Detection of Single-Molecule Optical Absorption at Room Temperature and Mechanistic Study of Transcriptional Bursting The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Chong, Shasha. 2014. Detection of Single-Molecule Optical Absorption at Room Temperature and Mechanistic Study of Transcriptional Bursting. Doctoral dissertation, Harvard University. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:12274110 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Detection of Single-Molecule Optical Absorption at Room Temperature and Mechanistic Study of Transcriptional Bursting A dissertation presented by Shasha Chong to The Department of Chemistry and Chemical Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Chemistry Harvard University Cambridge, Massachusetts April 2014 ©2014 - Shasha Chong. All rights reserved. Dissertation Advisor: Professor Xiaoliang Sunney Xie Shasha Chong Detection of Single-Molecule Optical Absorption at Room Temperature and Mechanistic Study of Transcriptional Bursting Abstract 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 iii 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. iv Contents Abstract ......................................................................................................................................... iii Table of Contents .......................................................................................................................... v Acknowledgements ....................................................................................................................... x Citation to Previously Published Work ................................................................................... xiii 1 Introduction .............................................................................................................................. 1 1.1 A Technical Introduction ...................................................................................................... 1 1.2 Pump-probe optical microscopy ........................................................................................... 1 1.3 Fluorescence Microscopy ..................................................................................................... 4 1.4 Structure of This Thesis ........................................................................................................ 9 References ................................................................................................................................. 12 2 Label-free imaging of biological chromophores ................................................................... 14 2.1 Background ......................................................................................................................... 15 2.1.1 Biological chromophores with undetectable fluorescence .......................................... 15 2.1.2 Stimulated emission ..................................................................................................... 15 2.1.3 Photothermal microscopy ............................................................................................ 16 2.2 Results and Discussion ....................................................................................................... 16 2.2.1 Principle of stimulated emission microscopy .............................................................. 16 2.2.2 Imaging biological chromophores with stimulated emission microscopy ................... 22 2.2.3 Principle of two-photon excited photothermal microscopy ......................................... 27 2.2.4 Imaging heme proteins with two-photon excited photothermal microscopy ............... 31 2.3 Materials and Methods ........................................................................................................ 35 v 2.3.1 Detailed apparatus of stimulated emission microscopy ............................................... 35 2.3.2 Detailed apparatus of two photon excited photothermal microscopy .......................... 37 2.3.3 Stimulated emission spectrum ..................................................................................... 38 2.3.4 Bacterial genetic constructs ......................................................................................... 39 2.3.5 Sample preparation for mammalian cell imaging ........................................................ 39 2.3.6 Sample preparation for animal tissue imaging ............................................................. 40 References ................................................................................................................................. 41 3 Detection of single-molecule optical absorption at room temperature with ground-state depletion microscopy .................................................................................................................. 44 3.1 Background ......................................................................................................................... 45 3.2 Results and Discussion ....................................................................................................... 47 3.2.1 Principle of ground-state depletion microscopy .......................................................... 47 3.2.2 Imaging gold nanoparticles with ground-state depletion microscopy ......................... 50 3.2.3 Detection of optical absorption from a single molecule at room temperature ............. 52 3.3 Conclusion .......................................................................................................................... 58 3.4 Materials and Methods ........................................................................................................ 59 3.4.1 Detailed apparatus of ground-state depletion microscopy ........................................... 59 3.4.2 Equations for modulation depth of the transmitted probe beam .................................. 60 3.4.3 Estimate of the ground-state depletion signal from a single gold nanoparticle ........... 62 3.4.4 Calculation of the ground-state depletion signal from a single Atto647N molecule ... 67 3.4.5 Calculation of shot noise .............................................................................................. 69 3.4.6 Modulation frequency dependence of ground-state depletion signals ......................... 69 3.4.7 Detectability of single-molecule ground-state depletion signals ................................. 71 vi References ................................................................................................................................. 72 4 A novel single-molecule assay to monitor real-time transcription on individual DNA templates