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.

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

This book provides systematic knowledge of basic principles in the design of fluorescence sensing and imaging techniques together with critical analysis of recent developments. Fluorescence is the most popular technique in chemical and biological sensing because of its ultimate sensitivity, high temporal and spatial resolution and versatility that enables imaging within the 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 detection of single molecules. Its application areas range from control of industrial processes to environment monitoring and clinical diagnostics. Being a guide for students and young researchers, it also addresses professionals involved in active basic and applied research. Making a strong link between education, research and product development, this book discusses prospects for future progress.

The increased use of fluorescence techniques is greatly enhanced by the improved instrumentation pioneered by inventive scientists and now made available commercially by several high-tech companies. Moreover, the design and development of many new molecular probes with higher selectivity for specific microenvironmental properties has stimulated many new researchers to employ fluorescence techniques for solving their problems. This topic book, the second in his series, reflects this exciting scientific progress and deals, among others, with new approaches and new probes in fluorescence spectroscopy, single molecule fluorescence, applications in biomembrane and enzyme studies and imaging of living cells.

The April 1997 conference held in Prague attracted the cream of primarily European and Russian researchers (with a handful from the US, primarily from the U. of Maryland School of Medicine) to the burgeoning biological and medical applications of innovative optical technology, particularly laser confocal fluorescence microscopy. Six invited review lectures illuminate these recent developments and their role in cell biology, including computer simulations of molecular behavior. The 34 other papers focus on the major themes of: new developments in fluorescence instrumentation; fluorescent probes; nucleic acid labels; and other fluorescent labels, markers, and fluorogenic substrates. Annotation copyrighted by Book News, Inc., Portland, OR

Abstract : Lysosome is an acidic membrane-bound organelle containing more than 70 hydrolytic enzymes that breaks down different biological macromolecules. Substantial lysosomal pH disruption can cause lysosome malfunction and consequently lead to lysosomal storage disease. Therefore, it is essential to precisely monitor lysosomal pH changes in order to explore cellular functions and get insightful understanding of physiological and pathological processes. Fluorescence imaging based on fluorescent probes is a powerful technique to monitor lysosomal pH changes because of advantageous features including operational simplicity, high sensitivity, non-invasive approach, and high spatial resolution. However, most of the reported lysosomal pH probes are based on Stokes-shift fluorescence with lower energy emission under higher energy excitation, and exhibit less than 600 nm absorption and emission wavelengths, which causes cellular and tissue photodamage and contains biological fluorescence background. In order to avoid these issues, we developed near-infrared fluorescent probes based on single-photon anti-Stokes fluorescence with near-infrared excitations and emissions. We significantly improved biocompatibility and water-solubility of fluorescent probes by introducing mannose residues to the fluorophores through oligo(ethylene glycol) tethered spacers for sensitive detection of lysosomal pH changes in two near-infrared channels. In order to take advantage of ratiometric and near-infrared imaging to overcome systematic errors of intensity-based fluorescent probes caused by probe concentration variation and uneven distribution, temperature, solvent polarity, and excitation light fluctuation, we developed ratiometric near-infrared fluorescent probes for ratiometric detection of lysosomal pH changes by introducing tetraphenylethene (TPE) dyes to hemicyanine dyes. Gradual lysosomal pH decreases result in gradual increases of hemicyanine fluorescence, and corresponding concomitant decreases of TPE fluorescence. The probes allow for development of various ratiometric near-infrared fluorescent probes for quantitative and comparative reliable analyses of cations, reactive nitrogen, oxygen and sulfur species by conjugating various biosensing groups into the near-infrared hemicyanine moieties.

The rapid progress in sensor science in recent years has resulted in the development of fluorescence probes with enhanced analytical capabilities. Because of the vast evolution of this research field, therefore, we have decided to combine all the research articles published in "Fluorescence Probes and Sensors" for a Special Issue (SI) book of Sensors which was focusing on the important role sensors play in "Fluorescent Probes and Sensors". Fluorescence novel Probes make an ideal candidate for promising applications in biological analytes and environmental monitoring. Fluorescent probes along with metal complexes have been developed as a new class of fluorophores with excellent properties. This book illustrating the suitability of newly developed sensors for fluorescent analysis applications, as well as describing novel applications of established sensors in solving real life analytical problems.

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.

Providing much-needed information on fluorescence spectroscopy and microscopy, this ready reference covers detection techniques, data registration, and the use of spectroscopic tools, as well as new techniques for improving the resolution of optical microscopy below the resolution gap. Starting with the basic principles, the book goes on to treat fluorophores and labeling, single-molecule fluorescence spectroscopy and enzymatics, as well as excited state energy transfer, and super-resolution fluorescence imaging. Examples show how each technique can help in obtaining detailed and refined information from individual molecular systems.