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Far-field optical imaging methods are essential for precise visualization of the dynamics of biomolecules and nanoparticles because they require no contact and make minimal intrusion to the sample. The research in the Fang Laboratory is aimed to open up new frontiers in chemical and biological discovery through the development and use of a novel optical imaging platform, which provides sub-diffraction-limited spatial resolution, high angular resolution, excellent detectability, and nanometer localization precision for single molecules and nanoparticles in live biological samples and microfluidic devices.
1. 3D Super-Resolution Fluorescence Microscopy: Total internal reflection fluorescence microscopy (TIRFM) has become an indispensable tool to study cellular organization and dynamic processes that occur near the cell culture and glass substrate interface. An automatic calibration and scanning prism-type TIRFM (Figure 1) has been constructed and tested for 10-nm vertical resolution in the Fang Laboratory. This new system is being employed for high precision tracking of non-blinking quantum dots and combined with other 2D super-resolution methods to achieve 10-20 nm resolution in 3D.
Figure 1. Schematic diagram of a home-built automatic calibration and scanning-angle prism-type total internal reflection fluorescence microscope (TIRFM).
2. Imaging Nanoparticles in Differential Interference Contrast (DIC) Microscopy: Nanoparticles are an increasingly important alternative to fluorescent molecules and quantum dots in biological and medical applications. In particular, noble metal particles are attractive optical labels because of their high absorption and scattering cross-sections at or near plasmon resonance wavelengths. A DIC microscope is being modified and enhanced for unambiguous identification of nanoparticle probes in complex environments and for nanoparticle-based multiplexing detection of biomolecules.
Figure 2. Unambiguous identification of gold nanoprobes in complex environments by turning "on" and "off" nanoprobes selectively.
3. Imaging Rotational Motions of Nanomachines in Live Cells: A cell can be conceived as a factory containing a hierarchical network of nanomachines. Translational motions of nanomachines can be readily revealed by a variety of single-particle/molecule tracking methods. However, rotational motions in live cells are much more difficult to resolve and still largely unknown due to technical limitations. A novel approach based on plasmonic nanorod probes and DIC microscopy is being developed to understand the characteristic rotational motions of nanomachines in live cells and engineered systems. The Fang Laboratory is particularly interested in rotational motions involved in endocytosis (e.g., dynamin) and intracellular transport (e.g., kinesin and dynein).
Figure 3. DIC images of two 25Ã--73 nm gold nanorods in different orientations at 540 and 720 nm illumination wavelengths.
4. Microfluidic Devices for Hi-Fidelity Optical Imaging: Fast developments of optical imaging techniques with high spatial and temporal resolution raise new challenges in the field of microfluidics to fabricate microchannels suitable for highly demanding single-molecule imaging and single-particle tracking experiments. The Fang Laboratory is developing the first high-fidelity optical imaging microfluidic platform to combine advanced optical imaging tools seamlessly with microfluidic devices' versatility and controllability over experimental conditions for time-dependent chemical/biological studies under external perturbation.
Figure 4. First fabrication procedure for the glass/PDMS hybrid microdevice suitable for Hi-Fi optical imaging.
5. Virtual Reality (VR) Display of Cells and Micro-structures: Immersive, highly visual, 3D VR environments have become increasingly important components in education, business, manufacturing, and medicine. Most current VR environments are computer simulated visual experiences. The goal of this proposed activity is to generate accurate VR environments directly from images of biological samples and engineered micro-structures on the Hi-Fi multi-modality optical imaging microfluidic platform. Students will then be able to explore and interact with 3D virtual cells/micro-structures.
Figure 5. 3D reconstruction of cell images. (A-C) Original DIC micrographs taken at 3 vertical positions. Over 200 frames can be acquired from a complete vertical scan. (D) Reconstructed 3D cell in Amira from the stack of DIC images. (E-G) Vertical cross sections of this 3D cell from the side indicated by the blue arrow. (H-J) Vertical cross sections from the other side. All cross sections are viewed with a linear perspective.