Fluorescent Hexaarylbiimidazole (HABI):Design,Synthesis And Application In Super-resolution Imaging

Electron microscopy is a very important and powerful tool in nano-scale imaging. Due to the nature of electron microscopy, it is not compatible with bio-samples and could only provide static images, which limits its application in bio-imaging greatly. In contrast, fluorescence microscopy possesses the advantage by its compatibility with living cells, which allows dynamic and minimally invasive imaging experiments. The spatial resolution of optical microscopy is～200nm, which lies in the wave nature of light. This has become a great obstacle of fluorescence microscopy for many years in practical applications. Recently, people have found some techniques to break the resolution limit of optical microscopy. We name these techniques as super-resolution imaging techniques. Of them, stochastic super-resolution imaging methods play a very important role (for example, STORM), which rely on the reversible fluorescence switching of the probes. The working principle of STORM is based on fluorescence switching of the probes under stimuli (light) and determination of the precise position (localization) of single emitters. Therefore, we prepare the dissertation based on designing and synthesizing fluorescent hexaarylbiimidazoles (HABI) and finally use it in super-resolution imaging. This dissertation comprises of the following five parts:In chapter1, we introduce the spatial resolution limit of conventional optical microscopy and the mechanism of super-resolution imaging techniques. And we emphasize the key role of fluorescent photoswitches played in stochastic super-resolution imaging methods. We also introduce all kinds of photochromic materials in brief. In contrast, we discuss the properties and history of HABI in great detail. We find that HABI is a T-type photochromic materials and the fading speed could be very fast to microseconds scale by proper chemical modifications. These characters are very suitable in stochastic super-resolution imaging techniques.In chapter2, we synthesized TPE-HABI, which comprises of the very famous AIE active materials, TPE and HABI by Suzuki-Miyaura couplings. Due to the AIE nature of TPE, TPE-HABI exhibits typical AIE properties and the fluorescence could be quenched reversibly by the radicals, which formed by the photo induced cleavages of HABI. The fluorescence quenching mechanism is proposed to be FRET. What’s more, we discover that the fluorescence maximum peak depends on the excitation wavelength. This phenomenon is rather rare for organic materials, and the reason behind it is still not clear. Finally, we applied TPE-HABI for super-resolution imaging in polymetrix. On the other hand, we point out the defects of TPE-HABI:a) the fading speed of radicals is slow, b) the fluorescence quantum yield of TPE-HABI is poor; c) the fatigue resistance of TPE-HABI is not very good.In chapter3, we try to solve problem of the poor fluorescence quantum yield of HABI at first. We propose that the relative low fluorescence quantum yield of TPE-HABI compared with TPE is because of the existence of photo induced electron transfer (PET) from the lone electron pair in imidazole rings to TPE. If we insert a non-conjugated linker to weaken or eliminate the PET process, the fluorescence quantum yield will improve greatly. With this idea in mind, we have designed, synthesized and investigated both the "conjugated" and "non-conjugated" Naphthalimide based HABIs. We find that the non-conjugated molecular design improves both the fluorescent quantum yield and "on/off’ratio greatly.In chapter4, we further discussed about the universality of the non-conjugated molecular design by introducing different emitting fluorophores. Using carbazole, naphthalimide and perylene monoimde, we have obtained deep blue (390nm), blue green (490nm) and far red (650nm) fluorescent HABIs. These non-conjugated HABIs are highly fluorescent with high "on/off’ratios, which greatly proves the universality of this idea. While on the other hand, we also point out that there is serious defect of these designed molecules. That is the fading speed of the colored species is very poor, which is even worse than that of the single HABI.In chapter5, we focus on improving the fading speed of the colored species, which exists in all the designing molecules above. With the non-conjugated between fluorophores and HABI in mind, we still using naphthalimide as fluorophore. Using the special structure of ethanolamine, the two triphenylimidazole are successfully inhibited in a single molecule. The half-life (t1/2) of the colored species has been improved from1365to only1.1s, which is1000times enhancement in fading speed. The complete fading could be realized in just few seconds! Meanwhile, we use this probe to investigate the self assembly of an amphipathy polymer (PS-PEO). Finally, we have obtained the detail self assembly dynamic processes of PS-PEO by super-resolution imaging method with the help of NI-N-HABI.