Fluorescence microscopy has allowed for decades of elegant and compelling biological discovery. However, the diffraction limit of light caps the spatial resolution of fluorescence microscopy to 250-300 nanometers. As the size of an average protein molecule is ~ 3 nm, diffraction-limited spatial resolution is often more than an order of magnitude larger than what is required to resolve nanoscale cellular structures and processes. With the advent of single-molecule localization microscopy (SMLM) and super-resolution microscopy (SRM), the spatial resolution of fluorescence microscopy has been improved more than 10-fold relative to conventional methods. Further, the fundamental principles of SMLM have engendered multiple other experimental methods to simultaneously probe a second informational domain, in addition to precise spatial information. For example, much of the work in this writing utilizes a functional SRM method known as single-molecule diffusivity mapping (SMdM), which will be overviewed in Chapter 1.3. We use SMdM to show that in the mammalian cell, the assembly and disassembly of the vimentin cytoskeleton is highly sensitive to the protein net charge state. Starting with the intriguing observation that the vimentin cytoskeleton fully disassembles under hypotonic stress yet reassembles within seconds upon osmotic pressure recovery, we pinpoint ionic strength as its underlying driving factor. Further modulating the pH and expressing vimentin constructs with differently charged linkers, we converge on a model in which the vimentin cytoskeleton is destabilized by Coulomb repulsion when its mass-accumulated negative charges are less screened or otherwise intensified. Additionally, we identify a key molecular player, DELE1, in relaying mitochondrial stress to the cytosol and triggering the integrated stress response. Then, using SMdM we corroborate the aforementioned finding and show that the intraorganellar diffusivity of both DELE1 and cytochrome c implies the presence of unique electrostatic interactions in the mitochondrial intermembrane space. Together, these studies represent some of the first applications of SMdM to study native cellular proteins rather than exogenous tracer proteins.

Optical methods, including light and fluorescence microscopies, have seen widespread

application in the characterization of materials and biology, but are not equally applicable to all

such systems. This dissertation aims to describe new developments in select optical methods, and

their subsequent application to answering scientific questions about material and biological

systems that were intractable using previous methodologies. We first develop a novel label-free

interference-based optical method for characterizing thin films on transparent substrates and

subsequently apply it to reveal reaction dynamics of graphene films in real time. We next

develop novel methods in a different optical technique, point-localization super-resolution

fluorescence microscopy. These include the application of spectrally-resolved super-resolution

microscopy to organic adlayers on surfaces and the development of a remote focusing technique

to image oblique planes through thick biological samples. We then combine our developments in

optical methods and super-resolution microscopy to describe a novel correlative super-resolution

imaging methodology, and overview the challenges and opportunities inherent to correlative

super-resolution methods. We conclude with concrete applications of super-resolution

fluorescence microscopy to biological systems and shed light on long-standing biological

questions. These include elucidation of the structure of meiotic chromosome axes in C. Elegans,

a mechanism for crossover-interference and crossover-regulation in the same meiotic

chromosomes, discovery of a novel structural arrangement for nuclear pore complexes in meiotic

cells, and discovery of a tube-shaped structure spanning the nucleus of breast cancer-like cells.

The advent of super-resolution microscopy over the past decade has excited many researchers. Single-molecule localization microscopy (SMLM; to include specific implementations including STORM, (f)PALM, and PAINT), a branch of super-resolution microscopy, offers outstanding spatial resolution of ~20 nm through single-molecule imaging. Another exciting but often overlooked feature of SMLM is that when properly designed, it may enable ultrahigh-throughput characterization of individual molecules for multiple physical parameters.

The first part of this dissertation describes the multidimensional characterization of single molecules through SMLM. We introduce spectrally-resolved SMLM, which captures the positions and spectra of single fluorescent molecules with ultra-high throughput. Combined with a solvatochromic fluorescent dye, we demonstrate spatial mapping of the local chemical environment for heterogeneous systems. We thus present polarity mapping of lipid membranes in live cells and discuss the origin of the heterogeneity. Difference between the plasma membrane and organelle membrane is visualized in live cells, along with their structures and dynamics. We also characterize the structure, polarity, and chemical compositions of adsorbed organic layers on a surface. Besides nanoscale structures of the surface adlayer, we reveal their spontaneous demixing on the surface. Finally, we develop a novel approach for the multidimensional characterization of single molecules through machine learning, in which the parameters of interest are directly extracted from the unmodified diffraction pattern of single fluorescent molecules. We thus demonstrate concurrent spectral separation and axial localization for two fluorescent dyes in fixed cells under SMLM settings.

In Part II, we discuss multiple strategies to improve the performance of optical microscopy, for which efforts we make unusual use of the exceptional mechanical, electrical, and optical properties of graphene, a single layer of bonded carbon atoms. We first take advantage of the superior conductivity of graphene to facilitate correlated SMLM and electron microscopy, in which graphene works as a protective and conductive layer to enable electron microscopy for wet cells. Both the molecular specificity from SMLM and the high spatial resolution from electron microscopy are thus conveniently achieved. We next demonstrate a novel electroporation-microscopy system based on graphene, which enables the spatially and temporally controlled delivery of fluorescent probes of various sizes into live cells with high efficiency. Remarkably, the superior optical and electrical properties of the graphene allow us to electroporate and image the cells in the same device with precise control and high resolution, thus broadening the palette of fluorescent probe for live cell SMLM. Lastly, we present new methodologies to achieve high-contrast and high-throughput microscopy for graphene itself on transparent substrates. Whereas traditional, transmission microscopy only provide ~3% optical contrast for each layer of graphene, we develop an approach based on interference reflection microscopy (IRM) to achieve >10-fold higher contrast on various inorganic and polymer transparent substrates, and demonstrate high-throughput fast imaging with diffraction-limited resolution.

Conventional fluorescence microscopy, despite its high sensitivity, lacks resolution at a scale comparable to biomacromolecules due to the diffraction of light. Single-molecule-based super-resolution microscopy (SRM), developed in the past 15 years, has enabled researchers to visualize the structural organization of the cell at molecular resolution with high specificity. Nevertheless, the power of single-molecule measurement in resolving inter-molecular heterogeneities has not been fully explored by the SRM field. There is plenty of room for expanding the scope of SRM to access measurement domains beyond biomolecular structures, such as the heterogeneity of physicochemical properties within the cell, a conceptual framework named “functional super-resolution microscopy” (f-SRM). This dissertation describes the efforts by the author and colleagues in developing f-SRM for living cells and applying f-SRM to discover previously unknown physicochemical heterogeneities within the cell. Part I of this dissertation describes the design, development, and application of f-SRM, which has revealed novel nanoscale properties of the cell invisible to conventional SRM. Specifically, I elaborate on the experiments of fluorescence emission spectrum-encoded single-molecule measurements of chemical polarity and molecular diffusivity at nanoscale resolution. Part II concerns a fluorescence excitation-based strategy for functional imaging, which led to the unexpected discovery of a tubular ER-Golgi intermediate compartment (t-ERGIC) in transporting a specific group of soluble proteins. In-depth biochemistry and cell biology analyses elucidated the biogenesis mechanism of the t-ERGIC involving the cargo receptor.

Single-molecule localization based super-resolution imaging methods have seen considerable

development since their inception over a decade ago. Such advances have enabled countless

biological discoveries and have positioned the field of single-molecule localization microscopy

(SMLM) at the forefront of biological research. This dissertation describes the expansion of

conventional SMLM methodologies to facilitate multicolor, true-color, and correlative imaging,

and details specific applications for each of these developments. First, we utilize a split-emission

optical setup to enable facile multicolor super-resolution imaging of 3 or more fluorophores.

Then, we develop a novel technique in which a dispersive prism is used to obtain the entire

fluorescence spectra of millions of single molecules. Finally, we apply nonrigid image

registration techniques to enable the correlative imaging of live and fixed Drosophila larvae. We

then describe the application of advanced multicolor imaging methods to the study of

fundamental biological processes. These include the discovery of a novel organization of

membrane ion channels in sperm cells, the identification of proteins responsible for force

generation in mitotic spindles, and the characterization of new structure-function relationships in

vesicular transport pathways such as COPII protein trafficking and autophagy.

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.

In 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.

Altogether, 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.

Tauopathies, such as Alzheimer’s Disease (AD) and Progressive Supranuclear Palsy (PSP), are characterized by the central nervous system accumulation of misfolded tau protein. Endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) may be cellular mechanisms that cause tauopathies. As one of three major branches of UPR, PERK was identified as a genetic risk factor for AD and PSP in several GWAS studies. Upon activation, PERK attenuates protein translation to restore ER homeostasis but prolonged PERK activation can induce apoptosis. Here, we studied the role of PERK in tau aggregation in human AD and PSP brain tissues and in a cell culture model. We prepared protein lysates from the hippocampus region of advanced AD patient brains (Braak 6) and normal brains (Braak 1). We found more tau aggregated protein in Braak 6 brains in comparison to Braak 1 brains by immunoblotting. We also found a statistically significant increase of phosphorylated PERK in Braak 1 brains in comparison to Braak 6 brains in AD patients. This finding suggests that PERK activity is inversely proportional to insoluble misfolded tau aggregation. We did not find any correlation between PERK-haplotype and PERK functional in AD brains. We also prepared protein lysates from 3 different anatomic regions from PSP patient brains (midbrain, frontal cortex, and occipital cortex). The PSP results were highly variable due to technical problems, and we did not identify any reproducible correlations between tau levels and phosphorylated PERK levels.We further studied how PERK and tau interact utilizing HEK293 cells stably expressing TauRD-YFP (Biosensor cells) developed by Marc Diamond’s group as a cellular model of tau protein aggregation. We activated or inhibited PERK activity in TauRD biosensor cells by treating them with pharmacochemical small molecule modulators (GSK2656157, GSK2606414, salubrinal, ISRIB). We monitored tau aggregation by the formation of YFP fluorescent puncta in the cells. We found an inverse association between PERK pathway activation and tau aggregation. In summary, our brain and cell culture findings implicate PERK as a regulator of tau protein accumulation. PERK pathway activation may have high therapeutic potential for tauopathy treatment.

The past decades have witnessed the exponential growth of studies utilizing optical techniques to characterize graphene functionalization. The promise and potential application of graphene and related materials are substantially expanded through chemical functionalization. However, due to the fact that graphene is a single layer of carbon atoms, it is difficult to study the in situ dynamics of graphene chemistry. Moreover, the inertness of the graphene basal plane has notably limited its viable chemical modification pathways. This dissertation describes efforts by the author and colleagues to overcome these limitations. Part I of this dissertation demonstrates the direct optical visualization of in situ dynamics of graphene chemistry through interference reflection microscopy. Specifically, we uncover the unique dynamics of the redox reaction, diazonium reaction and solution-enclosing blister generation process of substrate-supported graphene at high spatiotemporal resolution. Part II of this dissertation reports facile approaches to the chemical modifications of the inert graphene basal plane under ambient conditions. Optical characterizations techniques including interference reflection microscopy, transmission microscopy, fluorescence microscopy, and Raman spectroscopy are utilized here to help establish the successful modifications of graphene through our facile approaches. These approaches include direct azidation and chlorination of the graphene basal plane through the electrochemical oxidation of an aqueous sodium azide and sodium chloride solution as well as a photocatalytic approach for the facile azidation and chemical patterning of graphene surface.

Intracellular diffusion defines the kinetics and timescales of biological processes. However, due to the fast motion of biomolecules and the sub-micron scale of intracellular structures, elucidating how unbound molecules diffuse through the cell is challenging. Here, I introduce a recently developed techniques, single-molecule displacement/diffusivity mapping (SMdM), which capitalizes on super-resolution capabilities of single-molecule localization microscopy (SMLM), while uniquely maps out the motion and diffusivity of freely diffusing molecules at the nanoscale.In this dissertation, I present my recent efforts to transform SMdM into a powerful quantification tool by leveraging high-throughput single-molecule statistics. I first investigated enzymatic diffusion during catalysis challenging existing theories by utilizing high statistics of sub-millisecond displacements of unhindered single enzymes freely diffusing in common buffers to quantify D for four enzymes under catalytic turnovers to show no significant changes in D during catalysis, and additionally formulated how ∼ ±1% D precisions can be achieved. By similarly leveraging high-throughput single-molecule statistics to determine D, in the subsequent study, I showed that SMdM can characterize D up to 350 μm2/s for water-soluble dyes and small-molecule metabolites. I unveil that small-molecule intracellular diffusion is dominated by vast regions of high diffusivity ~60-70% of that in vitro, lifting a paradoxical speed limit of intracellular diffusion as suggested by previous studies. Next, I uncover strong in vitro charge-driven protein-protein interactions replicating the sign-asymmetric charge effects on intracellular diffusivity discovered in mammalian cells. By converting between D to molecular weight, we were provide novel insight into how positively charged protein at the limit of low diffuser concentration avoid aggregation and coacervation inside of cytoplasm, by modulating the concentration of negatively charged protein. Here we showed the formation in situ that positively charged diffusers drag a single monolayer of negatively charged macromolecules forming a net negatively charged complex, shedding new insight into mechanisms that drive charged-based diffusion interactions inside of the cell. In collaboration with Rebecca Heald’s group, I work with Xenopus laevis extracts to study how both charge and molecular size impacts diffusivity of macromolecules in membrane-less cytoplasm for a wide range of protein and demonstrate that negatively charged macromolecules diffuse approximately 45% slower in extract, but positively charged macromolecules diffuse substantially slower. We then demonstrate how nonspecific RNA interactions drive the solubilization of positively charged macromolecules, where depletion of RNA resulted in release of positively charged ribosomes that resulted in cytoplasmic aggregation. Lastly, we show that the filamentous actin cytoskeleton induces macromolecular crowding demonstrating nanoscale intracellular structures shape diffusion processes inside of the cell. Collectively, by uniquely mapping out fast diffusion at the single-molecule and super-resolution levels, my research sheds new insight on the fundamental laws governing molecular transport within living cells.