The constant evolution of optical microscopy over the past century has been driven by the desire to improve the spatial resolution and image contrast with the goal to achieve a better characterization of smaller specimens. In particular, the advanced nonlinear optical microscopies have unique advantages over traditional microscopy approaches: intrinsic three-dimensional (3D) imaging with 1μm lateral resolution, reduced photo-damage to tissue sample, decreased photo-bleaching to fluorescent molecule and deep penetration depth with the usage of near-infrared ultrafast lasers. The widely used modalities of nonlinear optical microscopy include two-photon excited fluorescence (TPEF), second harmonic generation (SHG), and coherent anti-stokes Raman scattering (CARS), providing morpho...[ Read more ]

The constant evolution of optical microscopy over the past century has been driven by the desire to improve the spatial resolution and image contrast with the goal to achieve a better characterization of smaller specimens. In particular, the advanced nonlinear optical microscopies have unique advantages over traditional microscopy approaches: intrinsic three-dimensional (3D) imaging with < 1μm lateral resolution, reduced photo-damage to tissue sample, decreased photo-bleaching to fluorescent molecule and deep penetration depth with the usage of near-infrared ultrafast lasers. The widely used modalities of nonlinear optical microscopy include two-photon excited fluorescence (TPEF), second harmonic generation (SHG), and coherent anti-stokes Raman scattering (CARS), providing morphological investigation of biological tissue. Instead of using artificially synthesized fluorescent probes, endogenous fluorescent molecules can be employed to achieve label-free imaging, avoiding any toxic effect induced by the foreign labels. Recently developed spectral and time-resolved fluorescence detection technology further enables monitoring the biochemical functions such as energy metabolism, protein alteration, cellular acidification etc. during the study of various biological processes.

My Ph.D. study focuses on developing new methodologies for the characterization of blood flow in microvasculature and the imaging of immune response. In particular, a new microvascular imaging approach based on the discovery of plasma autofluorescence in zebrafish is first demonstrated. An in vivo imaging flow cytometry using endogenous TPEF and its application in diagnosis of acute sterile inflammation is further discussed. Moreover, by using single short wavelength excitation at 650 nm, we can excite multi-color fluorescence signals of NADH, blood plasma and various fluorescent proteins. The scanning scheme developed in flow cytometry can be extended and applied to resolve the mixing dynamics of micro-flow in droplet-based microfluidic mixer, which has a considerable impact on the field of biomedical diagnostics and drug development, and are extensively applied in the food and chemical industries. Next, for the first time, by utilizing spectral and time-resolved endogenous fluorescence (reduced nicotinamide adenine dinucleotide (NADH), tryptophan and hemoglobin), we can differentiate different types of human blood cells in vitro, including erythrocyte, leukocyte and platelet. Morphological transformation and biochemical alteration such as energy metabolism in the innate immune response, bacterial infection, can be monitored under the cell culture model. To study the immune response in animal model in vivo, we induce tissue injury and bacterial infection in the skeletal muscle layer of zebrafish. Our findings show that time-resolved TPEF of NADH provides rich information to understand the energy metabolism of neutrophils during immune response while SHG of myosin thick filament generate imaging contrast to identify tissue injury. In the study, we also discovered two wild-type strains (DH5α & BL21) of bacteria Escherichia coli (E.coli) emitted distinct two-peak shape fluorescence spectrum under near-infrared excitation (720 nm). This finding enables label-free tracking bacteria (E.coli) in animal in vivo. Finally, a parallel work to monitor heavy metal pollution by using time-resolved chlorophyll fluorescence generated from aquatic algae is discussed.