A Novel Method for 3D Nanoscale Tracking of 100 nm Polystyrene Particles in Multi-Wavelength Evanescent Fields Microscopy – Absolute Difference Height Verification

Total internal reflection is an optical imaging technique for nanoparticle tracking and observation employing the scattered light from an evanescent field near the interface or reference surface. Generally, the nanoparticle behavior is the three-dimensional Brownian motion in an aqueous medium. The motion can be traced by an optical microscopy, but it cannot be traced by an electron microscopy technique. In the three-dimensional nanoparticle moving position, the X and Y positions are parallel to the surface, which can be traced by the general microscopy techniques. However, the height position Z of a nanoparticle perpendicular to the surface could not be traced without the longitudinal scanning method. Here, a novel method is proposed to investigate the 3D position of nanoparticles by applying multi-wavelength evanescent fields microscopy, which has a high spatial resolution in the Z -direction without longitudinal scanning. This paper focuses on the verification of measurement in the Z -direction. A piezoelectric actuator was employed to control the nanoparticle displacement in height Z . Standard polystyrene 100 nm particles were randomly adhered on a spherical tip that connected with the piezoelectric actuator. The spherical tip was essentially made from an optical adhesive ( n = 1.348) with a refractive index close to the water for decreasing the unnecessary signal from the tip-self during nanoparticle observation in the water. The proposed method could obtain the multi-wavelength scattering lights from the observed nanoparticles by an 8-bit color camera with higher than 50 frames per second recording to investigate the 3D nanoscale tracking. The X and Y positions of nanoparticles were determined by the centroid of the scattering light intensities. The height Z was determined from the logarithm ratios between the detected scattering light intensities of both wavelengths. The measurement repeatability of the absolute difference in height between nanoparticles could be measured less than ±16 nm by using the proposed method. The penetration height measurability range was approximated at 250 nm from the reference surface.