Biological Imaging With A New Light-emitting Probes

One of the current research interests in bioimaging is the ultra-sensitive detection of the targets in vivo or at the single-molecule scale.However,the signal from the targets labeled with fluorescent probes would probably be masked by the high autofluorescence from the endogenous components in biological samples.To solve this problem,it is necessary to develop some new luminescent labels to eliminate autofluorescence background in bioimaging.This thesis is focused on this project and composed of four parts.1.Organic Chemodosimeter for Fluorescence Imaging of Cu²⁺ in Live CellsBiological tissues have weak absorption of light in the red and near-infrared region. Therefore,a red fluorescence probe would greatly reduce the background fluorescence.In addition,visualizing the concentration and subcellular distribution of copper in physiological processes may greatly contribute to understanding its complex physiological functions and nosogenesis.A Rhodamine B derivative containing a highly electron-rich S atom has been synthesized as a red fluorescence turn-on chemodosimeter for Cu²⁺.As a result of Cu²⁺-promoted ring-opening,redox and hydrolysis reactions,comparable amplifications of absorption and fluorescence signals were observed upon addition of Cu²⁺,suggesting that the chemodosimeter effectively avoided the fluorescence quenching caused by the paramagnetic nature of Cu²⁺.Importantly,this compound can selectively recognize Cu²⁺ in aqueous media in the presence of other metal ions with high sensitivity(detection limit≤10 ppb) and a rapid response time(≤1 min). Moreover,by virtue of the chemodosimeter as fluorescent probe for Cu²⁺,confocal and two-photon microscopy experiments revealed a significant increase of intracellular Cu²⁺ concentration and the subcellular distribution of Cu²⁺,which was internalized into the living HeLa cells upon incubation in growth medium supplemented with 50μM CuCl₂ for 20 hours.2.Heavy-metal Complexes for Phosphorescence Imaging of Live CellsPhosphorescent heavy-metal complexes have relatively longer luminescence lifetimes(～μs) than those of endogenous fluorescent substances(～ns),and thus are appealing probes for completely avoiding background fluorescence in bioimaging through a time-gated technique.Being among the best class of phosphorescent heavy-metal complexes,iridium(â…¢) complexes exhibit many advantageous photophysical properties.To date,no luminescent staining of live cells using iridium(â…¢) complexes has been reported.Two cationic iridium(â…¢) complexes with bright green and red emissions were demonstrated as phosphorescent dyes for live cell imaging.In particular,their exclusive staining in cytoplasm,low cytotoxicity and reduced photobleaching,as well as cell membrane permeability,make the two complexes promising candidates for the design of specific bioimaging agents.Additionaly,an iridium(â…¢) complex with histidine-induced luminescence enhancement was demonstrated as a phosphorescent dye for exclusively staining the nuclei of cells.The advantage of phosphorescent complexes for bioimaging,the capability of visualizing nuclei of both live and fixed cells,as well as the short period time(～10 min) for staining promise wide applications in biological and medical studies.3.Rare-earth Nanophosphors for Laser Scanning Up-conversion Luminescence Microscopy(LSUCLM)Rare-earth up-conversion nanophosphors(UCNPs) as alternatives of conventional biological luminescent labels have attracted a tremendous amount of attention.We found that rare-earth nanophosphors exhibit a unique up-conversion luminescence mechanism and imaging modality and developed a new three-dimensional visualization method of laser scanning up-conversion luminescence microscopy(LSUCLM) with little photobleaching and no background fluorescence, by introducing a reverse excitation dichroic mirror and the confocal pinhole technique. Moreover,we demonstrated the up-conversion emission imaging of thin films containing embedded rare-earth nanophosphors and cells multilabeled with the nanophosphors and organic dyes.These data show that LSUCLM offers some distinct advantages,such as little photobleaching of both organic dyes and rare-earth nanophosphors,no background fluorescence from either endogenous fluorophores or colabeled fluorescent probes,and excellent compatibility with conventional confocal microscopy.4.¹⁸F-Labeled Rare-earth Nanophosphors for Dual-modality Positron Emission Tomography(PET) and Up-conversion Luminescence ImagingPET is a whole-body imaging technique with the highest sensitivity,while fluorescence imaging is widely used for cell and tissue imaging.Therefore,both radioactivity and fluorescence should ideally be combined into one probe for multi-level imaging. Radionuclide ¹⁸F^-has been incorporated into up-converting nanophosphors (UCNPs) in＞60%labeling yield by virtue of a simple,rapid and efficient strategy based on reaction between ¹⁸F^-and rare-earth elements.The effectiveness of ¹⁸F-labeled UCNPs for both PET and UCL imaging was futher evaluated by investigating the biodistribution of these nanoparticles using in vivo PET imaging and LSUCLM imaging experiments.The combination of in vivo PET and LSUCLM imaging indicates the potential of the ¹⁸F-labeled UCNPs for ultra-sensitive molecular imaging from the cellular scale to whole-body evaluation.