we now actively use them to study protein folding and transcription on the single-molecule level. In addition, we study the folding reaction energy landscape, pathways, conformational distributions and folding intermediates.

Biological Probes

Single-molecule experiments are currently performed predominantly in in-vitro environments. The ultimate challenge is to image cellular substructures, determine the relationships and dynamics of vesicles and organelles, describe existing conformational dynamics and biomolecular interactions, and localization all of the above in-vivo with single molecule sensitivity and nanometer-accuracy. This will allow the study of enzymes and multi-component molecular machines in their natural environment, with the signaling and regulation circuitry all wired-up.

Towards the accomplishment of this tremendously complex task, we are developing colloidal fluorescent semiconductor nanocrystals (a class of quantum dots) for biological labeling. Nanocrystals posses several properties that make them very attractive as fluorescent probes for in-vivo single-molecule experiments: broad excitation spectra, narrow emission spectra, precise-tunable emission peaks, long fluorescence lifetimes, and negligible photobleaching. We are developing organic coatings, bioconjugation schemes, targeting strategies, and unique instrumentation that take advantage of nanocrystals’ spectral properties. Recently, we have shown how these distinctive properties increase the resolution of fluorescence microscopy measurements down to the nanometer level using far-field optics. Multi-color imaging of luminescent nanocrystals.

We have also shown that their long fluorescence lifetime can be used to observe molecules and organelles in live cells without interference from autofluorescence background (a pre-requisite for single molecule detectability). We are currently pursuing applications in genomics (physical mapping, genome rearrangement), vesicle trafficking, cargo selection and in-vivo motility assays of molecular motors.

Conformational dynamics of biopolymers

The ability to watch one molecule at a time helps us obtain unique information on distribution functions of relevant observables, resolve subpopulations in heterogeneous samples, and record asynchronous time trajectories of observables that would otherwise be hidden.

For example, dynamic distance changes between two sites on a macromolecule (or between two different molecules) can be measured via single-pair fluorescence resonance energy transfer (spFRET) by following spectral changes in the emission of a single donor-acceptor pair. Orientational changes can be detected via single-molecule fluorescence polarization anisotropy (smFPA) by following changes in the dipole orientation of a rigidly-attached or tethered probe. Our group developed such tools and uses them to study protein folding and transcription on the single-molecule level. We study the folding reaction energy landscape, pathways, conformational distributions and folding intermediates.

The same tools are used to answer 25-years-old outstanding questions about the mechanisms of DNA transcription by the enzyme RNA polymerase. We are studying the relative motions between the enzyme and its DNA ‘molecular track’, the conformational motions in the enzyme itself, and conformational transitions in the DNA during the various steps of the transcription reaction.