Research On New Single-cell Imaging Methods

In part 1,luminol electrochemiluminescence(ECL)imaging was developed for the parallel measurement of active membrane cholesterol at single living cells,thus establishing a novel electrochemical detection technique for single cells with high analysis throughput and low detection limit.In our strategy,the luminescence generated from luminol and hydrogen peroxide upon the potential was recorded in one image so that hydrogen peroxide at the surface of multiple cells could be simultaneously analyzed.Compared with the classic microelectrode array for the parallel single-cell analysis,the plat electrode only was needed in our ECL imaging,avoiding the complexity of electrode fabrication.The optimized ECL imaging system showed that hydrogen peroxide as low as 10?M was visible and the efflux of hydrogen peroxide from cells could be determined.Coupled with the reaction between active membrane cholesterol and cholesterol oxidase to generate hydrogen peroxide,active membrane cholesterol at cells on the electrode was analyzed at single-cell level.The luminescence intensity was correlated with the amount of active membrane cholesterol,validating our system for single-cell cholesterol analysis.The relative high standard deviation on the luminescence suggested high cellular heterogeneities on hydrogen peroxide efflux and active membrane cholesterol,which exhibited the significance of single cell analysis.This success in ECL imaging for single-cell analysis opens a new field in the parallel measurement of surface molecules at single cells.In part 2,we performed 'wet etching' process on silicon wafer and manufactured nano-needle electrodes for localized ECL.The size of nano-needle electrode was about 700 nm and the electrochemical luminescence mapping(ECL-mapping)of luminol-hydrogen peroxide was applied for the study of ECL behavior and the diffusion studies.The results showed that ECL signal at electrode tip was higher than the flat regions,which exhibited that ECL signal was amplified at the tips of nano-needle electrodes.Besides,the diffusion distance of luminescence probe on the tip was characterized to be around 1?m.This phenomenon enhanced the luminescence and facilated the detection of weak ECL signals.This success in ECL-imaging of nano-needle electrodes could be applied in the follow-up studies about single cell ECL imaging.In part 3,a real-time surface plasma resonance microscope imaging(SPRI)monitoring device was established to analyze cell membrane cholesterol quantitatively.In our detection,filipin combined with membrane cholesterol specifically offering the alteration of SPR signal.The results showed that the area of cholesterol molecule on the cell membrane was determined to be 40 A2/molecule,and cholesterol distribution on cell surface was not uniform.The results matched with chemiluminescence imaging results about cholesterol distribution.The success of SPRI on single cell surface cholesterol analysis provided an effective technical method to understand the cell surface interaction between cholesterol and lipid molecules.In part 4,the pathway about the combination of apolipoprotein A-I(ApoA-I)with excess cholesterol in cells and the efflux out of cells in the form of high-density lipoprotein(HDL)was studied.The question whether the combination process occurs on the cell membrane or inside the cells was still controversial.Here,we used a 2 ?m diameter capillary containing fluorescent substrate and cholesterol oxidase to contact cell membrane,and the capillary was sealed by local cell membrane to isolation the solution.No fluorescence was observed at the tip of capillary exhibiting no HDL diffused from the sealed membrane in the presence of ApoA-I.When the capillary was about 5?m away from cell surface,the detection of HDL inside the capillary was achieved.The success of HDL analysis indicated that this method could contribute more in molecule efflux researches.In part 5,Co3O4@CDs nanoparticles were synthesized that could induce chemiluminescence with hydrogen peroxide.This new material had good cell permeability and low toxicity,which was loaded into the cells for the analysis of intracellular hydrogen peroxide.When the cells were stimulated by PMA to generate intracellular hydrogen peroxide,luminescent signal was detected by photomultiplier tube(PMT).Considering cancer cells usually had higher hydrogen peroxide level,this kind of material should be be used to detect and distinguish cancer cells from the cell population.