A majority of biological microscopy investigations involve the focusing of visible light with conventional lenses. Fluorescence microscopy is one of the most widely used tools in biology but its resolution has historically suffered from the diffraction limit to about 200 nm laterally and 800 nm axially. In the past decade, this resolution problem has been overcome by the rapidly emerging field of super-resolution microscopy. The first demonstrated super-resolution technique, STimulated Emission Depletion (STED) Microscopy, is the topic of this Dissertation. This Dissertation has two primary areas of focus: the design optimization of a STED microscope, covered in Chapters 2-4, and its application to super-resolution imaging in cells and tissues, covered in Chapters 4-6. Chapter 2 describes the STED apparatus and experimental methods used. This chapter covers the guiding principles behind the design of a STED microscope, which forms a basis for understanding the logic underlying the homebuilt STED microscope which was constructed for this research. This STED microscope has a typical resolution of approximately 60 nm (full-width-at-half-maximum) or 25 nm (sigma) and has the sensitivity to image single fluorophores. In Chapter 3, a framework for evaluating and optimizing STED performance in the presence of several key tradeoffs is presented. Chapter 4 describes both developments in STED Microscopy required to utilize far-red-emitting dyes and the challenges associated with performing super-resolution imaging in intact Drosophila tissue. In Chapter 5, the optimization of labeling density revealed the 9-fold symmetry of a centriole protein structure, an important organelle in cell development. In Chapter 6, Huntingtin protein aggregates are resolved beyond the diffraction limit in a cell model of the neurodegenerative Huntington's disease.

The development of live cell RNA imaging techniques will lead to the unraveling of many important biological processes. To achieve this goal, there have been three different strategies developed. They are the development of small molecule probes, nucleic acid probes, and green fluorescent protein (GFP) probes. In the following thesis, the pros and cons of each approach are discussed, followed by a proposal to resolve the limitations. In the small molecule case, a probe was developed that utilized a quenched sulforhodamine dye. It was designed so that its structure can be rationally modified from the initial lead compound. An aptamer sequence that activates the sulforhodamine probe with micro molar affinity was found by in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX), followed by fluorescence screening in E.coli. The rational modification of the structure of the initial sulforhodamine probe resulted in an overall 33-fold increase in binding affinity compared to the initial lead compound. Instead of the chemical modification of the lead compound, the small molecule's cell permeability and binding affinity to the target could be improved by linking to cell penetrating peptides (CPP). A CPP is a short peptide sequence composed of poly arginine amino acids which shows excellent cell uptake and affinity to RNA. However, the use of the CPP-linked dye in live cell imaging has been limited by strong signals in the endosome region. An attempt was made to overcome this difficulty by linking a quencher molecule to the dye-CPP via a disulfide bond, which only breaks when it enters the cytosol. For the nucleic acid probe, the major problem was its low cell permeability and low signal-to-background ratio due to the low copy number of mRNA targets within the cell. We made mutant Hammerhead ribozymes and embedded them in a non-coding region of the GFP expression vector that can be transfected to mammalian cells. This modified Hammerhead ribozyme acts as a logic gate, and the signal is amplified by the expression of GFP in the presence of the target mRNA. In vitro and in vivo results are discussed. Finally, a fragmented GFP system, the fluorescence of which could be recovered by binding to a specific RNA tag, was developed. The major problem for the GFP-mediated RNA imaging system was the low signal-to-background ratio from the GFP probe that is not bound to the RNA tag. To find the non-fluorescent GFP, the GFP was truncated from the C-terminus such that it loses its fluorescence with minimum loss of amino acids. An RNA sequence that has high affinity to this GFP was found by in vitro SELEX. The subsequent E.coli screening found an RNA sequence that reactivates the fluorescence of the GFP probe.

Fluorescence microscopy is one of the most widely used tools in cell biology due its intrinsically high detection sensitivity coupled with the ability to genetically label proteins and other cellular structures with fluorescent tags. However, the resolution of fluorescence microscopy has historically been limited to about 200 nm laterally and 800 nm axially because of the diffraction limit of visible light. In the past five years, imaging below the diffraction limit ("super-resolution imaging") by localizing single fluorophores, one at a time (1-3), has opened a wide a variety of new biological systems for study. This Dissertation is a collection of both techniques for two and three dimensional super-resolution imaging as well as applications in bacterial and yeast imaging. References 1. Betzig E, et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313: 1642-1645. 2. Hess ST, Girirajan TPK & Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91: 4258-4272. 3. Rust MJ, Bates M & Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3: 793-795.

Vesicle fusion is a central process in transport and communication in biology. In neuronal transmission, synaptic vesicles carrying neurotransmitters dock and fuse to the plasma membrane of the neuron, a process mediated by a combination of several membrane anchored and soluble proteins. Fusion results in the merger of the two apposing lipid bilayers, leading to the exchange of both the lipidic and aqueous components. The fusion reaction is thought to proceed through several stages: first, the membranes are brought into close proximity (docking), second, the outer leaflets mix, but the inner leaflets and contents remain separate (hemi-fusion), and finally, the inner leaflet and contents exchange (full fusion). Due to the complex nature of the fusion reaction and the multitude of proteins involved, the mechanism of the fusion reaction is not well understood. Simplified model systems for vesicle fusion can bring insight into the mechanism by studying the fusion reaction in a more defined and controllable system. This thesis describes a DNA-based model for the protein fusion machinery. Previously, DNA-lipids were used to tether lipid vesicles to glass-supported lipid bilayers. These vesicles could be observed by fluorescence microscopy, and are laterally mobile along the plane parallel to the supported bilayer. DNA-mediated docking between vesicles was characterized, but fusion was not observed due to the fact that the DNA partners were both coupled at the 5' end, so antiparallel hybridization holds the membranes apart. In this work, a new synthesis of DNA-lipid conjugates is described which allows coupling at both the 3' and 5' end of the DNA. Incorporation of complementary DNA-lipids coupled at opposite ends mediates fusion between lipid vesicles. Vesicle fusion was measured in bulk fluorescence assays (Chapter 2 and 3), by both lipid mixing and content mixing assays. The rate of vesicle fusion showed a strong dependence on the number of DNA per vesicle, as well as the sequence of the DNA. Consistent with previous results measured for the docking reaction, fusion was faster for a repeating DNA sequence than for a non-repeating sequence that required full overlap of the strands for hybridization. The role of membrane proximity on the rate of vesicle fusion was investigated in Chapter 3 by insertion of a short spacer sequence at the membrane-proximal end of fusion sequences. The length of the spacer sequence was varied between two and 24 bases, corresponding to length scales of approximately 1-12 nm. Fusion, as measured in bulk assays by lipid and content mixing, decreases systematically as the membranes are held progressively further apart, demonstrating a clear dependence of the rate of the fusion reaction on membrane proximity. While the bulk vesicle fusion assays showed that DNA-lipids can mediate vesicle fusion, these ensemble measurements convolve the multiple steps (docking, hemifusion, and full fusion) of the fusion reaction, complicating any kinetic analysis. In order to image individual vesicle fusion events between tethered vesicles, a new tethering strategy was developed (Chapter 4). This strategy exploits the dependence of DNA hybridization on salt by covalently attaching lipid vesicles to a glass-supported lipid bilayer, then triggering DNA-mediated docking and fusion by spiking the salt concentration. The kinetics of individual vesicle fusion events were subsequently measured using a FRET-based lipid mixing assay for many vesicles (Chapter 6). An analysis of the distribution of waiting times from docking to fusion indicated that this transition occurs in a single step. A second model membrane architecture was used to study individual fusion events between vesicles and a planar bilayer (Chapters 5 and 6). This architecture uses a DNA-tethered planar free-standing bilayer as the target membrane. The kinetics of individual vesicle fusion events to this membrane patch were also consistent with a single step process, as for vesicle to vesicle fusion. In this system, it was also possible to observe content transfer of vesicles containing a self-quenched aqueous dye (Chapter 5). By analyzing the diffusion profile of the dye, it was shown that the dye indeed is transferred into the region below the planar membrane patch, and is not released into the solution above the patch due to vesicle rupture or leakage.

The number of reports per year on single-molecule imaging experiments has grown roughly exponentially since the first successful efforts to optically detect a single molecule were completed over two decades ago. Single-molecule spectroscopy has developed into a field that includes a wealth of experiments at room temperature and inside living cells. The fast growth of single-molecule biophysics has resulted from its benefits in probing heterogeneous populations, one molecule at a time, as well as from advances in microscopes and detectors. There is a need for new fluorophores that can be used for single-molecule imaging in biological media, because imaging in cells and in organisms require emitters that are bright and photostable, red-shifted to avoid pumping cellular autofluorescence, and chemically and photophysically tunable. To this end, we have designed and characterized fluorescent probes based on a class of nonlinear-optical chromophores termed DCDHFs. This dissertation describes various physical and optical studies on these emitters, from sensing local environment to photoactivation. Chapter 1 is a general introduction to fluorescence and single-molecule spectroscopy and imaging. Single-molecule experiments in living cells are discussed and probes used for such experiments are summarized and compared. Chapter 2 explores the basic photophysics of the DCDHF fluorophores and some general methods of measuring relevant spectroscopic parameters, including photostability. Chapter 3 discusses the various approaches we have taken to modify particular properties by changing the fluorophore's structure. We have redesigned the DCDHF fluorophore into a photoactivatable fluorogen -- a chromophore that is nonfluorescent until converted to a fluorescent form using light -- described in Chapter 4. Finally, a different, chemical route to fluorescence activation is presented in Chapter 5. The remainder of the Dissertation is the Appendix and a full Bibliography. The Appendix includes a table of photophysical parameter for DCDHF fluorophore, various protocols used in the experiments discussed, MatLab codes, and NMR spectra.

Cell division is a major developmental event in the life cycle of a bacterial cell. Caulobacter crescentus division is asymmetric, producing daughter cells that differ in morphology and polar features: a sessile stalked cell and a motile swarmer cell that subsequently differentiates into a stalked cell. In this work we investigate the assembly of the Caulobacter cell division machinery (the divisome) using genetics, biochemistry, and microscopy. In Caulobacter, the cell division process requires a set of approximately twenty-three proteins localizing from the cytoplasm to the outer membrane. To understand divisome assembly as a function of the cell cycle, we generated fluorescent fusions to analyze the temporal regulation of 19 representative divisome and division-site localized proteins. In Chapter 2, we identified a series of stages and transitions in divisome assembly and the associated events yielding a comprehensive temporal picture of the process. The assembly interdependency for divisome formation in Caulobacter appears to involve cooperative rather than sequential recruitment, suggesting that it is a multiprotein subcomplex model. In Chapter 3, we describe our investigation of the Tol-Pal complex where we demonstrated that it plays a vital role for membrane integrity maintenance and that it is essential for viability. Cryo-electron microscope images of the Caulobacter cell envelope exhibited outer membrane disruption, and cells failed to complete cell division in TolA, TolB, or Pal mutant strains. The Tol-Pal complex is required to maintain the position of the transmembrane TipN polar marker, and indirectly the PleC histidine kinase, at the cell pole, but it is not required for the polar maintenance of other transmembrane and membrane-associated polar proteins tested. Thus, the Caulobacter trans-envelope Tol-Pal complex is a key component of cell envelope structure and function, mediating outer membrane constriction at the final step of cell division, as well as the positioning of a protein localization factor. In Chapter 4, we describe our examination of the FtsZ binding protein, ZapA. FtsZ is the most highly conserved divisome protein that polymerizes into a contractile ring near midcell, defining the future site of cell division. We showed that ZapA is required to maintain a normal cell length, and promotes Z ring assembly. The biochemical and functional studies suggest that Caulobacter ZapA is a positive regulator of Z-ring assembly. In summary, we have addressed three major stages in developments of the divisome in Caulobacter: Z-ring assembly, divisome maturation and outer membrane invagination. These experiments have provided a new understanding of how the Caulobacter cell temporally executes the cell division program to propagate reliably and how Caulobacter cell division is performed.

Since the first successful detection single molecules over two decades ago, single-molecule spectroscopy has developed into a burgeoning field with a wealth of experiments at room temperature and inside living cells. Probing asynchronous and heterogeneous populations in situ, one molecule at a time, is not only desirable, but critical for many biological questions. Further, super-resolution imaging based on sequential imaging of sparse subsets of single molecules, has seen explosive growth within the last five years. This dissertation describes both the application of live-cell single-molecule imaging as an answer to important biological questions, and development and validation of fluorescent probes for targeted super-resolution imaging.

Single-molecule, super-resolution fluorescence microscopy is a powerful technique for studying biological systems because it reveals details beyond the optical diffraction limit (on the 20-100 nm scale) such as structural and conformational heterogeneity. Further, single-molecule imaging measures distributions of behaviors directly through the interrogation of many individual molecules and reports on the nanoscale environment of molecules. Sub-diffraction imaging adds increased resolution to the advantages of fluorescence imaging over the techniques of atomic force microscopy and electron microscopy for studying biological structures, which include imaging of large fields of view in aqueous environments, specific identification of protein(s) of interest by fluorescent labeling, low perturbation of the system, and the ability to image living systems in near real-time (limited by the time required for super-resolution sequential imaging). This dissertation describes the application of single-molecule and super-resolution fluorescence imaging to studying the huntingtin (Htt) protein aggregates that are a hallmark of Huntington's disease and that have been implicated in the pathogenesis of the disease. The intricate nanostructures formed by fibrillar Htt aggregates in vitro and the sub-diffraction widths of individual fibers mark the amyloids as important targets for high-resolution optical imaging. The characterization of Htt aggregate species is critical for understanding the mechanism of Huntington's disease and identifying potential therapies. Following an introduction to single-molecule, super-resolution imaging and Huntington's disease in Chapter 1, Chapter 2 describes the single-molecule methods, experimental techniques, Htt protein sample preparations, and data analysis performed in this dissertation. Chapter 3 discusses the development of super-resolution imaging of Htt protein aggregates and the validation of the images by atomic force microscopy. Chapter 4 continues the study of Htt by one- and two-color super-resolution with imaging of Htt protein aggregates over time from the initial protein monomers to the large aggregate assemblies of amyloid fibers. In Chapter 5, I detail our progress to-date in studying the earliest stages of Htt aggregation using zero-mode waveguide technology. Chapter 6 concludes the dissertation with a discussion of the results from additional projects comprising the effect of chaperonin proteins on Htt aggregation, extension of super-resolution Htt imaging to three dimensions, and cellular imaging of Htt aggregates. The future directions for these exciting projects are summarized with the expectation that research efforts directed in these areas will contribute to our understanding of Htt aggregation and Huntington's disease.