New and powerful imaging techniques promise to make major contributions in studying biological processes that are localized in space, dynamic in time, and complex in nature. Our current research is focused on the following two major directions:

 

Next-generation live cell imaging for probing macromolecule dynamics in vivo

        If one could manage to visualize cellular activities unfold and evolve in living cells and organisms with ultimate single-molecule sensitivity, high specificity, millisecond time resolution, and nanometer spatial precision, many important biological problems could be addressed.

        On the physical instrumentation side, unfortunately, all the current fluorescence imaging modalities have limitations. For example, wide-field fluorescence microscopy lacks z- resolution; confocal microscopy suffers from low sensitivity, limited scanning speed, photobleaching and photodamage along the entire optical path; two-photon microscopy is also slow in speed, and exhibits serious nonlinear photobleaching at the focal plane; and total internal reflection fluorescence microscopy can only probe the thin surface. To this end, we are working to invent and develop novel imaging apparatus.

        On the molecular probe side, the recently emerging genetic tags, including optogenetic tools, photoswitchable and photoconvertible fluorescent proteins and hybrid genetic/chemical probes, exhibit unique photo-physical and bio-functional properties. To harness these advances, we are developing new concepts of live cell imaging and to apply them to compelling problems in cell biology and neurobiology. In particular, we are interested in monitoring DNA-protein interaction and protein dynamics in live cells and organisms.

 

Label-free chemical imaging of non-fluorescent  molecules with nonlinear Raman microscopy

        Visualizing the distribution, interaction and dynamics of small molecules, such as metabolites, neurotransmitters and drugs, is obviously important in understanding biological processes. However, this is technically challenging, as most small-molecules are non-fluorescent themselves and are extremely difficult to be labeled with relatively bulky fluorophores. 

                 

        Spontaneous Raman scattering microscopy provides specific vibrational signatures of chemical bonds (or a unique set of vibrational features) of small molecules, but is often hindered by poor sensitivity. Using two synchronized picosecond laser pulse trains at two different colors (pump and Stokes beams), we have recently developed a new multi-photon vibrational imaging technique based on the nonlinear spectroscopy of stimulated Raman scattering (SRS). The detection sensitivity and imaging speed of SRS imaging is orders of magnitude greater than that of spontaneous Raman microscopy, which is achieved by strong stimulated Raman amplification and high-frequency phase-sensitive detection scheme. Moreover, SRS microscopy has a major advantage over previous nonlinear Raman approaches such as CARS imaging in that SRS offers background-free, quantitatively linear and readily interpretable chemical contrast. As a label-free and sensitive imaging modality, SRS microscopy allows mapping of molecular species in 3D and the ability to follow their dynamics in living cells and organisms based on the wealth of Raman spectroscopy.

  

     Many exciting applications are under way. For example, we are now applying label-free SRS microscopy to visualize the content, distribution, composition and dynamics of lipid storage of a transparent model organism, C. elegans. When combined with powerful C.elegans molecular genetics, we hope to understand the genetic regulation of lipid metabolism and, more generally, how energy homeostasis is achieved at the organism's level in the presence of fluctuating environment. Multidisciplinary study along this line could shed light on the molecular basis of metabolic syndrome such as obesity.