Download or read book Terbium based Time gated F rster Resonance Energy Transfer Imaging for Evaluating Protein protein Interactions on Cell Membranes written by Stina Linden and published by . This book was released on 2015 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: This thesis investigates the use of time-gated FRET microscopy for detection of colocalization of two membrane proteins, E- and N-cadherin. These proteins are important for cell-cell contacts and have an important role in the epithelial to mesenchymal transition (EMT), a key process in cancer metastasis. In EMT cells lose their epithelial markers (such as E-cadherin) and gain mesenchymal markers (such as N-cadherin), increasing their motility and invasiveness, enabling escape from the primary tumor into the bloodstream as so called circulating tumor cells (CTCs). This manuscript focuses on the detection of CTCs that have undergone partial EMT, displaying a hybrid phenotype (epithelial-mesenchymal) and co-express E- and N-cadherin, by FRET co-localization studies on a model cell line. FRET (Förster resonance energy transfer) is a non-radiative energy transfer between two molecules that are in resonance and in close proximity (ca. 1-20 nm). A co-localization of E- and N-cadherin in clusters would therefore be detectable by FRET. The staining of the cadherins was done by using specific antibodies labelled with a long lifetime donor, the terbium complex Lumi4-Tb (TbL4) from Lumiphore, Inc., and various acceptors. The long lifetime donor and long lifetime sensitized acceptor emission (FRET) could be imaged in a time-gated microscopy setup. Time -gated imaging has several advantages compared to steady state imaging in terms of efficient background suppression in biological samples. The setup described in this manuscript is based on the use of an intensified CCD camera, a pulsed UV-laser excitation source, and a defined (μs) delay between excitation and image acquisition. In addition to the E- and N-cadherin FRET experiments the time-gated FRET imaging microscopy was used to investigate different biological samples (intracellular and membrane located). Although both protein markers could be successfully imaged on the same cells, FRET between E- and N-cadherin or E- and E-cadherin could not be detected. Control experiments with antibodies against the same primary antibody revealed strong time-gated FRET signals due to binding of both donor and acceptor antibodies to the same primary antibodies. The successful time-gated imaging of two different antibodies separated by a few nanometers demonstrates the feasibility of probing protein-protein interaction and co-localization at membranes using TbL4 based time-gated FRET imaging. Microsecond time-gated imaging is especially interesting for the investigation of protein-protein interactions in highly autofluorescent biological samples such as cancer tissues.
Download or read book Fluorescence Resonance Energy Transfer Studies of Protein Interactions written by Sarah Friede Martin and published by . This book was released on 2008 with total page 424 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book F rster Resonance Energy Transfer from Terbium Complexes to Quantum Dots for Multiplexed Homogeneous Immunoassays and Molecular Rulers written by David Karl Wegner and published by . This book was released on 2015 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Förster resonance energy transfer (FRET) is a non-radiative energy transfer from a donor to an acceptor in close proximity. Due to its extremely sensitive distance dependence in the 1 - 20 nm range, FRET plays an important role in nanobiotechnology. Thereby FRET can be used as signal transduction system but also for the distance estimation between donor and acceptor. The selected FRET acceptors in this work were semiconductor nanocrystals (quantum dots, QDs). This type of luminophore is well known for its superior photophysical properties. Their strong and broad absorption and their bright, narrow-band, and size-tunable photoluminescence (PL) emission make QDs ideally suited for FRET application. Combing QDs as FRET acceptors with luminescent terbium complexes (LTC) as FRET donors offers exceptionally large Förster distances of more than 10 nm. The Förster distance is characteristic of a FRET pair and is the distance at which the FRET efficiency equals 50 %. A large Förster distance is desirable as it offers the detection of biological interactions over large distances. LTC are suitable FRET donors for QDs because they provide long excited-state lifetimes in the millisecond range. This long PL decay time enables time-gated measurements for the suppression of autofluorescence and PL of directly excited QDs, which strongly increases the detection sensitivity. Additionally, the structured PL emission bands of LTCs together with the size-tunable PL emission bands of QDs make this FRET pair ideal for the application in multiplexed diagnostics, which is the measurement of multiple biomarkers in a single sample.The PhD thesis consists of two parts. In the first part the LTC-QD FRET pair was used within homogeneous FRET immunoassays for the detection of the biomarkers prostate specific antigen (TPSA), neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), and epidermal growth factor receptor (EGFR). The immunoassay sensitivity was optimized using different types of antibodies IgG, F(ab')2,F(ab), and for EGFR single heavy chain antibodies, which differ largely in their size. The use of small-volume serum samples and measurements on clinical as well customized fluorescence plate readers result in picomolar detection limits for all measured biomarkers. In addition to these QD-based in vitro diagnostic tests, a detailed study of the different FRET-systems using time-resolved spectroscopy was performed. The investigation revealed the influence of the different antibodies on distance, functionality, and sensitivity of the FRET immunoassays. The study was completed by the measurement of NSE and CEA in a duplexed format and real patient samples were investigated.The second part was to use FRET for nanometric distance measurements as molecular or spectroscopic ruler. Time-resolved FRET measurements enabled the calculation of the distance between donor and acceptor. Therefore two different binding strategies were investigated to establish a close proximity between the LTC-donor to the QD-acceptor, namely biotin-streptavidin recognition and polyhistidine mediated self-assembly. A detailed time-resolved study was performed of QDs with different sizes, shapes, and surface coatings in combination with LTC bound to three different host biomolecules, which also possessed different sizes, shapes, orientations, and binding conditions. The analysis of the multi-exponential decay curves of donor and acceptor allowed to obtain information about the size, shape, and biofunctionality of the investigated QD bioconjugates. The results were in agreement with other structural analysis methods, such as transmission electron microscopy (TEM) or dynamic light scattering (DLS), but with the advantage of a homogeneous measurement with three-dimensional resolution (not possible for TEM), without the inclusion of a hydration shell (drawback for DLS), and at low concentration in the same environment as used for the biological application.
Download or read book Fluorescent Protein Resonance Energy Transfer for High throughput Screening written by Annalee Whitefield Nguyen and published by . This book was released on 2006 with total page 314 pages. Available in PDF, EPUB and Kindle. Book excerpt: The desire to track proteins and monitor their functions inside of living cells has driven the development of new tools and techniques capable of making these measurements. Fluorescent protein Forster resonance energy transfer (FRET) is an attractive choice for probing intracellular events, but the low efficiency of most available pairs limits the utility of the technique. To address this problem, a quantitative evolutionary strategy using fluorescence-activated cell sorting was applied to optimize a cyan-yellow fluorescent protein pair for FRET. Multiple rounds of mutation, followed by sorting of the leading edge of the cell population enabled step-wise evolution of variants with improved FRET properties. The best pair of variants, CyPet-YPet, exhibited a seven-fold improvement in dynamic range over the parental pair when measured in vitro. Additionally, the optimized pair provided significantly improved resolution of FRET signal changes in bacterial, yeast, and mammalian cells using flow cytometry. The improvements in dynamic range were verified in applications including the detection of protease activities, protein-protein interactions, and small molecules.
Download or read book FRET F rster Resonance Energy Transfer written by Igor L. Medintz and published by Wiley-VCH. This book was released on 2013-12-23 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: FRET – Förster Resonance Energy Transfer Meeting the need for an up-to-date and detailed primer on all aspects of the topic, this ready reference reflects the incredible expansion in the application of FRET and its derivative techniques over the past decade, especially in the biological sciences. This wide diversity is equally mirrored in the range of expert contributors. The book itself is clearly subdivided into four major sections. The first provides some background, theory, and key concepts, while the second section focuses on some common FRET techniques and applications, such as in vitro sensing and diagnostics, the determination of protein, peptide and other biological structures, as well as cellular biosensing with genetically encoded fluorescent indicators. The third section looks at recent developments, beginning with the use of fluorescent proteins, followed by a review of FRET usage with semiconductor quantum dots, along with an overview of multistep FRET. The text concludes with a detailed and greatly updated series of supporting tables on FRET pairs and Förster distances, together with some outlook and perspectives on FRET. Written for both the FRET novice and for the seasoned user, this is a must-have resource for office and laboratory shelves.
Download or read book Advanced Fluorescence Microscopy written by Peter J. Verveer and published by Humana. This book was released on 2016-08-23 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume provides an overview of advanced fluorescence microscopy, covering a broad range of methods. Each chapter focuses on a different method and provides a practical guide for application in biological systems. 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 cutting-edge, Advanced Fluorescence Microscopy: Methods and Protocols seeks to provide scientists with methods for biological systems that are of interest.
Download or read book Advanced Photon Counting written by Peter Kapusta and published by Springer. This book was released on 2015-04-23 with total page 371 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume focuses on Time-Correlated Single Photon Counting (TCSPC), a powerful tool allowing luminescence lifetime measurements to be made with high temporal resolution, even on single molecules. Combining spectrum and lifetime provides a “fingerprint” for identifying such molecules in the presence of a background. Used together with confocal detection, this permits single-molecule spectroscopy and microscopy in addition to ensemble measurements, opening up an enormous range of hot life science applications such as fluorescence lifetime imaging (FLIM) and measurement of Förster Resonant Energy Transfer (FRET) for the investigation of protein folding and interaction. Several technology-related chapters present both the basics and current state-of-the-art, in particular of TCSPC electronics, photon detectors and lasers. The remaining chapters cover a broad range of applications and methodologies for experiments and data analysis, including the life sciences, defect centers in diamonds, super-resolution microscopy, and optical tomography. The chapters detailing new options arising from the combination of classic TCSPC and fluorescence lifetime with methods based on intensity fluctuation represent a particularly unique highlight.
Download or read book FRET and FLIM Techniques written by Theodorus W. J. Gadella and published by Elsevier. This book was released on 2011-07-29 with total page 603 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume reviews the techniques Förster Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM) providing researchers with step by step protocols and handy hints and tips. Both have become staple techniques in many biological and biophysical fields.
Download or read book Luminescent and Photoactive Transition Metal Complexes as Biomolecular Probes and Cellular Reagents written by Kenneth Kam-Wing Lo and published by Springer. This book was released on 2015-06-30 with total page 266 pages. Available in PDF, EPUB and Kindle. Book excerpt: The series Structure and Bonding publishes critical reviews on topics of research concerned with chemical structure and bonding. The scope of the series spans the entire Periodic Table and addresses structure and bonding issues associated with all of the elements. It also focuses attention on new and developing areas of modern structural and theoretical chemistry such as nanostructures, molecular electronics, designed molecular solids, surfaces, metal clusters and supramolecular structures. Physical and spectroscopic techniques used to determine, examine and model structures fall within the purview of Structure and Bonding to the extent that the focus is on the scientific results obtained and not on specialist information concerning the techniques themselves. Issues associated with the development of bonding models and generalizations that illuminate the reactivity pathways and rates of chemical processes are also relevant. The individual volumes in the series are thematic. The goal of each volume is to give the reader, whether at a university or in industry, a comprehensive overview of an area where new insights are emerging that are of interest to a larger scientific audience. Thus each review within the volume critically surveys one aspect of that topic and places it within the context of the volume as a whole. The most significant developments of the last 5 to 10 years should be presented using selected examples to illustrate the principles discussed. A description of the physical basis of the experimental techniques that have been used to provide the primary data may also be appropriate, if it has not been covered in detail elsewhere. The coverage need not be exhaustive in data, but should rather be conceptual, concentrating on the new principles being developed that will allow the reader, who is not a specialist in the area covered, to understand the data presented. Discussion of possible future research directions in the area is welcomed. Review articles for the individual volumes are invited by the volume editors. Readership: research scientists at universities or in industry, graduate students Special offer For all customers who have a standing order to the print version of Structure and Bonding, we offer free access to the electronic volumes of the Series published in the current year via SpringerLink.
Download or read book Modern Tools for Time Resolved Luminescence Biosensing and Imaging written by and published by Frontiers Media SA. This book was released on 2021-09-01 with total page 156 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Combinatorial Methods for Chemical and Biological Sensors written by Radislav A. Potyrailo and published by Springer Science & Business Media. This book was released on 2009-03-21 with total page 495 pages. Available in PDF, EPUB and Kindle. Book excerpt: Chemical sensors are in high demand for applications as varied as water pollution detection, medical diagnostics, and battlefield air analysis. Designing the next generation of sensors requires an interdisciplinary approach. The book provides a critical analysis of new opportunities in sensor materials research that have been opened up with the use of combinatorial and high-throughput technologies, with emphasis on experimental techniques. For a view of component selection with a more computational perspective, readers may refer to the complementary volume of Integrated Analytical Systems edited by M. Ryan et al., entitled “Computational Methods for Sensor Material Selection”.
Download or read book Nanotechnology Enabled Sensors written by Kourosh Kalantar-zadeh and published by Springer Science & Business Media. This book was released on 2007-09-19 with total page 502 pages. Available in PDF, EPUB and Kindle. Book excerpt: Nanotechnology provides tools for creating functional materials, devices, and systems by controlling materials at the atomic and molecular scales and making use of novel properties and phenomena. Nanotechnology-enabled sensors find applications in several fields such as health and safety, medicine, process control and diagnostics. This book provides the reader with information on how nanotechnology enabled sensors are currently being used and how they will be used in the future in such diverse fields as communications, building and facilities, medicine, safety, and security, including both homeland defense and military operations.
Download or read book Fluorescence in Industry written by Bruno Pedras and published by Springer. This book was released on 2019-06-14 with total page 422 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book gathers 12 outstanding contributions that reflect state-of-the-art industrial applications of fluorescence, ranging from the pharmaceutical and cosmetics industries to explosives detection, aeronautics, instrumentation development, lighting, photovoltaics, water treatment and much more. In the field of fluorescence, the translation of research into important applications has expanded significantly over the past few decades. The 18th volume in the Springer Series on Fluorescence fills an important gap by focusing on selected industrial applications of fluorescence, described in contributions by both industry-based researchers and academics engaged in collaborations with industrial partners.
Download or read book Fluorescence Spectroscopy and Microscopy written by Yves Engelborghs and published by . This book was released on 2014 with total page 816 pages. Available in PDF, EPUB and Kindle. Book excerpt: Reflecting the expanding field's need for reliable protocols, Fluorescence Spectroscopy and Microscopy: Methods and Protocols offers techniques from a worldwide team of experts on this versatile and vital subject. The topics covered fall into four broad categories: steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy, fluorescent probe development, and the various sub-categories of fluorescence microscopy, such as fluorescence recovery after photobleaching (FRAP), live cell FRET imaging (FRETim), fluorescence lifetime imaging (FLIM), fluorescence fluctuation spectroscopy (FFS), and single-molecule fluorescence spectroscopy (smFS). Written as a part of the popular Methods in Molecular Biology series, chapters include the kind of unambiguous detail and key implementation advice that proves essential for successful results.
Download or read book Introduction to Radiation Protection written by Claus Grupen and published by Springer Science & Business Media. This book was released on 2010-04-20 with total page 429 pages. Available in PDF, EPUB and Kindle. Book excerpt: This account of sources of ionizing radiation and methods of radiation protection describes units of radiation protection, measurement techniques, biological effects, environmental radiation and many applications. Each chapter contains problems with solutions.
Download or read book The Handbook of Nanomedicine written by Kewal K. Jain and published by Humana Press. This book was released on 2017-03-20 with total page 661 pages. Available in PDF, EPUB and Kindle. Book excerpt: Nanomedicine is defined as the application of nanobiotechnology in clinical medicine, which is currently being used to research the pathomechanism of disease, refine molecular diagnostics, and aid in the discovery, development, and delivery of drugs. In The Handbook of Nanomedicine, Third Edition, Prof. Kewal K. Jain updates, reorganizes, and replaces information in the comprehensive second edition in order to capture the most recent advances in this dynamic field. Important components of nanomedicine such as drug delivery via nanobiotechnology and nanopharmaceuticals as well as nanooncology, where the greatest number of advances are occurring, are covered extensively. As this text is aimed at nonmedical scientists, pharmaceutical personnel, as well as physicians, descriptions of the technology involved and other medical terminology are kept as clear and simple as possible. In depth and cutting-edge, The Handbook of Nanomedicine, Third Edition informs its readers of the ever-growing field of nanomedicine, destined to play a significant role in the future of healthcare.
Download or read book Characterization of Solid Surfaces written by Philip F. Kane and published by Springer Science & Business Media. This book was released on 2013-11-27 with total page 675 pages. Available in PDF, EPUB and Kindle. Book excerpt: Until comparatively recently, trace analysis techniques were in general directed toward the determination of impurities in bulk materials. Methods were developed for very high relative sensitivity, and the values determined were average values. Sampling procedures were devised which eliminated the so-called sampling error. However, in the last decade or so, a number of developments have shown that, for many purposes, the distribution of defects within a material can confer important new properties on the material. Perhaps the most striking example of this is given by semiconductors; a whole new industry has emerged in barely twenty years based entirely on the controlled distribu tion of defects within what a few years before would have been regarded as a pure, homogeneous crystal. Other examples exist in biochemistry, metallurgy, polyiners and, of course, catalysis. In addition to this of the importance of distribution, there has also been a recognition growing awareness that physical defects are as important as chemical defects. (We are, of course, using the word defect to imply some dis continuity in the material, and not in any derogatory sense. ) This broadening of the field of interest led the Materials Advisory Board( I} to recommend a new definition for the discipline, "Materials Character ization," to encompass this wider concept of the determination of the structure and composition of materials. In characterizing a material, perhaps the most important special area of interest is the surface.
