Fluorescence Imaging Of Subcellular Metal Ions Using Ion Selective Fluorescent Nanoparticles

Metal ions play a vital role in living organisms,not only participating in the organization of living tissue structures,but also playing a key role in the process of life metabolism.In living organisms,metal ions have a variety of existing forms and spatial distribution characteristics.Specific to life's basic building blocks,such as cells,metal ions in different cellular organelles have specific distributions.Moreover,there are various forms of existence such as free ions and protein bindings that assume a variety of different physiological functions within the cell.Therefore,the existence of different metal ions should be real-time distinguished at the subcellular level,which is of vital importance for the in-depth understanding of various physiological mechanisms of living organisms.At present,various methods for the detection of metal ions,such as AAS and ICP-MS for aqueous solutions,and Nano-SIMS and LA-ICP-MS with spatial resolution,have been well developed.Compared with these methods,fluorescence imaging can visualize spatial distribution of metal ions in living cells that is of great significance for the real-time detection of cellular physiological processes.However,the current fluorescence methods need specific probe design and complicated chemical synthesis.Therefore,developing a simple and universal fluorescence analysis method to realize real-time fluorescence of metal ions at the subcellular level in living cells is urgent.In this thesis,we use ion-selective fluorescent nanospheres to achieve this goal.Ion-selective fluorescent nanospheres are micelle particles composed of amphiphilic polymeric materials,protonated dyes,ionophores,and ion exchangers.Among them,protonated dyes act as fluorophores,and ionophores catch target ions.When the ionophores recognize ions,ion exchange causes deprotonation of the dyes,resulting in a decrease in its fluorescence intensity.The independence of the two components offers the various spectral regions and the target ions.Considering the easy commercialized dyes and ionophores,the micelle particles have the advantages of simplicity and versatility.In the first work,novel calcium-selective nanospheres incorporating Pluronic(?)F127 and(4-carboxybutyl)triphenylphosphonium bromide(TPP)as shell layers were designed to monitor the level of free calcium ion in mitochondria and lysosomes at living cells simultaneously.TPP as a target for mitochondria drove the nanospheres to bind intracellular mitochondria,while,the lipophilic F127 layer resulted in the partial accumulation of nanospheres in lysosomes.This dual feature of the shell layer led to the co-location of nanospheres in both mitochondria and lysosomes.Chromoionophore ?(ETH 5350)was chosen as the chromoionophore in the nanospheres that had different fluorescence lifetimes in either mitochondria or lysosomes,and therefore,the locations of the nanospheres at these two cellular compartments were identified.After the stimulation of cells using ionomycin,a burst of calcium concentration in mitochondria was observed that was associated with almost constant calcium concentration in lysosomes.The simultaneous recording of calcium ions in both of the compartments using fluorescence lifetime-solved nanospheres offered a special strategy to spatially monitor sub-cellular fluctuation of ions in living cells.In the second work,the ion-selective nanospheres were successfully applied to realize fluorescence imaging of the extracellular K+ near the cell membrane in real time.By the carboxylation of F127 at the nanosphere,the negatively charged particles are electrostatically repelled by the membrane with negative charge.This repulsion slows down the rate of cell endocytosis to the micelle particles,which allows the adsorption of particles at the cell membrane.Afer the K+ ion was added to the solution,the concentration of K+ in the vicinity of the cells was increased.It was observed that the micelle particles responded quickly to the change in the K+concentration associated with the drop of fluorescence intensity.This rapid response capability indicates that ion-selective nanospheres have the possibility of real-time detection of K+ released from the opening of potassium channels in cells.The potential ability might offer these micelles in the application prospect to understand membrane K+ transport mechanisms.