Contact & Support

Fluorescent Protein

RSS feed
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • Webinar: Introduction to Fluorescence Microscopy

    In this seminar we will provide an overview about the latest advances in fluorescence microscopy. You will learn how you can use widefield and confocal microscopes to help you understand life’s questions down to tiny details, at high speed and state-of-the-art image quality both in living and fixed samples.
    Read article
  • 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 resonance energy transfer (FRET).
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • Online Tool to Determine the Optimal Combination of Fluorescence Filter Cubes, Fluorophores and Light Sources

    To achieve optimal results in fluorescence microscopy, the light source, the fluorophores and the filter cubes have to match perfectly. A fluorescence microscope with a perfect fit of excitation and emission filter set is able to maximize the efficiency of excitation and emission of the fluorophores and thus provide crisp fluorescence images. The online tool Leica FluoScout™ helps users achieve excellent fluorescence imaging results by recommending the optimum filter cube and filter cube set for their choice of fluorophores.
    Read article
  • 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.
    Read article
  • Webinar: Fluorescent Probes and Digital Imaging: Where We Are and Where We're Going

    The discovery of green fluorescent protein a half century ago heralded a new and explosive era in microscopy, forever changing the landscape for biology imaging. The ability to fuse a genetically encoded fluorescent probe to an almost-unlimited variety of proteins has enabled scientists to investigate signaling pathways and the movement of intracellular proteins in living cells with unprecedented detail, particularly when coupled with powerful widefield fluorescence and confocal microscopy techniques.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • FLIM-FRET in Solutions

    FRET efficiency can be measured based on fluorescence lifetime microscopy (FLIM). FLIM-FRET allows analysis of molecular interactions both in vitro and in vivo. This article describes the use of FLIM in the time domain (TCSPC) to measure FRET in vitro in a biochemical assay using a Cerulean-Citrine construct.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 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).
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 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.
    Read article
  • 1
  • 2