Localized surface plasmon based super resolution imaging technique with nanostructures

Abstract

In this dissertation, I have investigated the plasmon enhanced total internal reflection fluorescence (TIRF) microscopy for improvement of imaging sensitivity and imaging resolution using various nanopatterns in total internal reflection fluorescence microscopy. First, I have found the feasibility of the evanescent field enhancement based on dielectric thin films and metal nanostructures in TIRF microscopy for sensitivity enhancement. The enhancement was associated with the overall field intensity amplification. To maximize the field intensity, multi-layers of dielectric films and metal nanopatterns are designed and fabricated using film evaporation and electron beam lithography, respectively. The sensitivity enhancement was confirmed with fluorescent beads and quantum dots attached on cancer cells. Especially, to find the optimum design parameters of plasmon enhanced TIRF microscopy using metal, plasmon momentum mismatches between near field and far field are also determined using rigorous coupled wave analysis. The numerical results are experimentally verified with fluorescent beads on various nanopatterns, such as nanowires and nanoislands. The results confirm that momentum mismatching when exciting plasmons can increase the consequent emission of fluorescence substantially. Consequently, I have introduced three kinds of plasmon-based techniques for super resolution imaging under diffracted-limited: 1) Surface plasmon-enhanced randomly activated (SUPRA) TIRF microscopy 2) nanoscale localization sampling technique and 3) plasmon based spatially activated light microscopy. For SUPRA-TIRF microscopy, I have investigated the imaging resolution enhancement by exciting randomly amplified local hot spots. The random hot spots are activated by chemically synthesized nanoislands. The distribution of hot spots can be adjusted for efficient excitation of fluorescent molecules. This technique was experimentally verified by imaging fluorescent beads and visualizing endocytosis of fluorescent adenoviruses. The results confirm the enhancement of resolution, which was more prominent at higher concentration of fluorescent molecules. For an NLS technique, periodic nanohole arrays that create locally amplified hot spots are fabricated. The localized near field hot spot temporally samples microtubular movement for enhanced spatial resolution. A four times improvement in spatial resolution compared to conventional TIRF microscopy is demonstrated. The resolution enhancement is achieved by imaging rhodamine-labeled microtubules that are sampled by the hot spots to provide sub-diffraction-limited images at 76 nm resolution in the direction of movement and 135 nm orthogonally. The intensity distribution produced by the NLS is measured to be broader than that of conventional imaging, which is consistent with the improvement of imaging resolution. NLS can be useful for moving objects that have a high labeling density of for performing fluctuation spectroscopy in small volumes, and may allow super-resolution on demand by customizing nanoantenna structures for specific resolution needs. On the other hands, PSALM is based on the spatially switched activation of local amplified hot spots under multiple light incidence conditions. The feasibility of the concept was demonstrated by imaging fluorescent beads on a two-dimensional gold nanowire of a 100-nm-wide grating ridge, the size of which is the measure of the imaging resolution. The result confirms the performance of PSALM for imaging the beads at a resolution below the conventional diffraction limit.Further studies of these techniques are expected to provide super resolution under 50 nm to observe and track extremely small molecules or proteins at/near cell membranes.