Harvard University Department of Physics

Super-resolution Optical Imaging
Optical microscopy is one of the most widely used imaging methods in biophysical and biomedical research. However, the spatial resolution of far-field optical microscopy, classically limited by the diffraction of light to ~300 nm, is substantially larger than typical molecular length scales in cells, leaving many biological problems beyond the reach of light microscopy. To overcome this limit, we have developed a new form of high resolution light microscopy, stochastic optical reconstruction microscopy (STORM). STORM uses photo-switchable fluorescent probes to temporally separate the otherwise spatially overlapping images of individual molecules, allowing the construction of high-resolution images. Using this concept, we have achieved three-dimensional, multicolor fluorescence imaging of molecular complexes, cells and tissues with ~20 nm lateral and ~50 nm axial resolutions. We are advancing STORM capabilities to ultimately enable real-time imaging of cells and tissues with resolution at the true molecular length scale. This new form of fluorescence microscopy allows molecular-interactions in cells and cell-cell interactions in tissues to be imaged at the nanometer scale. We are applying the STORM technology to cell biology and neurobiology.

Nucleic Acid – Protein Interaction
Many essential cellular reactions, such as DNA replication, transcription, messenger RNA editing, and protein synthesis, involve DNA-protein or RNA-protein complexes. Understanding nucleic acid-protein interactions is thus crucial for deciphering the molecular mechanisms underlying these central biological processes. We are using single-molecule fluorescence imaging to visualize the assembly process of these molecular complexes and the dynamic interactions between DNA, RNA and proteins within these complexes in real time. These experiments allow us to reveal transient states and multiple kinetic paths that are difficult to detect by classical ensemble experiments, to directly determine the relation between structural dynamics of these molecular complexes and their function, and thus to provide mechanistic understandings of these biomolecular processes. Using this approach, we are studying the assembly, the catalytic cycle, and the structure-function relation of nucleic acid interacting enzymes, such as telomerase and HIV reverse transcriptase.

Virus-cell Interactions
Viruses must deliver their genome into cells to initiate infection. This process is referred to as viral entry, a subject of fundamental importance as well as a therapeutic target for viral disease treatment. However, understanding viral entry mechanisms is challenging because of the involvement of multiple entry pathways and multiple steps on the pathway, each featuring dynamic interactions of the viruses with different cellular structures. What could then be a better way to study virus trafficking than taking a ride with the virus particle on its journey into the cell? To realize this goal, we have developed real-time imaging methods to track individual virus particles in live cells. This approach allows us to follow the fate of individual viruses, to dissect the entry pathways into microscopic steps, and to determine the molecular mechanism of each step. Using this approach, we are investigating the entry mechanisms of influenza virus and poliovirus, as well as related cellular trafficking pathways. Our research also extends to the assembly and budding mechanisms of viruses.