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.

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.