Development Of Novel Fluorescent Agents And Their Application For Bioimaging

Fluorescence imaging is prevalent in nearly every aspect of life sciences, serving as an indispensable tool in observing cell structures and understanding many fundamental processes thanks to its intrinsic advantages such as high sensitivity and low invasiveness. A key component for obtaining desired fluorescence images is choosing the right imaging agents. In this dissertation, new organic fluorescent imaging agents, exhibiting improved photophysical properties such as high brightness and resistance to photoleaching for bioimaging, were developed through rational design.Chapter1mainly focuses on the principle of fluorescence imaging, super-resolution fluorescence imaging, two-photon absorption and energy transfer."The principle of fluorescence imaging" includes theory of fluorescence, common fluorescence imaging system and frequently-used fluorescence imaging agents."Super-resolution fluorescence imaging" includes theories of some related imaging technologies and requirement of imaging agents."Two-photon absorption" refers to the design principle of molecules with large two-photon absorption cross-sections."Energy transfer" introduces the Forster resonance energy transfer (FRET) mechanism and the Dexter mechanism.In Chapter2, we report a design strategy that incorporates both rhodamine B (RhB) and difluoroboron dibenzoylmethane (BF2dbm) in the same polylactic acid (PLA) nanoparticles, which can be used for bioimaging. The nanoparticles not only possess advantages of good photostability and two-photon absorption, but also exhibit red-shifted emission characteristic of that from RhB (560-600nm). This compares to the blue-green emission (440-490nm) of BF2dbmPLA nanoparticles, which is not ideal for bioimaging due to its overlap with the autofluorescence and poor penetration depth within the biological systems. In addition, the nanoparticles have high biocompatibility due to the presence of PLA. The biological test shows the nanoparticles have low acute cytotoxicity and could be used for the bioimging of extremely sensitive neuronal cells.In Chapter3, we design a spiropyrane and rhodamine covalently linked dyad (Sp-RhB). The open-ring structure of photochromic Sp, i.e., the protonated merocyanine (McH) form, is expected to quench the fluorescence of rhodamine B through FRET. We hope to develop new super-resolution imaging agents based on this mechanism. The experiment result indicates the synthesized dyad can shift between a bright state and a dim one. However, the present result does not allow for super-resolution imaging as the contrast ratio is too low.In Chapter4, we design a series of fluorescent polymers containing a typical GFP chromophore derivative, where polymers are used to mimic the innate environment of the GFP chromophore. We reason that polymers of different chemical compositions may provide a suitable medium for the generation of an emissive state of the GFP chromophore. It was found that blue to green emissions were observed for certain polymer samples depending on the chemical and physical environment. To the best of our knowledge, this is the first observation of GFP chromophore in an artificial environment emitting in at a similar wavelength as well as decay rate compared to the naturally occurring GPF fluorescence. Theoretical calculations were then used to explain certain aspects of the experimental results.