Optimized Design Of Rhodamine Fluorophores For Single-Molecule Localization Super-Resolution Imaging

The rapid evolvement of super-resolution imaging techniques has revolutionized the biological studies in recent years.Super-resolution imaging provides unprecedented resolutions by breaking the diffraction limit of conventional fluorescent microscopies.The establishment of this technique relies on superior fluorophores,especially for localization-based super-resolution imaging which further requires sparse photoswitching behaviors of fluorophores in terms of both space and time.As core fluorophores for localization super-resolution imaging,rhodamines have been widely deployed for super-resolution imaging.However,these fluorophores exhibit suboptimal properties for these advanced imaging techniques.Therefore,it is necessity to update rhodamine fluorophores to facilitate high-quality super-resolution imaging through rationalized strategy of molecular design.Since the single-molecule characteristics predetermine the localization-based super-resolution imaging capabilities of a fluorophore,the establishment of standardized algorithm and the development of applicable software for analyzing single-molecule data emerge as a critical problem.In this dissertation,a single-molecule algorithm has been established,which is constituted by three components:1)the identification of single-molecule signals,which utilizes the super-resolution localization and molecular cluster methods for excluding spatially overlapped signals;2)the transition-state fitting of fluorescent trajectories,which adopts the hidden Markov model to analyze bright and dark states;3)the measurement of single-molecule photophysical properties,which defines the physical meanings of single-molecule characteristics.Following the established algorithm,a MATLAB based software with GUI interface has been developed for automatically analyzing single-molecule characteristics.The development of algorithm and software for single-molecule analysis would set a stage for evaluating single-molecule photophysics in the future.Ring-closed forms of rhodamine spiroamides are photoactivated to highly fluorescent ring-opened forms,providing sparse dark to bright transforms in a controllable fashion,suitable for photoactivated localization imaging.However,one of its key properties,the duration of on-states,i.e.on time,has never been optimized previously.In this dissertation,a molecular design strategy is developed to lengthen the on time of photoactivaed zwitterionic state through an intramolecular acid microenvironment,and a new rhodamine spiroamide,Rh-Gly,is obtained by introducing a carboxylic acid group in proximate to the lactam location.Photoactivation and pKa studies substantiate the the stabilization effect of adjacent acid group.The established single-molecule algorithm and software enable the measurement of single-molecule photophysics of fluorophores developed through the acidic strategy.Single-molecule results demonstrate that acid strategy lengthens the on time from～30 ms to～60 ms,and predict that these fluorophores permit high-quality super-resolution imaging.Furthermore,through Rh-Gly and its probes derived following acid strategy,high-quality super-resolution imaging of mitochondria in living cells is obtained at～50 nm spatial resolution in 10 s,and super-resolution imaging of nucleus H2B proteins in living cell and microtubule filaments in fixed cells are realized.The development of this strategy would benifit the future development of high-performance rhodamine spirolactams for photoactivation localization microscopies as well as insipire the improvement of other fluorophores.Conventional rhodamines,like tetramethylrhodamine(TMR),show suboptimal fluorescence in aqeous solution.The nonradiative process of these fluorophores probably correlates to the formation of twisted intramolecular charge transform(TICT)state.In this dissertation,a quaternary piperazine strategy is developed to suppass the TICT formation and improve the brightness of rhodamine fluorophores.The quaternary piperazine substituted rhodamine,MPR(Φ＝0.93;ε=8.7 × 104 L×mol-1×cm-1),exhibites over two-fold brightness enhancement compared to its analog TMR(Φ=0.47;ε=7.8 × 104 L×mol-1×cm-1)in ensemble spectrum studies.Single-molecule photophysic properties of MPR and TMR are measured through the priorly established single-molecule algorithm and software.The results demonstrate that MPR shows increased single-molecule brigthness and holds an enhanced capability of super-resolution imaging.Moreover,through biological functional derivatives of MPR,these quaternary piperazine substituted rhodamines enable super-resolution imaging of microtubules in fixed cells and plasma membrane or lysosomes in living cells.Rhodamine fluorophores,developed from the two new molecular designs in this dissertation,would serve as scaffolds for the development of super-resolution imaging probes,promoting the future envolvement of super-resolution techniques.