Probe Design And Single Cell Measurement Based On Single Particle Energy Transfer

Cells are the basic structural and functional units of lives.Understanding the transport mechanism of matter and energy in single-cell will expand our knowledge on the production mechanism of disease and even the origin of life.Therefore,in the past few decades,single-cell analysis has achieved rapid development.Among them,the optical analysis method has been widely concerned because it’s non-destructive and in situ analysis of living cells and exhibits high temporal and high spatial resolution.The development of optical analysis is highly dependent on the development of luminescent probes.Because of the wide emission peak,the common luminescent probes easily lead to spectral crosstalk in the multi-channel analysis.Although the existing nano-laser probe based on Spaser（Surface Plasmon Amplification by Stimulated Emission of Radiation）seems to solve the above problem,it is difficult to be compatible with the existing optical analysis instruments due to its high lasing threshold.And such a harsh lasing condition can easily cause cell damage and affect the accuracy of the measurement results.Besides,in the aspect of single cell thermal analysis,although a lot of work on the temperature distribution in single cell has been reported,the fluctuation range of temperature distribution in single cell is still controversial.At the same time,the research on single cell heat transfer is almost blank.Therefore,we developed a three-level Spaser luminescence probe with a low threshold and constructed a transient microscope system,based on the energy transfer principle of single Au nanoparticles.Au nanoparticles were used as thermal disturbance probes to measure the heat transfer characteristics in single cells.The main research contents are summarized as follows:1.Preparation and characterization of a three-level spaser for next-generation luminescent nanoprobe.By using a dye with three energy levels as the gain medium,we developed a Spaser based nano-luminescence probe with a three-level system,utilizing the energy transfer from the polarized dipole of dye to the Au nano-particle resonator.The emission line width of the prepared plasmonic nano-laser probe is about 3 nm,which is much smaller than that of the common fluorescent probe.The lasing emission lifetime is about 100μs,which can easily avoid the interference of the intracellular luminescent background.And the lasing threshold is about 1 m J·cm^-2,which is compatible with conventional biological analysis instruments（such as the confocal microscope,etc.）.Based on this probe,we then constructed a Spaser-STED super-resolution microscope.The spatial resolution of the microscope is about 74 nm,which is much better than that of the confocal microscope（286 nm）.The developed Spaser probe exhibits enormous potential as the next generation luminescent probe for single-cell analysis.2.Construction of a transient microscope system for measuring the heat transfer coefficients.The microscope system is using a transient microscopic imaging method based on pump-probe detection technology,which possesses high temporal and spatial resolution and exhibits broad prospects in applications.We used gold nanoparticle as the thermal disturbance probe to measure the heat transfer coefficient of the medium by utilizing the energy transfer of the laser-heated gold nanoparticle to the surrounding medium.The spatial resolution of the microscope we constructed is based on the thickness of the thermal diffusion layer in the surrounding medium of the gold nanoparticle probe.In principle,the thickness of the thermal layer is related to the pump laser power density（LPD）and thermal diffusion time.By optimizing the pump LPD,the thermal field can be reduced to less than 100 nm.The time resolution of the microscope is about 5μs（time gate width）,which have achieved the highest time resolution so far,compared with the previous steady-state or quasi-steady-state temperature measurement techniques.Then,the transient heat transfer microscope system was used to measure the heat transfer coefficients of pure water and glycerol medium.The results indicate that the transient heat transfer measurement system has good accuracy and precision（the total system error is about 10%）.The combination of near diffraction limit imaging ability and microsecond level time-resolved ability provides a unique advantage for the revealing of ultra-fast thermodynamic events at the cellular or subcellular level.3.Measurement of heat transfer properties within single cells.We have studied the heat transfer characteristics of five different cell lines,including human He La,MCF-7 and MCF-10A cells,chicken fibroblasts,and bullfrog fibroblasts.We found that the distribution of heat transfer coefficient is uneven in these five kinds of cells,and the distribution range of heat transfer coefficient in living cells is wider than that in fixed cells.Then,we also explored the relationship between the heat transfer characteristics of the single cells and the ambient temperature.And we found an“active”regulation of heat dissipation characteristic in the living homothermal animal cells,that is,the heat transfer coefficient in the cell increases sharply when the ambient temperature is higher than the optimum culture temperature of the cell.Although the heat dissipation coefficient of living cold-blooded animal（bullfrog）cells also increases with the increasing of ambient temperature,there is no such a saltation phenomenon.Moreover,when the homothermal animal cells are fixed,the“active”regulatory property is disappeared.Based on this,we think that the homothermal animals may have an“active”heat dissipation regulation at the single cell level,which will change its heat transfer coefficient according to the ambient temperature to ensure the heat balance of the cells.According to the results of cellular metabonomics analysis,we have proposed a closed-loop control theory for the regulation of heat dissipation characteristics of human cells:when the external temperature is higher than the body temperature,the intracellular tricarboxylic acid（TCA）cycle weakens leading to the decrease of intracellular heat production.At the same time,the intracellular arginine metabolism increases resulting in the production of a large number of endothelial relaxing factor（NO）,which will stimulate the cells to accelerate heat dissipation.