A variety of contemporary analytical platforms in technical and biological applications take advantage of labeling the objects of interest with fluorescent or luminescent tracers. Luminescent tracers take advantage of the unique property of some lanthanide metals to absorb and emit light. Long lifetime of lanthanide emission allows temporal gating of the signal, which avoids the short-lived background of interfering sample components. This property in combination with large Stokes shift contributes to extreme sensitivity of detection (ca. 10−13-10−14 M), which makes lanthanide-based probes suitable for large variety of challenging tasks ( e.g., intracellular detection of single DNA/RNA, or protein molecules, microbial pathogen detection in human specimens, tracing analysis, etc.). Luminescent probes include antenna fluorophore that absorbs light and transfers the excitation energy to a lanthanide metal tethered to antenna through chelation. The probe also contains crosslinking group that allows covalent labeling of the molecule of interest. Despite great potentials of lanthanide-based tracers the wide spread of the technology is impeded by very high price of commercially available probes to their complex structure. The goal is to develop novel approaches for the synthesis of lanthanide probes with improved quantum efficiency. This work discusses development of new strategies for synthesis of antena-fluorophores, development of new methods for introduction of the crosslinking groups in the luminescent probes and elucidates the mechanisms of chemical reactions leading to principal synthetic intermediates. New quinoline and quinolone based fluorescent compounds were synthesized, whose light emission can be conveniently tuned by simple structural modifications. Developed probes represent high-quantum yield, large Stokes shift fluorophores with amine-reactive and click-reactive groups convenient for conjugation. Some of these compounds can be used as sensitizers for lanthanide emission in design of highly sensitive luminescent probes. Obtained probes demonstrate efficient derivatization reactions allowing introduction of amine- or click-reactive crosslinking groups into the fluorophores. The reactivity of synthesized compounds is confirmed in reaction with low molecular weight nucleophiles as well as with click-reactive DNA-oligonucleotide counterparts. These reactive derivatives can be used for covalent attachment of the fluorophores to various biomolecules of interest including nucleic acids, proteins, live cells and small cellular metabolites. Synthesized compounds were characterized using N MR, steady-state and time-resolved fluorescence spectroscopy, as well UV absorption spectroscopy. This work also discusses the development of fluorescent derivatives of the antifungal drugs, posaconazole and caspofungin, for diagnostic imaging of fungal cells. Invasive fungal infections (IFI's) are a growing threat to human health particularly, with increase in the number of immune-compromised patient population, such as organ transplant recipients, al logeneic B MT, hematologic cancers; AIDS etc. The diagnosis of fungal infections is a complicated task and requires a combination of clinical observations, laboratory investigation, and radiological or other diagnostic imaging methods. Only 25% of I FI cases are diagnosed pre-mortem due to the current diagnostic challenges, which justifies the development of express diagnostic procedures. To address the issue fluorescent derivatives of the antifungal drugs, posaconazole and caspofungin, were synthesized. The fluorescent derivatives retained strong and highly specific binding to their cellular targets rendering the cells fluorescent. This new affinity-based approach strongly facilitates the detection of fungal pathogen thereby overcoming the current diagnostic challenges and can be used for clinical diagnostics. The power of this approach is not limited to fungal pathogens, but in fact represents a broader platform useful for detection of other classes of infections, both in the biological specimens and in the whole body using contemporary 3-D imaging approaches like fluorescence based in vivo imaging, luminescence imaging, positron emission tomography approach, and computer-assisted X-ray tomography.

The use of fluorescent and luminescent probes to measure biological function has increased dramatically since publication of the First Edition due to their improved speed, safety, and power of analytical approach. This eagerly awaited Second Edition, also edited by Bill Mason, contains 19 new chapters and over two thirds new material, and is a must for all life scientists using optical probes.The contents include discussion of new optical methodologies for detection of proteins, DNA and other molecules, as well as probes for ions, receptors, cellular components, and gene expression. Emerging and advanced technologies for probe detection such as confocal laser scanning microscopy are also covered. This book will be essential for those embarking on work in the field or using new methods to enhance their research.TOPICS COVERED:* Single and multiphoton confocal microscopy* Applications of green fluorescent protein and chemiluminescent reporters to gene expression studies* Applications of new optical probes for imaging proteins in gels * Probes and detection technologies for imaging membrane potential in live cells* Use of optical probes to detect microorganisms* Raman and confocal raman microspectroscopy* Fluorescence lifetime imaging microscopy* Digital CCD cameras and their application in biological microscopy

Fluorescent nucleic acid probes, which use energy transfer, include such constructs as molecular beacons, molecular break lights, Scorpion primers, TaqMan probes, and others. These probes signal detection of their targets by changing either the intensity or the color of their fluorescence. Not surpr- ingly, these luminous, multicolored probes carry more flashy names than their counterparts in the other fields of molecular biology. In recent years, fluor- cent probes and assays, which make use of energy transfer, have multiplied at a high rate and have found numerous applications. However, in spite of this explosive growth in the field, there are no manuals summarizing different p- tocols and fluorescent probe designs. In view of this, the main objective of Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols is to provide such a collection. Oligonucleotides with one or several chromophore tags can form fluor- cent probes capable of energy transfer. Energy transport within the probe can occur via the resonance energy transfer mechanism, also called Förster tra- fer, or by non-Förster transfer mechanisms. Although the probes using Förster transfer were developed and used first, the later non-Förster-based probes, such as molecular beacons, now represent an attractive and widely used option. The term “fluorescent energy transfer probes” in the title of this book covers both Förster-based fluorescence resonance energy transfer (FRET) probes and probes using non-FRET mechanisms. Energy transfer probes serve as molecule-size sensors, changing their fluorescence upon detection of various DNA reactions.

This volume introduces different concepts and methods of detecting RNA in biological material in a variety of model systems. The chapters in this book discuss methods that will answer numerous biological questions that arise in the study of RNAs. Some of the topics covered in this book are single mRNA molecule detection in embryos and neurons; detection of mRNA and associated molecules by ISH-IEM on frozen sections; optimizing molecular beacons for intracellular analysis of RNA; imaging translation dynamics of single mRNA molecules in live cells; preparation of high-throughput sequencing libraries; and capturing RNA binding proteins in embryos and in cell-culture. 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. Cutting-edge and comprehensive, RNA Detection: Methods and Protocols is a valuable resource for novel and experiences scientist in the expanding field of RNAs.

Optical probes, particularly the fluorescent varieties, enable researchers to observe cellular events in real time and with great spatial resolution. Optical Probes in Biology explores the diverse capabilities of these powerful and versatile tools and presents various approaches used to design, develop, and implement them. The book examines the use

Biological O2 sensing probes and measurement techniques were first introduced in the late 80s. In the last 3-5 years they have undergone major development that have made them available and affordable for a broad range of applications in various disciplines of the life and biomedical sciences. These new chemistries and technologies, which are significantly different from the majority of other fluorescence-based probes and detection techniques, have already demonstrated their high utility. This book will provide a systematic overview of the existing and emerging O2 sensing technologies in their different modifications, a practical guide to their rational selection and use, and examples of biological applications/case studies, including details on how to set up and conduct such experiments, troubleshoot and interpret the data.

Chemical Tools for Imaging, Manipulating, and Tracking Biological Systems: Diverse Methods for Optical Imaging and Conjugation, Volume 639, the latest release in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. Chapters in this new release include Fluorogenic detection of protein aggregates in live cells using the AggTag method, Synthesis and Application of Ratiometric Probes for Hydrogen Peroxide Detection, Chemical Tools for Multicolor Protein FRET with Tryptophan, Fluorescing Isofunctional Ribonucleosides for Adenosine Deaminase Activity and Inhibition, Temporal profiling establishes a dynamic S-palmitoylation cycle, Solvation-guided design of fluorescent probes for discrimination of amyloids, and much more. Provides the authority and expertise of leading contributors from an international board of authors Presents the latest release in the Methods in Enzymology series Includes the latest information on retinoid signaling pathways

Biological Nanostructures and Applications of Nanostructures in Biology: Electrical, Mechanical, and Optical Properties contains reviews and discussions of contemporary and relevant topics dealing with the interface between the science and technology of nanostructures and the science of biology. Moreover, this book supplements these past groundbreaking discoveries with discussions of promising new avenues of research that reveal the enormous potential of emerging approaches in nanobiotechnology. The topics include: - Biomedical applications of semiconductor quantum dots, - Integrating and tagging biological structures with nanoscale quantum dots, - Applications of carbon nanotubes in bioengineering, - Nanophysical properties of living cells, - Bridging natural nanotubes with fabricated nanotubes, - Bioinspired approaches to building nanoscale devices and systems, - Hairpin formation in polynucleotides. This state-of-the-art survey of key developments in nanotechnology - as they apply to bioengineering and biology - is essential reading for all academics, biomedical engineers, medical physicists, and industry professionals wishing to take advantage of the latest developments and highly-promising discoveries in nanoscience underlying applications in bioengineering and biology.

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.

Fluorescence is the most popular technique in chemical and biological sensing and this book provides systematic knowledge of basic principles in the design of fluorescence sensing and imaging techniques together with critical analysis of recent developments. Its ultimate sensitivity, high temporal and spatial resolution and versatility enables high resolution imaging within living cells. It develops rapidly in the directions of constructing new molecular recognition units, new fluorescence reporters and in improving sensitivity of response, up to the detection of single molecules. Its application areas range from the control of industrial processes to environmental monitoring and clinical diagnostics. Being a guide for students and young researchers, it also addresses professionals involved in basic and applied research. Making a strong link between education, research and product development, this book discusses prospects for future progress.