NEW IMAGING METHODS FOR FLUORESCENT LABELING AND STUDIES OF GENOMIC INTERACTIONS AT A SINGLE GENE LOCUS
Understanding how cells function at the molecular level is the major goal of modern biological research. Fluorescence microscopy provides a direct means of visualizing cellular structures, enzymatic reactions, chromosomal interactions as well as gene regulation at a single-cell level. An obstacle in microscopy is diffraction; which limits the resolution of conventional fluorescence microscopy to ~200 nm laterally and ~500 nm axially. More recently, researchers have developed new modes of microscopy that provide “super-resolution”, that is, they circumvent the diffraction limit. Using these new imaging techniques, we can now resolve structures at resolutions that are an order of magnitude improvement over the diffraction limit. This thesis presents several new imaging tools and methods we have developed in our lab to facilitate specific biological studies at high resolution at the single-cell level. Since the invention of super-resolution imaging techniques, the development of labeling methods for fluorescent imaging of non-fluorescent cellular structures has been a rate-limiting step. Here, I describe a novel labeling method developed for super-resolution imaging of cellular structures that utilizes RNA aptamers for labeling proteins with super-resolution compatible dyes. The aptamer tightly binds to a common protein tag - green fluorescent protein (GFP). Since there are so many cell-lines already created that express GFP tagged proteins, this new labeling method can be used to study many proteins of interest without selecting and verifying the binding of a new aptamer to the protein we want to study. I demonstrate the feasibility of this new labeling method by imaging proteins on the plasma membrane as well as proteins inside cells. Another technique we are developing is for use in studies of chromosomal interactions. There are assumptions about how genes are regulated through enhancer-promoter interactions; however, these assumptions are difficult to verify due to difficulties in visualizing the specific processes involved and difficulties in accurately identifying the regulatory sequences and proteins involved. In our new method, we combine locus-specific fluorescent labeling, 2-photon localized crosslinking and sequencing to help us achieve the goal of investigating chromosomal interactions. Lastly, I investigated the use of NADH imaging to studying how a cancer cell’s metabolic state may change as the cell squeezes through confined regions in a model system of cancer cell extravasation, which will provide new insights into cancer cell metastasis. In the study, I use microfluidic devices with constrictions of varying widths to mimic the type of constrictions cells experience as they leave and enter vessels. Since Nicotinamide adenine dinucleotide (NADH) is a cofactor involved in many metabolic reactions and is auto-fluorescent when excited with UV light (or 700 nm two-photon excitation), fluorescence lifetime and fluorescence anisotropy imaging techniques were used to monitor the metabolic state of the cells by measuring cellular NADH levels.
Zipfel, Warren R.
Lis, John T.; Feigenson, Gerald W.
Doctor of Philosophy
dissertation or thesis