| Molecule is regarded as the basic carrier of chemical property of matter. So in the process of exploring the mysteries of life, mankind always has a dream of in vivo, in situ, real-time studying the interaction between matter at the single-molecule level. As the rapid development of single-molecule optical technology, the dream has been turned into reality in recent years. Fluorescence correlation spectroscopy(FCS) is a typical single-molecule optical technology. In the FCS system, there exists a tiny detection area(usually less than 10-15 L) built by the confocal of a laser and a pinhole. Fluorescent molecules or particles move into or out of this area due to Brownian motion or chemical reaction, resulting in the fluctuation of the fluorescence intensity. The fluctuation signal is collected by a sensitive detector with high temporal resolution and then auto-correlated or cross-correlated to detect the dynamic information of the molecule or particles. As one progenitor of the field of single-molecule optical technology, FCS has become a routine method to study the dynamic behaviors of single fluorescent molecule since first introduced 40 years ago. At present, FCS is widely used in biology and medicine with its excellent temporal resolution and high statistical confidence. For example, to investigate the conformational dynamics of molecule, signal identification, biochemical reaction kinetics and so on.However, current FCS technology still suffers from inherent limitations and faces new challenges. First, the traditional fluorescent probes used in FCS, including organic fluorescent dyes, fluorescent protein, quantum dots, etc., undergo fluorescence decay or even bleaching under high-intensity or long-term illumination. In addition, the fluorescence of quantum dot always blinks. The fluorescence intensity variation of the probes caused by their own properties overlays the fluorescence fluctuation caused by their dynamic behaviors, which can seriously interferes with the detection. The probes that exhibit fluorescence decay can hardly stand long-term measurement that is required in the research on biological systems. Second, the rapid development in the field of biology and medicine brings new challenges to the analytical chemist. The complex and heterogeneous biological systems require high-throughput and multi-channel detection methods. The present FCS method employs a confocal optical configuration and commonly uses a photomultiplier tube or an avalanche diode(APD) as the detector, so it is impossible to conduct high-throughput and multi-channel measurement but only single-window detection can be realized.In order to solve the two scientific problems mentioned above, we developed two new fluctuation correlation spectroscopy methods in this dissertation. The metal nanoparticles were employed to replace the traditional fluorescent molecules as the optical probe. Total internal reflection(TIR) illumination and dark field microscopy were creatively introduced as new optical configurations, and the high sensitive electron multiplying charge-coupled device(EMCCD) was equipped as the detector. The main contributions are as follows:(1) We established spatially resolved scattering correlation spectroscopy(SRSCS) using a TIR configuration. First, we synthesized stable silver nanoparticles of uniform size and employed their strong scattered light as the detection signal. A homemade and millimeter-scale hole, which replaced the emitting filter in TIR configuration, was used to efficiently separate the scattered light of nanoparticles from the background laser as well as its reflected beam. An EMCCD was utilized to collect the signal as an array detector. Using this new fluctuation correlation spectroscopy method, we studied the dynamic behaviors of silver nanoparticles with the size of 16 ± 2 nm and found that SRSCS was sensitive to the variation of the concentration and diffusion coefficient of nanoparticles with the correlation coefficient of 0.998 and 0.988, respectively. The signal to noise ratio was 14.4. Experimental results demonstrated that SRSCS was a high sensitive, high-throughput and multi-channel technique to detect the dynamic information of nanoparticles, including concentration, diffusion coefficient and so on. Furthermore, we investigated the effect of certain experimental factors on the measurement. The results measured from 200000 frames was proved to be reliable.(2) We built tempo-spatially resolved scattering correlation spectroscopy under dark-field illumination(DFSCS). Dark field microscope was simple, low cost and easy to use, and its experimental setup was achieved by the introduction of a dark-field condenser to the frequently-used bright-field microscope. We realized the fluctuation correlation spectroscopy on dark field microscopy for the first time. Since the dark field illumination was a new configuration of fluctuation correlation spectroscopy, the theoretical model of DFSCS was first deduced. The reliability and adaptability of the model were tested by both simulated results and experimental data. The concentration and diffusion coefficient of nanoparticles obtained by Monte Carlo simulation were very close to their corresponding set value with the relative error of 4%. We also proposed the evaluation methodology of the statistical accuracy of DFSCS. In addition, the sensitivity of the new method was studied in theory as well as in experiments. DFSCS was sensitive to the variation of the concentration and diffusion coefficient of nanoparticles with the correlation coefficient of 0.972 and 0.993, respectively. The signal to noise ratio was 17.6. The measurement results of gold nanoparticles verified that DFSCS was sensitive to the dynamics of nanoparticle, such as concentration and diffusion coefficient. The time resolution of DFSCS was 0.5 ms per frame, and 500000 frames were collected for each measurement. So DFSCS had a higher statistical accuracy.(3) We applied DFSCS to investigate dynamic behaviors of nanoparticles in live cells. The 60 nm diameter gold nanoparticles were modified by a cancer treatment drug named Herceptin, which acted as delivery agent to help the uptake of nanoparticles into Si Ha cells. The intracellular nanoparticles were distributed in cell membrane or organelles specifically and emitted strong scattered light under the dark field microscope. The scattered signal was detected by DFSCS to study the dynamic behaviors of intracellular nanoparticles located at various areas as well as characterize the heterogeneous intercellular environment. The diffusion coefficient of intracellular nanoparticles were 0.056 to 0.235 2μm s, which was an indication of the difference among diffusion rates of nanoparticles located at various position. Our experimental results manifested that DFSCS was a multi-channel method that could detect different micro zones simultaneously. We also found that the intracellular environment was an extremely heterogeneous system. With the help of the diffusion law, we verified the existence of micro domains in the cell which hinder the diffusion of nanoparticles. |
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