Super-resolution and super-localization microscopy: a novel tool for imaging chemical and biological processes

Dong, Bin
Major Professor
Wenyu Huang
Committee Member
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Optical microscopy imaging of single molecules and single particles is an essential method for studying fundamental biological and chemical processes at the molecular and nanometer scale. The best spatial resolution (~ λ/2) achievable in traditional optical microscopy is governed by the diffraction of light. However, single molecule-based super-localization and super-resolution microscopy imaging techniques have emerged in the past decade. Individual molecules can be localized with nanometer scale accuracy and precision for studying of biological and chemical processes.

The obtained spatial resolution for plant cell imaging is not yet as good as that achieved in mammalian cell imaging. Numerous technical challenges, including the generally high fluorescence background due to significant autofluorescence of endogenous components, and the presence of the cell wall (> 250 nm thickness) limit the potential of super-resolution imaging in studying the cellular processes in plants. Here variable-angle epi-fluorescence microscopy (VAEM) was combined with localization based super-resolution imaging, direct stochastic optical reconstruction microscopy (dSTORM), to demonstrate imaging of cortical microtubule (CMT) network in the Arabidopsis thaliana root cells with 20 – 40 nm spatial resolution for the first time. With such high spatial resolution, the subcellular organizations of CMTs within single cells, and different cells in many regions along the root, were analyzed quantitatively.

Nearly all of these technical advances in super-localization and super-resolution microscopy imaging were originally developed for biological studies. More recently, however, efforts in super-resolution chemical imaging started to gain momentum. New discoveries that were previously unattainable with conventional diffraction-limited techniques have been made, such as a) super-resolution mapping of catalytic reactions on single nanocatalysts and b) mechanistic insight into protein ion-exchange adsorptive separations. Furthermore, single molecules and single particles were localized with nanometer precision for resolving the dynamic behavior of single molecules in porous materials. This work uncovered the heterogeneous properties of the pore structures. In this dissertation, the coupling of molecular transport and catalytic reaction at the single molecule and single particle level in multilayer mesoporous nanocatalysts was elucidated. Most previous studies dealt with these two important phenomena separately. A fluorogenic oxidation reaction of non-fluorescent amplex red to highly fluorescent resorufin was tested. The diffusion behavior of single resorufin molecules in aligned nanopores was studied using total internal reflection fluorescence microscopy (TIRFM).

To fully understand the working mechanisms of biological processes such as stepping of motor proteins requires resolving both the translational movement and the rotational motions of biological molecules or molecular complexes. Nanoparticle optical probes have been widely used to study biological processes such as membrane diffusion, endocytosis, and so on. The greatly enhanced absorption and scattering cross sections at the surface plasmon resonance (SPR) wavelength make nanoparticles an ideal probe for high precision tracking. Furthermore, gold nanorods (AuNRs) were used for resolving orientation changes in all three dimensions. The translational and rotational motions of AuNRs in glycerol solutions were tracked with fast imaging rate up to 500 frames per second (fps) in reflected light sheet microscopy (RLSM). The effect of imaging rates on resolving details of single AuNR motions was studied.