Fluorescence Applications In Biotechnology And Life Sciences PdfBy Jessey M. In and pdf 27.03.2021 at 14:47 9 min read
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The phenomenon of coral fluorescence in mesophotic reefs, although well described for shallow waters, remains largely unstudied.
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Biomedical Applications for Spectroscopy
Evgenia Matveeva. Download PDF. A short summary of this paper. His research in biotechnology has concentrated on development and application of fluorescence techniques for analysis of the identity, vitality and adaptability of microbial, plant and animal cell systems, with major efforts in elucidating roles of cell membranes in environmental adaptation of yeasts.
He currently focuses on fermentation processes in winemaking. His interests also include enhancement of biochemical and biophysical education. He specialises in fluorescence and phosphorescence spectroscopy with a particular focus on the structure and properties of free radicals. His interests also include photochemistry, astrophysics and mass spectrometry. His key field of expertise is Ultrafast Spectroscopy and Photochemistry. With his group he investigates light induced processes in a variety of molecular systems including synthetic polymers, biological macromolecules and supramolecular assemblies.
He is also a Senior Lecturer and expert in optical microscopy and imaging, with special interests in quantitative imaging, fluorescence microscopy, live cell imaging and various forms of 3D microscopy.
His interests include the structure, function, development and application of novel genetically encoded fluorophores with a particular focus on the application of such probes to investigations into the role and mechanism of autophagy.
She explores fluorescent proteins in corals and studies their properties and biological role. She has been involved in quantum dot research for over 10 years. In his work he focuses on scanning cytometry and quantitative approach to and confocal microscopy. He is involved in research projects on molecular mechanism of programmed cell death in cancer and tissue development, roadblocks in the secretory pathway of filamentous fungi, processes involved in the remodelling of complex tissue of intestinal mucosa, cell interactions with various titanium-based alloys in transplantology, pathogeninduced virus and bacteria changes in the cell cytoskeleton and characterization of the luminescence properties of newly developed semiconductors.
His interests include the extraction of quantitative information from digital images of biological origin and the development of new microscopy techniques. He has a strong background in chemometrics and bioinformatics, and his current interests lie in several areas, including unmixing of hyperspectral images, analysis of microarray data, and biomarker discovery. He has many years experience in developing and applying sophisticated unmixing algorithms for spectroscopic and hyperspectral data, initially for mineral exploration applications, but more recently in biological and pharmaceutical applications.
Dr Marko Nykanen Medical School, University of Tampere, Finland develops advanced imaging technologies especially for subcellular and molecular analyses. He has over 12 years hands-on experience in various light LM and electron EM microscopies. He has been involved with ultrastructural studies and biological imaging both in academia and in the industry.
They are interested in various aspects of the cell biology of the malaria parasite during its intraerythrocytic life stage. They have utilised a variety of fluorescencebased live cell methods, including the photobleaching techniques described in this chapter, to examine macromolecular dynamics and protein trafficking in these cells. He is an acknowledged expert in the field of time-gated luminescence detection and its application in microscopy and flow cytometry.
He holds key patents in flow cytometry, fluorophore chemistry and biological instrumentation. A key aim of Dr. Connally's research is the development of ultra-sensitive luminescence based techniques for pathogen detection. BTF provides the world's most precise reference standards for microbiological testing. Flow cytometry is the underpinning technology for making most of the BTF products and Dr Vesey with his team has considerably pushed its boundaries.
His research is concerned with chemical processes that occur on ultrafast femtosecond, picosecond and nanosecond time-scales.
He develops ultrafast laser spectroscopic techniques that are applied to the study of photophysical processes occurring in macro and supra-molecular systems, and adapted to microspectroscopic studies. His research is concerned with developing new microscopy techniques for high resolution imaging of biological samples.
He is also involved in femtosecond spectroscopy experiments investigating the photophysics and photochemistry of many novel materials and biological samples.
His research is directed at developing advanced optical imaging systems based on spectral and time-resolved techniques that are capable of extracting the most valuable diagnostic information possible from fluorescence signals. He applies these systems to studies of breast cancer and pharmaceutics with the ultimate aim of translating these devices into the clinical setting. He has over 15 years experience in developing and applying advanced techniques of single molecule fluorescence spectroscopy and imaging.
In particular, his group has developed high-precision double-focus fluorescence correlation spectroscopy, fluorescence lifetime correlation spectroscopy, and defocused image analysis of molecular orientation. He is a worldrecognized leader in a cross-disciplinary field of fluorescence, also renowned for his work in multi-photon spectroscopy and microscopy including the effect of metal nanoparticles.
He has an unusually broad research background arising from long involvement with protein chemistry, protein-ligand interaction and protein thermodynamics, applications of advanced fluorescence spectroscopy to study molecular processes in biological systems.
He pioneered the studies of an important fluorescence effects mediated by ultrathin metal films, fluorescence enhancement and the Surface Plasmon Coupled Emission SPCE. These technologies create revolutionary opportunities for designing novel fluorescence-based assays and detection systems with much higher sensitivity and minimal need for sample pre-purification.
He holds several patents and authored and co-authored well in excess of hundred publications in the area of advanced fluorescence technologies. Her research focuses on unraveling the organization of cell membrane, and understanding the role of lipid raft domains in signal transduction processes.
Her to date most important contribution to the field has been the establishment of fluorescent microscopy techniques to visualize and quantify lipid structure in cell membranes. His research interests include biological membranes and membrane proteins, regulation and mechanism of charge transport across biological membranes and voltage-sensitive fluorescent dyes.
Her expertise spans the fields of biophotonics, optical characterisation, ultrasensitive detection of analytes, biosensing, bioimaging. Her research lies at the interface of materials science, photonics and biotechnology and she is drawing on her earlier achievements in materials science and ultrasensitive optical characterisation. Her past most significant body of work focused on optical characterisation of trace impurities and defects in materials and the development of cathodoluminescence as a diagnostic tool for quantitative analysis and applied to a wide variety of materials.
Current work focuses on an innovative ultrasensitive surface plasmon resonance sensing system for the application in bioassays capable of sensing ultrasmall volumes.
She published over papers in refereed journals. The Network brings together about academics and industry representatives. This book is one of the Network initiatives Preface here goes my preface Immunoassays can also be used to identify and measure antibodies. The basic principle of immunoassays relies on the specificity of the antibody-antigen reaction.
The antibody is a protein immunoglobulin molecule, produced by the immune system in humans and animals in response to a foreign substance antigen. Antibodies belong to a type of protein called immunoglobulins; the most common one is immunoglobulin G IgG. IgG is an Y-shaped large protein molecular weight about kD composed of two main structural and functional regions??. The Fc region Fragment, crystallizable , located at the stem of the "Y" is composed of two heavy chains. Its role involves binding to various cell receptors and complement proteins.
The Fc region is a region of constant structure within an antibody class. The second, called the Fab region Fragment, antigen binding which is located at each end of the forked portion of the "Y" on the antibody is composed of one constant and one variable domain of each of the heavy and the light chain. These domains shape the antigen binding site at the amino terminal end of the monomer.
The variable domains at the two chains bind the epitope on their specific antigens. It is important to note that Fab contains the antigen Ag binding site that varies between different antibodies. Each antibody recognizes a particular foreign antigen and binds to it. Among different antigens we find relatively simple substances including small molecules such as drugs or pesticides, or a complex ones such as proteins or a viruses. Although many types of immunoassays are available on the market, the fundamental principles and components of an immunoassay are common.
In order to carry out an immunoassay we must be carry out the calibration using appropriate standards, we need to select an appropriate antibody and choose the detection mode, which are described below.
Standards and calibration: Standards or calibrators are known concentrations of the analyte that are used to generate a standard curve [1,2]. In order to produce such standard curve, the "signal" detected from the assay is plotted versus the known "concentration" of the standard and a best-fit line or curve is drawn.
The concentration of the analyte can then be determined by measuring the "signal" in an unknown sample and using the standard curve to deduce the concentration of the analyte. Antibody: Depending on the specific assay format, there may be one or more antibodies involved into binding to the analyte in the sample .
Antibodies are called polyclonal if produced by antigen injection into experimental animal, or monoclonal if produced by cell fusion and cell culture techniques.
Monoclonal antibodies have better specificity and more reproducible properties from batch to batch, and are preferred in immunoassays. Detection mode: The detection mode is defined by the character of the reporter label used in the immunoassay.
All immunoassays require the use of labelling in order to visualise and measure the amount of antigen or antibody present. A label is a molecule that will react as part of the assay, so a change in signal can be measured in the solution.
There are a number of different labels, such as substances that produce light luminophores , radioactive isotopes, or enzymes producing colour or fluorescence, used for quantification of a sample's response. Depending on the assay format, these labels can be located on the antigen or the antibody. Here we will focus on immunoassays utilizing fluorescent labels. The bound antigen is separated from the excess analyte which did not bind to the antibody. The amount of the analyte in the unknown sample is inversely proportional to the amount of labelled antigen, which can be measured in a fluorometer or spectrophotometer.
In the one step competitive format Figure 1. In the two-step competitive format, the antibody is first incubated with specimen containing antigens of interest; then in the second step, labelled antigen is added.
Two step competitive assay formats usually provide improved assay sensitivity by a factor of several compared to one step assay formats. In non-competitive immunoassays, or "sandwich" immunoassays, the analyte is "sandwiched" between two antibodies, the capture antibody and the detection antibody Figure 1. Typically, the capture antibody is coated to a solid phase such as a well , and the detection antibody which should be present in excess is labeled with a fluorophore, radioactive label such as I , or an enzyme.
As the amount of analyte antigen is increased, the amount of labelled antibody-antigen complex also increases. Thus, the amount of analyte in an unknown sample is directly proportional to the amount of labeled detection antibody measured by the detection system. Noncompetitive assay formats can also utilize either one step or two step methods, as with the competitive assay. The two step assay format employs washing steps in which the sandwich binding complex is isolated and washed to remove excess unbound labeled reagent and any other interfering substances.
These steps are omitted in one step assays.
Many biomedical research applications exploit the natural fluorescence response of amino acids, the essential building blocks of all proteins. These protein fluorescence responses to light have been used for everything from pharmaceutical manufacturing to cancer treatments, and even biowarfare defense. Exploring this biomedical spectroscopy niche is a dive into the deep end of cutting-edge science. Spectroscopy is an essential technology that makes these biomedical applications, and so many others, possible. Avantes, a leader in the development of high-sensitivity, high-resolution spectrometers, is the trusted choice for hundreds of researchers and original equipment manufacturers in biomedical applications. Proteins, the complex organic compounds made of chains of amino acids, are the most abundant organic molecule in all living things on Earth. These molecules play many roles within cells and organisms.
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The development of super-resolved fluorescence microscopy, for which the Nobel Prize was awarded in , has been a topic of interest to physicists and biologists alike. It is inevitable that numerous questions in biomedical research cannot be answered by means other than direct observation. In this review, advances to fluorescence microscopy are covered in a widely accessible fashion to facilitate its use in decisions related to its acquisition and utilization in biomedical research. The advent of optical microscopy has had a significant impact on the life sciences specially in visualizing the molecular architecture of the cell 1 — The development of fluorescent probes has allowed observations at the single molecule level 12 whilst the recent introduction of super resolution microscopy has allowed imaging of an array of nanoscopic biological complexes with unprecedented resolution 1 , 5 — 7 , 9. In this review, advanced fluorescence microscopy techniques are discussed in an application-oriented fashion.
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Download the PDF version. Introduction The application of fluorescence polarization offers unique advantages over conventional fluorescence imaging and quantitation. In fluorescence polarization studies, fluorophores tied to biological samples or chemical compounds help elucidate the underlying mechanism of interest. As opposed to the standard fluorescence techniques, fluorescence polarization allows for fast and accurate quantitative measurements with relatively simple instrumentation compared to sophisticated research instruments.
Protein Detection and Identification
Biotechnology is a broad area of biology , involving the use of living systems and organisms to develop or make products. Depending on the tools and applications, it often overlaps with related scientific fields. In the late 20th and early 21st centuries, biotechnology has expanded to include new and diverse sciences , such as genomics , recombinant gene techniques, applied immunology , and development of pharmaceutical therapies and diagnostic tests. The term biotechnology was first used by Karl Ereky in , meaning the production of products from raw materials with the aid of living organisms. The wide concept of biotechnology encompasses a wide range of procedures for modifying living organisms according to human purposes, going back to domestication of animals, cultivation of the plants, and "improvements" to these through breeding programs that employ artificial selection and hybridization.
E-mail: dklim korea. E-mail: blseong yonsei. A facile method for the quantification of native state protein is strongly required to accurately determine the amount of expressed protein of interest. Here we report a simple bead-based assay, which can sensitively quantify the amount of native state green fluorescent protein using Ni-NTA nickel-nitrilotriacetic acid -modified microbead particles. The bead-based method is simple and straightforward to perform and it showed a highly sensitive capability to detect the expressed fluorescent protein because of the enriched fluorescent protein on the beads.
Fluorescence, the absorption and re-emission of photons with longer wavelengths, is one of those amazing phenomena of Nature. Its discovery and utilization had, and still has, a major impact on biological and biomedical research, since it enables researchers not just to visualize normal physiological processes with high temporal and spatial resolution, to detect multiple signals concomitantly, to track single molecules in vivo , to replace radioactive assays when possible, but also to shed light on many pathobiological processes underpinning disease states, which would otherwise not be possible. Compounds that exhibit fluorescence are commonly called fluorochromes or fluorophores and one of these fluorescent molecules in particular has significantly enabled life science research to gain new insights in virtually all its sub-disciplines: Green Fluorescent Protein.
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