Detection of Single-Molecule Optical Absorption at Room Temperature and Mechanistic Study of Transcriptional Bursting

Advances in optical imaging techniques have allowed quantitative studies of many biological systems. This dissertation elaborates on our efforts in both developing novel imaging modalities based on detection of optical absorption and applying high-sensitivity fluorescence microscopy to the study of biology.;Although fluorescence is the most widely used optical contrast mechanism in biological studies because of its background-free detection, many intracellular molecules are intrinsically non-fluorescent and difficult to label without disturbing their natural functions. Therefore, label-free imaging methods based on contrast mechanisms other than fluorescence are highly desirable. In the first part of this dissertation, we explore new methods for sensitive detection of optical absorption. Chapter 2 describes stimulated emission microscopy and two-photon excited photothermal microscopy. We demonstrate label-free imaging of non-fluorescent biological chromophores with high sensitivity and three-dimensional optical sectioning capability. In Chapter 3, we introduce ground-state depletion microscopy and demonstrate room-temperature detection of the absorption signal from a single molecule. This measurement represents the ultimate detection sensitivity of nonlinear optical spectroscopy at room temperature.;In the second part of my thesis, I focus on transcription of DNA. Transcription is the first step in gene expression and essential for all cell functions. Recent experiments have shown that transcription of highly expressed genes occurs in stochastic bursts. But the origin of such ubiquitous phenomenon was unknown. We investigate the mechanism in bacteria through a series of in vitro and live-cell experiments based on high-sensitivity fluorescence microscopy. Chapter 4 describes a novel high-throughput in vitro single-molecule assay to follow real-time transcription on individual DNA templates. Using this assay, we show in Chapter 5 that positive supercoiling buildup on a DNA segment by transcription slows down transcription elongation and eventually stops transcription initiation. Transcription can be resumed upon gyrase binding to the DNA segment. Furthermore, using single-cell mRNA counting fluorescence in situ hybridization (FISH) assay, we find the extent of transcriptional bursting depends on the intracellular gyrase concentration. Together, these findings prove that transcriptional bursting of highly expressed genes in bacteria is primarily caused by reversible gyrase dissociation from and rebinding to a DNA segment, changing the supercoiling level of the segment.