Leica Science Lab - Tag : Fluorescent Protein https://www.leica-microsystems.com//science-lab/tag/tags/fluorescent-protein/show/Tag/ Article tagged with Fluorescent Protein en-US https://www.leica-microsystems.com/20208 Quantitative Fluorescence FLIM FRET and Biosensors: Versatile Tools for Biomedical Research Fluorescence Lifetime Imaging (FLIM) in combination with Förster Resonance Energy Transfer (FRET) has proven to be very beneficial for investigations in biomedical research for a wide range of structural elements and dynamic changes in cells. FRET allows to monitor molecular interactions, as the FRET signal depends strongly on the distance of the two FRET partners. This allows to investigate interaction of molecules, like ligand-receptor pairs, protein-protein interactions or interactions of effectors with DNA. https://www.leica-microsystems.com//science-lab/flim-fret-and-biosensors-versatile-tools-for-biomedical-research/ Sun, 10 Jun 2018 22:00:00 +0000 Dr. Rolf T. Borlinghaus https://www.leica-microsystems.com/19773 Fluorescence Microscopy Basics in Microscopy The Fundamentals and History of Fluorescence and Quantum Dots At some point in your research and science career, you will no doubt come across fluorescence microscopy. This ubiquitous technique has transformed the way in which microscopists can image, tag and trace anything from whole organisms to single proteins and beyond. In this article, we will examine what is meant by "fluorescence", the history and basic physics behind its definition, the discovery and application of Green Fluorescent Protein (GFP) and a look at the rapidly expanding field of fluorescent probes including Quantum Dots. https://www.leica-microsystems.com//science-lab/the-fundamentals-and-history-of-fluorescence-and-quantum-dots/ Wed, 15 Nov 2017 16:44:00 +0000 PhD Martin Wilson https://www.leica-microsystems.com/19635 Fluorescence Microscopy Live-Cell Imaging Stereo Microscopy Widefield Microscopy Real Time Observation of Neutrophil White Blood Cell Recruitment to Bacterial Infection In Vivo The zebrafish (Danio rerio) is an emerging vertebrate model organism to study infection. The transparent larva comprises a fully functional innate immune system and enables live imaging of fluorescent immune cells in transgenic animals. Zebrafish infection models have been developed for both the human bacterial pathogen Shigella flexneri and the natural fish bacterial pathogen Mycobacterium marinum. Importantly, whilst S. flexneri causes acute infection and is typically used as an inflammatory paradigm, M. marinum causes a chronic disease similar to tuberculosis in humans. Here, we use real time fluorescence microscopy to image transgenic zebrafish larvae with neutrophils (granulocyte white blood cells) expressing the green fluorescent protein eGFP. https://www.leica-microsystems.com//science-lab/real-time-observation-of-neutrophil-white-blood-cell-recruitment-to-bacterial-infection-in-vivo/ Thu, 27 Jul 2017 11:20:00 +0000 MSc Alexandra R. Willis, MSc Vincenzo Torraca, PhD Serge Mostowy, PhD James DeRose https://www.leica-microsystems.com/19429 Fluorescence Microscopy What is Photomanipulation? The term photomanipulation describes a wide range of techniques that enable the microscopist the transition from passive observer to instigator of events by offering a way of interacting with their sample via targeted illumination. Typically researchers are trying to observe specific processes of interest in order to understand the underlying biological process. Microscopists are often forced to hunt through large populations of cells or acquire hours of time laps footage before they’re able to observe events of interest and in many cases it’s simply not possible to observe certain processes using conventional microscopy techniques alone. Photomanipulation tools enable the microscopist to initiate biological events, precisely adjusting sample labeling, biological activity, local chemical environments and in some instances physically destroy parts of their specimen. https://www.leica-microsystems.com//science-lab/what-is-photomanipulation/ Mon, 15 May 2017 07:59:00 +0000 Dr. Peter Laskey, Dr. Christoph Greb, Dr. Oliver Schlicker https://www.leica-microsystems.com/6395 Fluorescence Microscopy Photoactivatable, photoconvertible, and photoswitchable Fluorescent Proteins Fluorescent proteins (FPs) such as GFP, YFP or DsRed are powerful tools to visualize cellular components in living cells. Nevertheless, there are circumstances when classical FPs reach their limits. Watching dedicated, spatially limited protein populations of a certain protein of interest is impossible with common FPs, since they are expressed throughout the entire cell. At this point photoactivatable, photoconvertible and photoswitchable fluorescent proteins enter the stage. The members of this fluorescence toolkit can be activated from a non-fluorescent state, they can change their emission spectrum, or they are even able to be reversibly switched "on and off". With the help of these “optical highlighters”, researchers can track a distinct protein population over time by activating respectively converting their fluorescence with a spatially defined light beam of a given wavelength. https://www.leica-microsystems.com//science-lab/photoactivatable-photoconvertible-and-photoswitchable-fluorescent-proteins/ Thu, 04 May 2017 11:31:00 +0000 Dr. Christoph Greb https://www.leica-microsystems.com/19348 Neuroscience Live-Cell Imaging Clap On, Clap Off. Protein On, Protein Off Unlike genetic or pharmacological manipulation, light-controlled proteins respond immediately, can be temporally and spatially triggered, are reversible, and are specific to the protein of interest. However, designing and using light-controllable proteins often requires expertise and specialized equipment. Now, two recently published articles in Science describe generalizable methods for making photo-controllable proteins. https://www.leica-microsystems.com//science-lab/clap-on-clap-off-protein-on-protein-off/ Tue, 02 May 2017 22:27:00 +0000 https://www.leica-microsystems.com/13942 Fluorescence Microscopy Quantitative Fluorescence Video Talk by Roger Tsien: Fluorescent Protein Indicators In this talk, Roger Tsien discusses how fluorescent proteins have been turned into indicators for a wide variety of biological molecules, including pH, ions, redox potential, and signaling molecules like phosphoinositides. The talk also covers reporters used to measure the activity of enzymes like kinases, phosphatases, and proteases. It covers both single proteins whose intensity or wavelength change, as well as reporters using Förster resonance energy transfer (FRET). https://www.leica-microsystems.com//science-lab/video-talk-by-roger-tsien-fluorescent-protein-indicators/ Tue, 16 Aug 2016 10:29:00 +0000 PhD Roger Y. Tsien https://www.leica-microsystems.com/13935 Fluorescence Microscopy Video Talk by Roger Tsien: Fluorescent Proteins Live cell imaging has been revolutionized by the discovery of the green fluorescent protein (GFP). This lecture covers the history of GFP, how it folds and becomes fluorescent, how it has been mutated to produce additional colors (blue, cyan, yellow), and the discovery of red fluorescent proteins from corals. It also covers novel photoswitchable and photoactivatible fluorescent proteins, whose color can be changed by light, and new infrared fluorescent proteins. https://www.leica-microsystems.com//science-lab/video-talk-by-roger-tsien-fluorescent-proteins/ Wed, 07 Oct 2015 16:55:00 +0000 PhD Roger Y. Tsien https://www.leica-microsystems.com/16035 Fluorescence Microscopy Live-Cell Imaging Basics in Microscopy Fluorescent Proteins Illuminate Cell Biology Green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria and GFP-like fluorescent proteins from other animals have had an important role in the technical innovations that have driven these advances. This poster provides a comprehensive user's guide to fluorescent proteins and sensors , their key properties and the cell biological questions to which they can be applied. https://www.leica-microsystems.com//science-lab/fluorescent-proteins-illuminate-cell-biology/ Fri, 18 Sep 2015 09:03:00 +0000 https://www.leica-microsystems.com/15700 Super-Resolution Live-Cell Imaging CRISPR-Cas9 CRISPR/Cas9-mediated Endogenous Protein Tagging for RESOLFT Super-Resolution Microscopy of Living Human Cells Overexpression is a notorious concern in conventional and especially in super-resolution fluorescence light microscopy studies because it may cause numerous artifacts including ectopic sub-cellular localizations, erroneous formation of protein complexes, and others. Nonetheless, current live cell super-resolution microscopy studies generally rely on the overexpression of a host protein fused to a fluorescent protein. https://www.leica-microsystems.com//science-lab/crisprcas9-mediated-endogenous-protein-tagging-for-resolft-super-resolution-microscopy-of-living-human-cells/ Wed, 22 Jul 2015 16:24:00 +0000 https://www.leica-microsystems.com/15458 Confocal Microscopy Fluorescence Microscopy Improving Axial Resolution in Confocal Microscopy with New High Refractive Index Mounting Media Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. https://www.leica-microsystems.com//science-lab/improving-axial-resolution-in-confocal-microscopy-with-new-high-refractive-index-mounting-media/ Thu, 11 Jun 2015 13:04:00 +0000 https://www.leica-microsystems.com/15509 CLEM Correlative In-Resin Super-Resolution and Electron Microscopy Using Standard Fluorescent Proteins We introduce a method for correlative in-resin super-resolution fluorescence and electron microscopy (EM) of biological structures in mammalian culture cells. Cryo-fixed resin embedded samples offer superior structural preservation, performing in-resin super-resolution, however, remains a challenge. https://www.leica-microsystems.com//science-lab/correlative-in-resin-super-resolution-and-electron-microscopy-using-standard-fluorescent-proteins/ Thu, 04 Jun 2015 10:28:00 +0000 https://www.leica-microsystems.com/13911 Super-Resolution Live-Cell Imaging Nanoscale Protein Diffusion by STED-Based Pair Correlation Analysis We describe for the first time the combination between cross-pair correlation function analysis (pair correlation analysis or pCF) and stimulated emission depletion (STED) to obtain diffusion maps at spatial resolution below the optical diffraction limit (super-resolution). Our approach was tested in systems characterized by high and low signal to noise ratio, i.e. Capsid Like Particles (CLPs) bearing several (>100) active fluorescent proteins and monomeric fluorescent proteins transiently expressed in living Chinese Hamster Ovary cells, respectively. https://www.leica-microsystems.com//science-lab/nanoscale-protein-diffusion-by-sted-based-pair-correlation-analysis/ Fri, 21 Nov 2014 09:36:00 +0000 Paolo Bianchini https://www.leica-microsystems.com/14080 Fluorescence Microscopy Live-Cell Imaging Spectral and Structural Comparison Between Bright and Dim Green Fluorescent Proteins in Amphioxus The cephalochordate Amphioxus naturally co-expresses fluorescent proteins (FPs) with different brightness, which thus offers the rare opportunity to identify FP molecular feature/s that are associated with greater/lower intensity of fluorescence. Here, we describe the spectral and structural characteristics of green FP (bfloGFPa1) with perfect (100%) quantum efficiency yielding to unprecedentedly-high brightness, and compare them to those of co-expressed bfloGFPc1 showing extremely-dim brightness due to low (0.1%) quantum efficiency. https://www.leica-microsystems.com//science-lab/spectral-and-structural-comparison-between-bright-and-dim-green-fluorescent-proteins-in-amphioxus/ Thu, 28 Aug 2014 15:54:00 +0000 https://www.leica-microsystems.com/12428 Confocal Microscopy Fluorescence Microscopy Colonization of Potato Rhizosphere by GFP-Tagged Bacillus subtilis MB73/2, Pseudomonas sp. P482 and Ochrobactrum sp. A44 Shown on Large Sections of Roots Using Enrichment Sample Preparation and Confocal Laser Scanning Microscopy The ability to colonize the host plants’ rhizospheres is a crucial feature to studyin the case of Plant Growth Promoting Rhizobacteria (PGPRs) with potential agricultural applications. In this work, we have created GFP-tagged derivatives of three candidate PGPRs: Bacillus subtilis MB73/2, Pseudomonas sp. P482 and Ochrobactrum sp. A44. https://www.leica-microsystems.com//science-lab/colonization-of-potato-rhizosphere-by-gfp-tagged-bacillus-subtilis-mb732-pseudomonas-sp-p482-and-ochrobactrum-sp-a44-shown-on-large-sections-of-roots-using-enrichment-sample-preparation-and-confocal-laser-scanning-microscopy/ Fri, 02 May 2014 15:04:00 +0000 https://www.leica-microsystems.com/11000 Confocal Microscopy Neuroscience Live-Cell Imaging Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification. https://www.leica-microsystems.com//science-lab/imaging-pheromone-sensing-in-a-mouse-vomeronasal-acute-tissue-slice-preparation/ Fri, 02 May 2014 14:24:00 +0000 https://www.leica-microsystems.com/12779 Super-Resolution Sample Preparation for GSDIM Localization Microscopy – Protocols and Tips The widefield super-resolution technique GSDIM (Ground State Depletion followed by individual molecule return) is a localization microscopy technique that is capable of resolving details as small as 20 nanometers. GSDIM is suitable for a wide range of samples. https://www.leica-microsystems.com//science-lab/sample-preparation-for-gsdim-localization-microscopy-protocols-and-tips/ Tue, 01 Apr 2014 12:40:00 +0000 Dr. Marko Lampe, Dr. Christoph Greb https://www.leica-microsystems.com/9350 Fluorescence Microscopy Confocal Microscopy Live-Cell Imaging Handbook of Optical Filters for Fluorescence Microscopy Fluorescence microscopy and other light-based applications require optical filters that have demanding spectral and physical characteristics. Often, these characteristics are application-specific and an optic that might be appropriate and optimal for one is both inappropriate and sub-optimal for another. https://www.leica-microsystems.com//science-lab/handbook-of-optical-filters-for-fluorescence-microscopy/ Tue, 06 Aug 2013 07:59:00 +0000 BSc. Jay Reichman https://www.leica-microsystems.com/5831 Fluorescence Microscopy Widefield Microscopy Fluorescent Proteins – Introduction and Photo Spectral Characteristics The prospects of fluorescence microscopy changed dramatically with the discovery of fluorescent proteins in the 1950s. The starting point was the detection of the jellyfish Aequorea victoria green fluorescent protein (GFP) by Osamo Shimomura. Hundreds of GFP mutants later, the range of fluorescent proteins reaches from the blue to the red spectrum. https://www.leica-microsystems.com//science-lab/fluorescent-proteins-introduction-and-photo-spectral-characteristics/ Mon, 09 Apr 2012 22:00:00 +0000 Dr. Christoph Greb https://www.leica-microsystems.com/4713 Fluorescence Microscopy Modern Fluorescent Proteins and their Biological Applications Here we present two review articles on fluorescent proteins and their biological applications. These first article reviews our current knowledge of blue, green, and red chromophore formation in permanently emitting FPs, photoactivatable FPs, and fluorescent timers. The second article focuses on novel monomeric RFPs and their application for studying gene expression, nuclear localization, and dynamics using advanced imaging. https://www.leica-microsystems.com//science-lab/modern-fluorescent-proteins-and-their-biological-applications/ Fri, 18 Nov 2011 12:50:00 +0000 https://www.leica-microsystems.com/4392 Laser Microdissection Cell Cultures and Laser Microdissection Many of the discoveries that are now being made in cell division and differentiation, the relationships between single cells and cell organelles, treatments of cells with pharmaceutic substances, etc. would not be possible without live cell cultures. Allowing morphological and biochemical observations of single cells under different experimental conditions, they provide a unique source of information. https://www.leica-microsystems.com//science-lab/cell-cultures-and-laser-microdissection/ Fri, 07 Oct 2011 09:15:57 +0000 M.Sc., Cornelia Gilbrich-Wille https://www.leica-microsystems.com/10866 CLEM Fluorescence Microscopy EM Sample Preparation A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce ‘"miniSOG"’ (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. https://www.leica-microsystems.com//science-lab/a-genetically-encoded-tag-for-correlated-light-and-electron-microscopy-of-intact-cells-tissues-and-organisms/ Sun, 04 Sep 2011 19:37:00 +0000 https://www.leica-microsystems.com/4182 Fluorescence Microscopy Basics in Microscopy An Introduction to Fluorescence Fluorescence is widely used in microscopy and an important tool for observing the distribution of specific molecules. Most molecules in cells do not fluoresce. They therefore have to be marked with fluorescing molecules called fluorochromes. https://www.leica-microsystems.com//science-lab/an-introduction-to-fluorescence/ Tue, 31 May 2011 13:29:00 +0000 Wymke Ockenga https://www.leica-microsystems.com/10321 Quantitative Fluorescence A mTurquoise-Based cAMP Sensor for Both FLIM and Ratiometric Read-Out Has Improved Dynamic Range FRET-based sensors for cyclic Adenosine Mono Phosphate (cAMP) have revolutionized the way in which this important intracellular messenger is studied. The currently prevailing sensors consist of the cAMP-binding protein Epac1, sandwiched between suitable donor- and acceptor fluorescent proteins (FPs). https://www.leica-microsystems.com//science-lab/a-mturquoise-based-camp-sensor-for-both-flim-and-ratiometric-read-out-has-improved-dynamic-range/ Fri, 29 Apr 2011 14:47:00 +0000 https://www.leica-microsystems.com/3514 Confocal Microscopy Quantitative Fluorescence White Confocal Choose Your Excitation Wavelength Although time correlated single photon counting (TCSPC) is the method of choice for fluorescence lifetime quantification, it requires dedicated instrumentation including a pulsed laser source, a photon counting card, and a fast detector. https://www.leica-microsystems.com//science-lab/choose-your-excitation-wavelength/ Tue, 12 Apr 2011 22:00:00 +0000 Ph.D. Corentin Spriet, Ph.D. Aymeric Leray, Dave Trinel, Franck Riquet, Laurent Héliot https://www.leica-microsystems.com/10323 Quantitative Fluorescence ATP Changes the Fluorescence Lifetime of Cyan Fluorescent Protein via an Interaction with His148 Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent proteins in more detail. Using different donor and acceptor pairs we found that ATP only affected the CFP-YFP based versions. https://www.leica-microsystems.com//science-lab/atp-changes-the-fluorescence-lifetime-of-cyan-fluorescent-protein-via-an-interaction-with-his148/ Fri, 05 Nov 2010 16:34:00 +0000 https://www.leica-microsystems.com/2730 Multiphoton Microscopy Confocal Microscopy Deep Tissue Imaging Developmental biology using Multiphoton microscopy with OPO. To gain new insight into the fundamental control of cell response to physical changes and to study the dynamics and roles of biological flow during the development of the zebrafish, Dr. Julien Vermot established his lab last year at the Institute of Genetics and Molecular and Cellular Biology (IGBMC) in Strasbourg, France. https://www.leica-microsystems.com//science-lab/deep-tissue-imaging/ Mon, 01 Nov 2010 23:00:00 +0000 PhD Julien Vermot, Dr. Andrea Mülter https://www.leica-microsystems.com/2731 Confocal Microscopy Multiphoton Microscopy What is an OPO? Multiphoton microscopy with OPO: imaging with excitation wavelengths up to 1.300 nm. Because light scattering is dependent on the wavelength, better tissue penetration can be achieved by using longer excitation wavelengths. This is where excitation with infrared light, two-photon processes, and the OPO (optical parameter oscillator) can dramatically improve image quality. https://www.leica-microsystems.com//science-lab/what-is-an-opo/ Mon, 01 Nov 2010 23:00:00 +0000 Dr. Bernd Sägmüller, Dr. Andrea Mülter https://www.leica-microsystems.com/9421 White Confocal Confocal Microscopy The First Supercontinuum Confocal that Adapts to the Sample Until now, biological and medical research fluorescence imaging in multi-user facilities or institutes has been limited by the type or number of dyes that could be excited. The Leica TCS SP5 X supercontinuum confocal unites the broadband capabilities of the Leica TCS SP5 AOBS® and the freedom and flexibility to select any excitation line within the continuous range of 470 to 670 nm. https://www.leica-microsystems.com//science-lab/the-first-supercontinuum-confocal-that-adapts-to-the-sample/ Fri, 22 Aug 2008 16:40:00 +0000 PhD Kees Jalink, Prof. Alberto Diaspro, Valentina Caorsi, Paolo Bianchini, Dr. Rolf T. Borlinghaus, Scott Young https://www.leica-microsystems.com/10325 Quantitative Fluorescence A Comparison of Donor-Acceptor Pairs for Genetically Encoded FRET Sensors: Application to the Epac cAMP Sensor as an Example We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and acceptor fluorescent proteins separated by a short linker. https://www.leica-microsystems.com//science-lab/a-comparison-of-donor-acceptor-pairs-for-genetically-encoded-fret-sensors-application-to-the-epac-camp-sensor-as-an-example/ Wed, 02 Apr 2008 16:09:00 +0000