Study On Speckle And Signal Background Contrast Of Single Nanoparticle Interference Scattering Microscopy

Interference scattering microscopy(i SCAT)is a far-field optical imaging method based on light scattering.The detection principle is that the scattered light of the particle to be measured and the reference light of the same path interfere with each other.The particle signal intensity of pure scattering detection is proportional to the sixth power of the particle diameter(D~6),while the particle signal of i SCAT detection is proportional to the third power of the particle diameter(D~3),so as the size of nanoparticles continues to decrease,i SCAT highlights higher detection sensitivity.Compared with fluorescence microscopy,the scattering signal of Rayleigh scatterer does not have problems of flicker,saturation and bleaching,so this technology can detect the scatterer in real time for a long time,with higher positioning accuracy and imaging quality,and can track single particles with high space-time accuracy and detect the physiological and dynamic processes of proteins in biological cells.As a special Rayleigh scatterer,gold nanoparticles(GNPs)have strong scattering signals under plasmon resonance conditions,so they are excellent biomarkers.In biomarker applications,the size of the marker itself should be as small as possible to minimize the impact on the performance and behavior of the marked object.At present,the most common size of GNPs as markers are 20 nm.One of the most important reasons for this may be that the random speckles produced by the sample substrate limit the signal resolution.Based on this,this thesis has carried out research on single particle i SCAT from the following three aspects,which provides a research basis for using smaller size GNPs as markers in the future.We first studied the effect of substrate morphology on i SCAT,and the relationship between the intensity of speckle and the surface roughness.We mainly compared the results of i SCAT detection of GNPs on the surface of coverslip and mica.Since the surface roughness of mica is less than that of coverslip,the experimental results show that GNPs with a diameter of 10 nm can be detected on the surface of mica,while only GNPs with a diameter of 20 nm were detected on the surface of coverslip.It indicates that the smaller the surface roughness of the substrate,the weaker the random speckles generated on the substrate surface,so the lower the background fluctuation,the more conducive to the detection of small-sized GNPs.Second,we studied the influence of the substrate material on the i SCAT of GNPs.We mainly compared the i SCAT detection results for the GNPs on the silicon substrate and on the surface of the coverslip or mica.Due to the high reflectivity of the silicon surface,the intensity of the reflected light on the surface of the silicon substrate is stronger than that of coverslip or mica,and the signal contrast of the scatterer detected by i SCAT is inversely proportional to the reflected light on the surface of the substrate,so the detection result of the signal contrast of GNPs on the surface of silicon substrate is lower than that of coverslip or mica.Due to the small roughness of the silicon substrate surface,the experiment successfully detected GNPs with a diameter of 10 nm.Finally,we studied the enhancement of the contrast of i SCAT signals.Because the signal contrast of the scatterer detected by i SCAT is inversely proportional to the reflected light of the substrate surface,it is designed to use a metal mask circle to attenuate the reflected light of the substrate surface in the detection optical path,thereby enhancing the signal contrast of the detected GNPs.The diameter of the metal mask circle used in this paper belongs to the micron level.The experimental results show that after the metal mask circle is used to attenuate the reflected light of the substrate surface,the signal contrast of the detected GNPs can be enhanced,and the imaging quality of the GNPs is also improved;And the thicker the metal mask circle thickness,the more obvious the effect of the GNPs signal contrast enhancement.The research in this paper shows that i SCAT has high detection sensitivity and can detect GNPs with a diameter of 10 nm on the surface of mica and silicon substrates.Designing a method of using a metal mask to circularly attenuate the reflected light on the substrate surface in the detection optical path can enhance the signal contrast of the GNPs.