Development Of Monochromatic Dark-Field Microscopy And Its Applications In Single Nanoparticle Electrochemistry

With the development of single molecule,single particle and single cell research,the object of chemical research has gradually advanced from the macroscopic homogeneous system to the micro-heterogeneous system.Dark-field microscopy is one of the most important technologies to support this chemical research.Here,dark-field microscopy images the Rayleigh scattering signal of the sample,which uses oblique illumination to avoid interference from incident light,and thus it has an excellent signal-to-background ratio.In the obtained optical images,the region with the sample has a strong scattering intensity,and the region without the sample exhibits a darker background.In the past two decades,the most typical research object of single nanoparticles-based dark-field imaging has been noble metal nanomaterials such as silver and gold.There are some reasons.First,the local surface plasmon resonance(LSPR)effect of noble metal nanomaterials has significantly enhanced the scattering cross sections,and thus it is easily detectable.Conventional dark-field microscopy makes it easy to image single nanoparticles of 30-40 nm.After optical system optimization,the detection capability can be increased to below 10 nm.Second,owing to the LSPR effect,single nanoparticles have strong characteristic scattering peaks.Moreover,the scattering spectrum contains a wealth of information on the chemical composition,geometry,surface state and dielectric environment,and thus it has important research value.Therefore,the dark-field imaging technology based on noble metal nanomaterials has been widely used and developed in the fields of nanoplasmonics,optical sensing,nanoelectrochemistry,and cell imaging.However,there are still some bottlenecks and limits in dark-field microscopy.First,due to the relatively low time resolution,dark-field microscopy is rarely used for analysis of fast kinetics processes at microsecond to millisecond level.In general,a scattering spectrum in dark-field microscopy is used as a criterion for qualitative and quantitative analysis.And a spectral acquisition in grating-type imaging spectrometer needs a time ranging from a few hundred milliseconds to a few seconds,making it difficult to meet the needs of microsecond-scale reaction kinetics studies.Secondly,almost all single nanoparticles dark-field scattering studies are based on noble metal nanomaterials,which greatly limits the application range of dark-field microscopy.Due to the above technical limitations,the research progress of single nanoparticles dark-field imaging in recent years has slowed down.Based on the above understanding,a monochromatic dark-field microscopy with adjustable wavelength and microsecond time resolution was built.Using this new device,the electrochemical impedance spectroscopy of single nanoparticles was measured in the frequency range of 1 Hz-30 kHz,which extends the single nanoparticles electrochemical study from the traditional potential scanning mode to the high-frequency alternating current(AC)modulation mode for the first time.Thus,it can be used to develop single nanoparticle sensing based on impedance analysis.In addition,the introduction of optical superlocalization algorithm and Fourier transform strategy into single nanoparticle electrochemical studies revealed that the optical scattering center of a single nanoparticle reflects the center of its electron density,rather than the generally geometric center.Finally,the resonance Rayleigh scattering effect of pure absorbing materials was used to study the change of the scattering spectra of single Prussian blue nanoparticles in the electrochemical redox process.The research objects of dark-field microscopy were extended from traditional noble metal nanomaterials to inorganic coordination compound,which injects new vitality into the single nanoparticles dark-field imaging.The specific research content of this thesis is summarized as follows:1.Development of a monochromatic dark-field microscopyIn conventional dark-field microscopy,a white light source is used as excitation source and grating-type spectral imaging is used to measure the scattering spectra of single nanoparticles.The lower time resolution makes it unsuitable for the fast-electrochemical processes study.A monochromatic dark-field microscopy was built.It could be used to achieve spectral acquisition by scanning the wavelength of the excitation source.At the same time,the scattering intensity was measured by a single point detector to increase the time resolution to the microsecond level.And single gold nanorods were used as optical probe to evaluate the performance of this developed device.The results show that this new device has excellent dark-field imaging quality and good optical system stability.In single nanoparticle electrochemical studies,the voltage response sensitivity of single gold nanorods is up to-10 mV,which is an order of magnitude higher than that obtained-100 mV in grating-type dark-field microscopy.2.Determination the electrochemical impedance spectroscopy of single nanoparticlesBy using the developed monochromatic dark-field microscopy,the electrochemical impedance spectroscopy of single nanoparticles was first measured.By applying a sinusoidal modulation voltage at a certain frequency to the electrochemical system,there is a periodic change in the electron density in single gold nanorods,which is manifested as a periodic change of the scattering intensity under monochromatic illumination.By gradually changing the modulation frequency in the range of 1 Hz-30 kHz,the amplitude and phase of the scattering intensity of a single gold nanorod were plotted against the frequency to obtain the electrochemical impedance spectrum of the single gold nanorod.Based on the impedance spectrum,the results reveal that the electrochemical charging and discharging process of single nanoparticles was affected by ion migration in the electrolyte in the low frequency region(<100 Hz).In the high frequency region(>100 Hz),the non-Faradaic charging and discharging is a pure double-layer process without the effect of the electrolyte.Thus,the calculated surface capacitance of single gold nanorods was 36.6±11.8?F/cm2,which is closer to the true capacitance of gold.The electrochemical impedance spectroscopy of single nanoparticles not only helps to understand the electrochemical charging and discharging process of the nanoscale interface,but also helps to develop new sensors.3.Tracking the scattering centroid shift of single nanoparticles during electrochemical charging and dischargingOn the image plane of a dark-field microscopy,the Rayleigh scattering spot of a single nanoparticle follows the two-dimensional Gaussian distribution with a full width at half maximum(FWHM)of about 300 nm.It is generally believed that the center of the scattering spot(called the "optical scattering center")reflects its geometric centroid through the superlocalization algorithm analysis.In this work,the Fourier transform and the superlocalization algorithm were combined to track the migration path of the optical scattering center of a single nanoparticle during periodic electrochemical charging and discharging.The introduction of the Fourier transform resulted in a detection sensitivity of 0.1 nm,which is several times higher than that of the conventional optical centroid tracking method.The results show that in the process of electrochemical charging,as electron injection occurs,the uneven distribution of electrons on the surface of single nanoparticles caused the movement of the optical scattering center.The average displacement of dozens of single nanoparticles was about 0.4 nm,and the direction of movement has no correlation with the direction of its geometric long axis.This result indicates that the optical scattering center of single nanoparticles actually reflects the center of its electron density,not the geometric centroid.This work not only provides a new perspective for studying the interaction between photons and electrons at the sub-nanoparticle level,but also provides a highly sensitive research strategy for other fast and reversible chemical reactions.4.Resonance Rayleigh scattering spectra of single Prussian blue nanoparticlesBased on the light absorption properties of Prussian blue nanomaterials,the resonance Rayleigh scattering spectra of single Prussian blue nanoparticles were studied by dark-field microscopy.The results show that Prussian blue nanoparticles scattered red light in an aqueous solution,and its maximum scattering wavelength is consistent with its absorption wavelength.Similar to the LSPR effect of noble metal nanomaterials,the resonance Rayleigh scattering behavior of single Prussian blue nanoparticles was affected by the refractive index of the surrounding environment.As the refractive index increased,the scattering intensity of single Prussian blue nanoparticles decreased and its scattering spectra were blue-shift.Moreover,the electrochemical redox process of single Prussian blue nanoparticles was further studied in real-time.During the reduction process,the scattering intensity of Prussian blue nanoparticles gradually decreased.During the oxidation process,the scattering intensity of Prussian blue nanoparticles gradually increased.This process was reversible.In this work,the typical inorganic complex material is taken as an example to introduce the dark-field imaging,which broadens the research object of dark-field microscopy,and there is of great significance to promote the applications of dark-field microscopy in single nanoparticles measurement and imaging.In summary,from the perspective of single nanoparticles dark-field imaging technology:1)The developed monochromatic dark-field microscopy in this work has a microsecond time resolution,which is especially suitable for the study of rapid reaction kinetics;2)Innovatively combining the superlocalization algorithm with the Fourier transform not only improves the sensitivity of the optical centroid analysis method,but also provides a new analysis technique for dark-field imaging;3)By introducing the principle of resonant Rayleigh scattering in dark-field imaging for the first time,which broadens the fields of applications of dark-field microscopy.From the scientific point of view of nanoelectrochemistry,the established a theoretical model of single nanoparticles electrochemical impedance spectroscopy expands the electrochemical research mode of single nanoparticles,and it is expected to develop new biosensing strategy.In addition,the relationship between the disclosed optical scattering center and the electron density distribution can deepen the understanding of photon-electron interactions in the LSPR effect.