Fluorogenic probes, small-molecule sensors that unmask brilliant fluorescence upon exposure to specific stimuli, are essential tools for chemical biology. Probes that detect enzymatic activity can be used to illuminate the complex dynamics of biological processes at a level of spatiotemporal detail and sensitivity unmatched by other techniques. This dissertation describes the development of new fluorophore chemistries to expand our current fluorogenic probe toolkit and the subsequent application of these probes to study dynamic cell transport processes. Chapter 1. Enzyme-Activated Fluorogenic Probes for Live-Cell and In Vivo Imaging. Chapter 1 reviews recent advances in enzyme-activated fluorogenic probes for biological imaging, organized by enzyme classification. This review surveys recent masking strategies, different modes of enzymatic activation, and the breadth of current and future probe applications. Key challenges, such as probe selectivity and spectroscopic requirements, are described in this chapter along with therapeutic and diagnostic opportunities that can be accessed by surmounting these challenges. Chapter 2. Electronic and Steric Optimization of Fluorogenic Probes for Biomolecular Imaging. In many fluorogenic probes, the intrinsic fluorescence of a small-molecule fluorophore is masked by ester masking groups until entry into a cell, where endogenous esterases catalyze the hydrolysis of esters, generating fluorescence. The susceptibility of masking groups to spontaneous hydrolysis is a major limitation of these probes. Previous attempts to address this problem have incorporated auto-immolative linkers at the cost of atom economy and synthetic adversity. In this chapter, I report on a linker-free strategy that employs adventitious electronic and steric interactions in easy-to-synthesize probes. I find that halogen-carbonyl n-->[pi]* interactions and acyl group size are optimized in 2',7'-dichlorofluorescein diisobutyrate. This probe is relatively stable to spontaneous hydrolysis but is a highly reactive substrate for esterases both in vitro and in cellulo, yielding a bright, photostable fluorogenic probe with utility in biomolecular imaging. Chapter 3. Cellular Uptake of Large Monofunctionalized Dextrans. Dextrans are a versatile class of polysaccharides with applications that span medicine, cell biology, food science, and consumer goods. In Chapter 3, I apply the electronically stabilized probe described in Chapter 2 to study the cellular uptake of a new type of large monofunctionalized dextran that exhibits unusual properties: efficient cytosolic and nuclear uptake. This dextran permeates various human cell types without the use of transfection agents, electroporation, or membrane perturbation. Cellular uptake occurs primarily through active transport via receptor-mediated processes. These monofunctionalized dextrans could serve as intracellular delivery platforms for drugs or other cargos. Chapter 4. Paired Nitroreductase-Probe System to Quantify the Cytosolic Delivery of Biomolecules. Cytosolic delivery of large biomolecules is a significant barrier to therapeutic applications of CRISPR, RNAi, and biologics such as proteins with anticancer properties. In Chapter 4, I describe a new paired enzyme-probe system to quantify cytosolic delivery of biomolecules-a valuable resource for elucidating mechanistic details and improving delivery of therapeutics. I designed and optimized a nitroreductase fusion protein that embeds in the cytosolic face of outer mitochondrial membranes, providing several key improvements over unanchored reporter enzymes. In parallel, I prepared and assessed a panel of nitroreductase-activated probes for favorable spectroscopic and enzymatic activation properties. Together, the nitroreductase fusion protein and fluorogenic probes provide a rapid, generalizable tool that is well-poised to quantify cytosolic delivery of biomolecules. Chapter 5. Future Directions. This chapter outlines several future directions for expanding the scope of fluorogenic probes and developing new biological applications. Additionally, Chapter 5 is followed by an appendix describing a tunable rhodol fluorophore scaffold for improved spectroscopic properties and versatility. Overall, the work described in this thesis illustrates the power of enzyme-activated fluorogenic probes to provide fresh insight into dynamic biological processes, with direct implications for improved therapeutic delivery.

Although dimerization is common process in protein biology enabling to discover new functionalities. Inspired by nature, we used dimerization approach to expand the toolbox of bright fluorogenic probes for selective imaging of live-cell biological events. The strategy was based on intramolecular self-aggregation of dyes, in particular homodimerization of the fluorophore via short linkage and tethered targeted ligand. We developed a family of fluorogens based of rhodamine scaffolds spanning their fluorescence from green to far-red region, named Gemini. In collaboration, we developed a bright photostable fluoromodule, Gemini-651/o-Coral, for RNA live-cell imaging. Systematic analysis of structure-activity relationship in dimerization probe design enabled the development of the probe for discrimination of cancer cells expressing biotin receptors on the cell surface. While being modular, dimerization-caused quenching approach provides bright activated fluorescence response of probes to biological event of interest. We showed its broad applicability in field of semisynthetic RNA/peptide-based technologies and targeted protein imaging. Further expansion of dimerized fluorogenic probes will significantly contribute to understanding the complexity of biological processes in living systems.

Mots-clés de l'auteur: Fluorophores ; Fluorogenicity ; Fluorogenic probes ; Live-cell imaging ; STED ; Centrosomes ; Plk4.

This detailed volume provides in-depth protocols for protein labeling techniques and applications, with an additional focus on general background information on the design and generation of the organic molecules used for the labeling step. Chapters provide protocols for labeling techniques and applications, with an additional focus on general background information on the design and generation of the organic molecules used for the labeling step. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Site-Specific Protein Labeling: Methods and Protocols provides a comprehensive overview on the most relevant and established labeling methodologies, and helps researchers to choose the most appropriate labeling method for their biological question.

This open access book describes marked advances in imaging technology that have enabled the visualization of phenomena in ways formerly believed to be completelyimpossible. These technologies have made major contributions to the elucidation of the pathology of diseases as well as to their diagnosis and therapy. The volume presents various studies from molecular imaging to clinical imaging. It also focuses on innovative, creative, advanced research that gives full play to imaging technology inthe broad sense, while exploring cross-disciplinary areas in which individual research fields interact and pursuing the development of new techniques where they fuse together. The book is separated into three parts, the first of which addresses the topic of visualizing and controlling molecules for life. Th e second part is devoted to imaging of disease mechanisms, while the final part comprises studies on the application of imaging technologies to diagnosis and therapy. Th e book contains the proceedings of the 12th Uehara International Symposium 2017, “Make Life Visible” sponsored by the Uehara Memorial Foundation and held from June 12 to 14, 2017. It is written by leading scientists in the field and is an open access publication under a CC BY 4.0 license.

Ultrafast Dynamics at the Nanoscale provides a combined experimental and theoretical insight into the molecular-level investigation of light-induced quantum processes in biological systems and nanostructured (bio)assemblies. Topics include DNA photostability and repair, photoactive proteins, biological and artificial light-harvesting systems, plasmonic nanostructures, and organic photovoltaic materials, whose common denominator is the key importance of ultrafast quantum effects at the border between the molecular scale and the nanoscale. The functionality and control of these systems have been under intense investigation in recent years in view of developing a detailed understanding of ultrafast nanoscale energy and charge transfer, as well as fostering novel technologies based on sustainable energy resources. Both experiment and theory have made big strides toward meeting the challenge of these truly complex systems. This book, thus, introduces the reader to cutting-edge developments in ultrafast nonlinear optical spectroscopies and the quantum dynamical simulation of the observed dynamics, including direct simulations of two-dimensional optical experiments. Taken together, these techniques attempt to elucidate whether the quantum coherent nature of ultrafast events enhances the efficiency of the relevant processes and where the quantum–classical boundary sets in, in these high-dimensional biological and material systems. The chapters contain well-illustrated accounts of the authors’ research work, including didactic introductory material, and address a multidisciplinary audience from chemistry, physics, biology, and materials sciences. The book is, therefore, a must-have for graduate- and postgraduate-level researchers who wish to learn about molecular nanoscience from a combined spectroscopic and theoretical viewpoint.

Geared towards research scientists in structural and molecular biology, biochemistry, and biophysics, this manual will be useful to all who are interested in observing, manipulating and elucidating the molecular mechanisms and discrete properties of macromolecules.

Stimulated Raman Scattering Microscopy: Techniques and Applications describes innovations in instrumentation, data science, chemical probe development, and various applications enabled by a state-of-the-art stimulated Raman scattering (SRS) microscope. Beginning by introducing the history of SRS, this book is composed of seven parts in depth including instrumentation strategies that have pushed the physical limits of SRS microscopy, vibrational probes (which increased the SRS imaging functionality), data science methods, and recent efforts in miniaturization. This rapidly growing field needs a comprehensive resource that brings together the current knowledge on the topic, and this book does just that. Researchers who need to know the requirements for all aspects of the instrumentation as well as the requirements of different imaging applications (such as different types of biological tissue) will benefit enormously from the examples of successful demonstrations of SRS imaging in the book. Led by Editor-in-Chief Ji-Xin Cheng, a pioneer in coherent Raman scattering microscopy, the editorial team has brought together various experts on each aspect of SRS imaging from around the world to provide an authoritative guide to this increasingly important imaging technique. This book is a comprehensive reference for researchers, faculty, postdoctoral researchers, and engineers. - Includes every aspect from theoretic reviews of SRS spectroscopy to innovations in instrumentation and current applications of SRS microscopy - Provides copious visual elements that illustrate key information, such as SRS images of various biological samples and instrument diagrams and schematics - Edited by leading experts of SRS microscopy, with each chapter written by experts in their given topics

Fluorescence Microscopy is a precise and widely employed technique in many research and clinical areas nowadays. Fluorescence Microscopy In Life Sciences introduces readers to both the fundamentals and the applications of fluorescence microscopy in the biomedical field as well as biological research. Readers will learn about physical and chemical mechanisms giving rise to the phenomenon of luminescence and fluorescence in a comprehensive way. Also, the different processes that modulate fluorescence efficiency and fluorescence features are explored and explained.

Life scientists believe that life is driven, directed, and shaped by biomolecules working on their own or in concert. It is only in the last few decades that technological breakthroughs in sensitive fluorescence microscopy and single-molecule manipulation techniques have made it possible to observe and manipulate single biomolecules and measure their individual properties. The methodologies presented in Single Molecule Techniques: Methods and Protocols are being applied more and more to the study of biologically relevant molecules, such as DNA, DNA-binding proteins, and motor proteins, and are becoming commonplace in molecular biophysics, biochemistry, and molecular and cell biology. The aim of Single Molecule Techniques: Methods and Protocols is to provide a broad overview of single-molecule approaches applied to biomolecules on the basis of clear and concise protocols, including a solid introduction to the most widely used single-molecule techniques, such as optical tweezers, single-molecule fluorescence tools, atomic force microscopy, magnetic tweezers, and tethered particle motion. Written in the highly successful Methods in Molecular BiologyTM series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and accessible, Single Molecule Techniques: Methods and Protocols serves as an ideal guide to scientists of all backgrounds and provides a broad and thorough overview of the exciting and still-emerging field of single-molecule biology.