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Quantitative Fluorescence

The phenomenon of fluorescence has revolutionized research in biology and medicine. More than 100 years ago, Paul Ehrlich began to develop specific chemical staining methods and the latest development of light-controlled proteins (optogenetics) will surely not mark the end of this progression. A historically continuous trend is the quest for measuring and quantifying data, with or without generation of images as data source. Many techniques have been introduced that are based on fluorescence, but not necessarily require image formation. The laboratory jargon refers to this methods as “F-techniques”: FLIM, FCS , FCCS, FRET, FRAP… to mention just the most important. Read the articles in this topic, to find out more details on these modern methods.

  • Lifetime – a Proper Alternative

    „Way too complicated!“ - the notorious feedback when it comes to fluorescence lifetime measurements. This will change now! New technologies and new concepts for data evaluation, all implemented in the new Leica SP8 FALCON, render fluorescence lifetime imaging (FLIM) as fuss-free as ordinary confocal imaging. And by the way: with Leica FALCON you can record frames 10 times faster compared to the classical standard. And three (or more) dimensional image stacks or time series are generated in a snap. Four channels simultaneously? No problem! And of course there are tunable excitation wavelength both visible with white light lasers (WLL) and infrared (the latter for multiphoton microscopy). That should be reason enough to delve into fluorescence lifetime imaging. The picture shows a lifetime image of a mouse embryo. Recorded in 722 stitched tiles and fitted for four separate characteristic times. Recording time ca 1 hour – compared to ca 1 day with the classical approach.
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  • 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).
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  • STED-FLCS: An Advanced Tool to Reveal Spatiotemporal Heterogeneity of Molecular Membrane Dynamics

    Heterogeneous diffusion dynamics of molecules play an important role in many cellular signaling events, such as of lipids in plasma membrane bioactivity. However, these dynamics can often only be visualized by single-molecule and super-resolution optical microscopy techniques. Using fluorescence lifetime correlation spectroscopy (FLCS, an extension of fluorescence correlation spectroscopy, FCS ) on a super-resolution stimulated emission depletion (STED) microscope, we here extend previous observations of nanoscale lipid dynamics in the plasma membrane of living mammalian cells.
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  • Individual Macromolecule Motion in a Crowded Living Cell

    There is solid evidence for analyzing fluorescence correlation and dual color fluorescence crosscorrelation spectroscopy data ( FCS and dual color FCCS) in cellular applications by equations based on anomalous subdiffusion. Using equations based on normal diffusion causes artifacts of the fitted biological system response parameters and of the interpretations of the FCS and dual color FCCS data in the crowded environment of living cells. Equations based on normal diffusion are not valid in living cells. The original article embraces the status of the experimental situation and touches obstacles that still hinder the applications of single molecules in the cellular environment.
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  • FRAP with TCS SP8 Resonant Scanner

    Fast FRAP experiments need a sufficient number of measurement points for meaningful interpretation and fitting analysis. To study very fast translocational processes, the use of a resonant scanner (RS) is preferred. The advantage in using FRAP with the RS is that statistics are much better in experiments that require fast acquisition: If the half time of recovery is about 0.5 sec you may have only about 3 to 4 data points using the conventional scanner, whereas with the resonant scanner you can get about 20 data points.
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  • Webinar: Dissecting Protein Dynamics in Living Cells by FRAP

    This webinar presented by Dr Marco Fritzsche, University of Oxford, and Jennifer Horner, PhD, Leica Microsystems, you will learn about how to use Fluorescence Recovery After Photo-bleaching (FRAP) microscopy to study protein dynamics.
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  • A Straightforward Approach for Gated STED-FCS to Investigate Lipid Membrane Dynamics

    Recent years have seen the development of multiple technologies to investigate, with great spatial and temporal resolution, the dynamics of lipids in cellular and model membranes. One of these approaches is the combination of far-field super-resolution stimulated-emission-depletion (STED) microscopy with fluorescence correlation spectroscopy ( FCS ). STED- FCS combines the diffraction-unlimited spatial resolution of STED microscopy with the statistical accuracy of FCS to determine sub-millisecond-fast molecular dynamics with single-molecule sensitivity.
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  • Cortical Actin Networks Induce Spatio-temporal Confinement of Phospholipids in the Plasma Membrane – A Minimally Invasive Investigation by STED-FCS

    Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking.
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  • A Lipid Bound Actin Meshwork Organizes Liquid Phase Separation in Model Membranes

    The eukaryotic cell membrane is connected to a dense actin rich cortex. We present FCS and STED experiments showing that dense membrane bound actin networks have severe influence on lipid phase separation. Our results reveal a mechanism how cells may prevent macroscopic demixing of their membrane components, while at the same time regulate the local membrane composition.
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  • How to Measure FRET

    Here, I will expand, including what to measure when doing FRET. There are a number of approaches to FRET quantification: 1. Sensitized Emission – This two-channel imaging technique uses an algorithm that corrects for excitation and emission crosstalk.
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  • You May Not Know Theodor Förster but You Know His Work: FRET

    If you think FRET stands for Fluorescence Resonance Energy Transfer, you are wrong … in good company but wrong. FRET actually stands for Förster Resonance Energy Transfer. Find out why and more about FRET in this article.
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  • Interview with Dr. Gertrude Bunt and Prof. Fred S. Wouters on the FOM 2015

    Only a few days to go before the start of Focus on Microscopy 2015 in Göttingen, Germany. This year’s FOM is being organized by Dr. Gertrude Bunt and Prof. Dr. Fred S. Wouters from the University Medical Center, Göttingen, in cooperation with Prof. Dr. G.J. (Fred) Brakenhoff, University of Amsterdam, The Netherlands.
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  • How to Choose the Right Confocal Microscope for Your Lab?

    Confocal microscopy has come a very long way since its invention more than a half-century ago. Today, with novel technology driven by leading imaging companies, it has become the standard for fluorescence microscopy. Choosing the right confocal microscope for your specific research requires the appropriate mix of features related to resolution, sensitivity, and speed.
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  • ICln: A New Regulator of Non-Erythroid 4.1R Localisation and Function

    To optimise the efficiency of cell machinery, cells can use the same protein (often called a hub protein) to participate in different cell functions by simply changing its target molecules. There are large data sets describing protein-protein interactions ("interactome") but they frequently fail to consider the functional significance of the interactions themselves.
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  • Novel Fluorescent Carbonic Nanomaterials for Sensing and Imaging

    Small brightly fluorescent carbon nanoparticles have emerged as a new class of materials important for sensing and imaging applications. We analyze comparatively the properties of nanodiamonds, graphene and graphene oxide ‘dots’, of modified carbon nanotubes and of diverse carbon nanoparticles known as ‘C-dots’ obtained by different methods.
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  • Step by Step Guide for FRAP Experiments

    Fluorescence recovery after photobleaching (FRAP) has been considered the most widely applied method for observing translational diffusion processes of macromolecules. State of the art laser scanning microscopes such as the Leica TCS SP8 have the advantage of using a high intensity laser for bleaching and a low intensity laser for image recording. The LAS AF application wizard offers different ways to carry out a FRAP experiment.
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  • Imaging Enzymes at Work

    For the understanding of functions of proteins in biological and pathological processes, reporter molecules such as fluorescent proteins have become indispensable tools for visualizing the location of these proteins in intact animals, tissues, and cells. For enzymes, imaging their activity also provides information on their function or functions, which does not necessarily correlate with their location. Metabolic mapping enables imaging of activity of enzymes.
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  • Widefield Calcium Imaging with Calcium Indicator Fura2

    In eukaryotic cells Ca2+ is one of the most widespread second messengers used in signal transduction pathways. Intracellular levels of Ca2+ are usually kept low, as Ca2+ often forms insoluble complexes with phosphorylated and carboxylated compounds. Typically cytosolic Ca2+ concentrations are in the range of 100 nM. In response to stimuli Ca2+ may either be released from external medium or internal stores to raise the Ca2+ concentration.
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  • Integrative Open-Source Software for Image Analysis in Biology

    Imaging techniques are indispensable in many fields of life sciences today. With state-of-the-art optics and metrology, they provide hundreds of gigabytes of still images and videos. Correspondingly, there is a growing need for complex software solutions to ensure that the amounts of generated data can be automatically managed, processed and analyzed – and shared online with a large group of users. The combination of individual open-source software projects is proving especially useful for solving such complex image analysis problems.
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  • Label-free FLIM

    Many biological samples exhibit autofluorescence. Its often broad spectra can interfere with fluorescent labeling strategies. This application letter demonstrates how autofluorescence can serve as an intrinsic contrast in fluorescence lifetime imaging microscopy (FLIM) resulting in multi-color image stacks.
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  • FRET with FLIM

    FLIM combines lifetime measurements with imaging: lifetimes obtained for each image pixel are color-coded to produce additional image contrast. Thus, FLIM delivers information about the spatial distribution of a fluorescent molecule together with information about its biochemical status or nano-environment. A typical application of FLIM is FLIM-FRET. FRET is a well-established technique to study molecular interactions. It scrutinizes protein binding and estimates intermolecular distances on an Angström scale as well.
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  • Fluorescence Correlation Spectroscopy

    Fluorescence correlation spectroscopy ( FCS ) measures fluctuations of fluorescence intensity in a sub-femtolitre volume to detect such parameters as the diffusion time, number of molecules or dark states of fluorescently labeled molecules. The technique was independently developed by Watt Webb and Rudolf Rigler during the early 1970s.
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  • Quantitative Fluorescence

    Seeing is believing – and measuring is knowing. Microscopes generate images that are not only used for illustration, but are also subject to quantification. More advanced techniques use illumination patterns (without image formation) or do not generate an image at all – but are still microscopical techniques. These F-techniques are becoming increasingly important in current biosciences.
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  • The New Repository on the Block

    The need for data validation and accessibility has never been greater than it is today. We are inundated with information from a multitude of resources, but how can we easily evaluate the accuracy of that data? In the past, the peer review process provided this and was often run by publishers.
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  • FLCS – Advances in Fluorescence Correlation Spectroscopy

    The characterization of substances at the single molecule level has become part of the standard repertoire of scientific research institutes. One of the most common methods is Fluorescence Correlation Spectroscopy ( FCS ), which can be used to examine the dynamics and concentration of fluorescent molecules in solution.
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  • Fluorescence Recovery after Photobleaching (FRAP) and its Offspring

    FRAP (Fluorescence recovery after photobleaching) can be used to study cellular protein dynamics: For visualization the protein of interest is fused to a fluorescent protein or a fluorescent dye. A region of interest (ROI) can be monitored applying a high amount of light to bleach the fluorescence within the ROI. The following illumination with low light conditions provides insight into the redistribution of molecules via recovery of fluorescence.
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  • Förster Resonance Energy Transfer (FRET)

    The Förster Resonance Energy Transfer (FRET) phenomenon offers techniques that allow studies of interactions in dimensions below the optical resolution limit. FRET describes the transfer of the energy from an excited state of a donor molecule to an acceptor molecule. Unlike absorption or emission of photons, FRET is a non-radiative energy exchange and consequently not a variation of light-matter interactions.
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  • 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).
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  • 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.
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  • 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.
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