The micro-viscosity and molecular crowding experienced by specific proteins can regulate their dynamics and function within live cells. Unfortunately, conventional GFP cannot report on the local viscosity, because their chromophores are shielded within the protein β-barrel. Interestingly, we discovered that the bright-dark photoswitching kinetics of the chromophore inside a photochromic fluorescent protein is slowed down by increasing medium viscosity outside the protein, possibly due to that the chromophore’s cis-trans isomerization is accompanied by conformational “breathing” of the β-barrel (Proc. Natl. Acad. Sci. 2012). Thus, this unique genetically encoded micro-viscosity reporter provides protein-specific information about the crowded intracellular environments.  Recently, we extended this idea to photoswitchable organic fluorophores by developing a hybrid genetic-chemical molecular rotor probe whose fluorescence lifetime can report protein-specific micro-environments in live cells (Chem. Commun. 2012). We currently are utilizing these new probes to monitor the progression of protein aggregation in live cells.

Super-nonlinear fluorescence microscopy for high-resolution deep tissue imaging

        It is highly desirable to be able to optically probe biological activities deep inside live organisms. By employing a spatially confined excitation via a nonlinear transition, two-photon fluorescence microscopy has become indispensable for imaging scattering samples. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation intensity.

        To extend the above fundamental imaging-depth limit, we have recently developed a number of super-nonlinear fluorescence microscopy techniques with high-order nonlinearity.  Most notably, stimulated emission reduced fluorescence (Biomed. Opt. Express 2012), multiphoton activation and imaging (MPAI) and multiphoton deactivation and imaging (MPDI) of photo-activatable (Opt. Express 2012) or photo-switchable (Biomed. Opt. Express 2012) probes have been demonstrated on tissue phantoms. Due to the long-lived nature of these switchable states, the incident photons can operate in a sequential manner, and the nonlinearity effect could accumulate (up to sixth order) as the population is being cycled through these states. Conceptually different from conventional multiphoton processes mediated by transient virtual states, our strategy constitutes a new class of fluorescence microscopy with high-order nonlinearity that is mediated by real population transfer (J. Phys. Chem. Lett. 2012). We are now in the process of applying these new techniques on mice brain in vivo.

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 (Curr. Opin. Chem. Biol. 2011).

     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 (Nature Methods 2011). Multidisciplinary study along this line could shed light on the molecular basis of metabolic syndrome such as obesity.