The Science and Art of Super-Resolution Microscopy

It was the great and mythical Sherlock Holmes who famously said, “It has long been an axiom of mine that the little things are infinitely the most important”. Though we cannot see down to the molecular scale by eye, these fundamental building blocks of life and chemistry are of enormous interest to researchers and laypeople alike. Light microscopes constitute the oldest and most versatile tools to see what the human eye cannot; even so, the size of the smallest objects observable with such instruments was constrained to a “diffraction limit” by physical laws relating to the wave-nature of light. At first glance, attempting to circumvent the diffraction limit of light to improve the resolution of optical microscopes would seem an ill-advised endeavor, like trying to invent a perpetual motion device or time travel machine - fundamentally doomed by the laws of nature. Nonetheless, groundbreaking work that would have galvanized the likes of Mr. Holmes has recently transformed light microscopy into fluorescence nanoscopy, with orders of magnitude improved resolution over its technological predecessor. This nascent field, christened super-resolution microscopy (SRM), enables researchers to bring our understanding of physiological and chemical processes into much sharper focus./nIn the brief timespan since the invention of super-resolution techniques, remarkable advances involving them have been achieved: from improvements in methodological capabilities to nanoscopic discoveries within biological and non-biological systems. The work in this dissertation represents my own modest contribution to this burgeoning field. The narrative begins with a discussion of the historical and theoretical bases for SRM via single-molecule localization approaches. I then detail my part in the characterization and successful development of analytical methods that correlate SRM modalities with other imaging techniques. These methods include (1) graphene-enabled correlative SRM-SEM and (2) a correlative microscopy-spectroscopy technique we have termed spectrally-resolved STORM. We demonstrate that our multimodal approaches have substantial capacity for revealing a much more detailed and holistic picture of nanoscale features in living systems, and furthermore are remarkably facile to implement compared to similar techniques. The subsequent study makes use of SRM to study fluorescently-tagged graphene nanoribbons and carbon nanotubes with the aim of contributing to their eventual application in novel electronic devices. We find that SRM can reveal nanoscale features in complicated bundles of these graphenic materials, even in the context of suboptimal photophysical behavior on the part of the fluorophore. The remaining chapters are dedicated to my and my collaborators’ research into a number of biological systems. We describe the discovery of novel, periodic cytoskeletal nanoarchitectures of various stem cells as they develop into cells of the central nervous system. We also discuss the application of SRM to help understand the metabolic origins of Huntington’s Disease pathophysiology by quantifying changes in mitochondrial size and electron transport chain protein expression. Finally, we chronicle the membrane ultrastructure of platelets and their progenitor cells during development, and find significant points of disparity in comparison to closely-related erythrocytes./nAltogether, the explorations described in this opus underscore the exciting potential of SRM to reveal the formerly invisible and answer the formerly obscure. They furthermore lay the foundation for future methodological improvements and scientific discoveries. With such extraordinary tools at our disposal, the future of research in all arenas looks bright indeed.

Advisor: Xu, Ke