Leica Science Lab - Tag : FLIM https://www.leica-microsystems.com//science-lab/tag/flim/ Article tagged with FLIM en-US https://www.leica-microsystems.com/72831 Quantitative Imaging Multiphoton Microscopy Accessing the Metabolic Information of Stem Cells Cancer often comes with changes in the metabolism of cells. Monitoring the metabolic state using fluorescence lifetime imaging microscopy (FLIM) is a good way to assess these changes. In some cases, endogenous signals, e.g., from NAD/NADH, can be exploited, while in other cases the resulting information is not sufficient. In this publication, the mitochondrial membrane potential (MMP) was studied using different fluorescent probes (SYTO dyes and TMRM) in various samples, including organoids derived from mouse intestine. https://www.leica-microsystems.com/science-lab/accessing-the-metabolic-information-of-stem-cells/ Fri, 18 Jun 2021 10:04:00 +0000 Corporate Communications https://www.leica-microsystems.com/72738 Confocal Microscopy How to Quantify Changes in the Metabolic Status of Single Cells Metabolic imaging based on fluorescence lifetime provides insights into the metabolic dynamics of cells, but its use has been limited as expertise in advanced microscopy techniques was needed. Now, STELLARIS 8 DIVE FALCON makes metabolic imaging accessible to every scientist, thanks to the integration of phasor analysis to easily use fluorescence lifetime information. https://www.leica-microsystems.com/science-lab/how-to-quantify-changes-in-the-metabolic-status-of-single-cells/ Thu, 27 May 2021 10:52:00 +0000 Corporate Communications https://www.leica-microsystems.com/72465 Confocal Microscopy Quantitative Imaging Label-free Differentiation of Microplastics Phasor analysis for fluorescence lifetime imaging microscopy (FLIM) is a powerful and fitting-free tool that provides a 2D graphical assessment of pixel-accurate lifetime distributions in a sample image. In biology and biomedical research, this tool is used in a variety of applications, such as protein-protein interactions, signaling events in cells, or to distinguish spectrally overlapping fluorophores. In this study, we show that phasor analysis is also suitable for the identification and differentiation of anthropogenic materials, such as autofluorescent microplastics. https://www.leica-microsystems.com/science-lab/label-free-differentiation-of-microplastics/ Wed, 12 May 2021 13:20:00 +0000 PhD Adrian Monteleone https://www.leica-microsystems.com/71573 Quantitative Imaging Adding Dimensions to Multiplex Molecular Imaging Molecular imaging of living specimens offers a means to draw upon the growing body of high-throughput molecular data to better understand the underlying cellular and molecular mechanisms of complex events ranging from embryonic development to disease processes. However, imaging approaches are challenged by unavoidable tradeoffs between spatial resolution, temporal resolution, field of view and the limited photon budget. https://www.leica-microsystems.com/science-lab/adding-dimensions-to-multiplex-molecular-imaging/ Thu, 15 Apr 2021 12:23:00 +0000 Professor Scott Fraser https://www.leica-microsystems.com/45279 Confocal Microscopy Quantitative Imaging How FLIM Microscopy helps to detect microplastic pollution The use of autofluorescence in biological samples is a widely used method to gain detailed knowledge about systems or organisms. This property is not only found in biological systems, but also anthropogenic materials, such as plastics, can emit autofluorescence. Measuring the temporal resolution of this emitted autofluorescence with fluorescence lifetime imaging microscopy (FLIM) generates data on fluorescence decay, i.e. the fluorescence lifetime (τ). Our study showed that the fluorescence lifetime can be used for a label-free characterization of plastics (microplastics). https://www.leica-microsystems.com/science-lab/how-flim-microscopy-helps-to-detect-microplastic-pollution/ Mon, 28 Dec 2020 09:50:00 +0000 PhD Adrian Monteleone https://www.leica-microsystems.com/44422 Confocal Microscopy Live-Cell Imaging Advanced Quantitative Fluorescence Microscopy to Probe the Molecular Dynamics of Viral Entry Viral entry into the host cell requires the coordination of many cellular and viral proteins in a precise order. Modern microscopy techniques are now allowing researchers to investigate these interactions with higher spatiotemporal resolution than ever before. Here we present two examples from the field of HIV research that make use of an innovative quantitative imaging approach as well as cutting edge fluorescence lifetime-based confocal microscopy methods to gain novel insights into how HIV fuses to cell membranes and enters the cell. https://www.leica-microsystems.com/advanced-quantitative-fluorescence-microscopy-to-probe-the-molecular-dynamics-of-viral-entry/ Wed, 11 Nov 2020 09:48:00 +0000 PhD Ben Libberton https://www.leica-microsystems.com/30582 Super-Resolution Time-resolved STED microscopy Introduced more than 30 years ago, stimulated emission depletion (STED) microscopy has raised to a standard and widely used method for imaging in the life sciences. Thanks to continuous technological progress, STED microscopy can now provide effective sub-diffraction spatial resolution, while preserving most of the useful aspects of fluorescence microscopy, such as optical sectioning, molecular specificity and sensitivity, and live-cell compatibility. https://www.leica-microsystems.com/science-lab/time-resolved-sted-microscopy/ Thu, 03 Sep 2020 12:04:00 +0000 Ph.D. Giuseppe Vicidomini https://www.leica-microsystems.com/26832 Quantitative Imaging Imaging Intracellular Temperature using Fluorescence Lifetime Imaging Microscopy (FLIM) This video shows the presentation of Dr. Kohki Okabe given at the 9th Congress of the Federation of Asian and Oceanian Physiological Society (FAOPS) held in Kobe, Japan on 30 March 2019. Dr. Okabe is an Assistant Professor and JST-PRESTO Researcher with the Laboratory of Bioanalytical Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo. https://www.leica-microsystems.com/science-lab/imaging-intracellular-temperature-using-fluorescence-lifetime-imaging-microscopy-flim/ Thu, 07 Nov 2019 09:10:00 +0000 PhD Kohki Okabe, Dr. Giulia Ossato https://www.leica-microsystems.com/26677 Quantitative Imaging SP8 FALCON: a novel concept in fluorescence lifetime imaging enabling video-rate confocal FLIM SP8 FALCON (FAst Lifetime CONtrast) is a fast and completely integrated fluorescence lifetime imaging microscopy (FLIM) confocal platform. SP8 FALCON delivers video-rate FLIM with pixel-by-pixel quantification, thanks to a novel concept for measuring fluorescence lifetimes built on fast electronics and sensitive spectral hybrid detectors. Photon arrival times are recorded at count rates typical for standard confocal imaging. The system has ultra-short dead time and powerful built-in algorithms for data acquisition and analysis. The deep integration of FLIM into the confocal platform provides easy access to complex FLIM experiments. https://www.leica-microsystems.com/science-lab/a-novel-concept-in-fluorescence-lifetime-imaging-enabling-video-rate-confocal-flim/ Thu, 17 Oct 2019 09:37:00 +0000 Dr. Luis Alvarez, Bernd Widzgowski, Bram van den Broek, Dr. Giulia Ossato, PhD Kees Jalink, Dr. Lioba Kuschel, Dr. Julia Roberti, Frank Hecht https://www.leica-microsystems.com/26294 Quantitative Imaging Development of fluorescence lifetime imaging microscopy (FLIM) and its relevance for functional imaging Prof. Ammasi Periasamy, Director, Keck Center for Cellular Imaging, University of Virginia, was interviewed by Dr. Giulia Ossato, Product Manager functional imaging, during Leica Microsystems Meets Science 2019 in Mannheim, Germany. They had an inspiring chat about fluorescence lifetime imaging microscopy (FLIM). The technological development of FLIM and its relevance for functional imaging in terms of studying redox states was discussed. Prof. Periasamy also talked about his research on metabolism and dysfunction in live cells. He stated that one of the central developments in the last few decades is the application of lifetime contrast to gather information about molecular interactions and function. https://www.leica-microsystems.com/science-lab/development-of-fluorescence-lifetime-imaging-microscopy-flim-and-its-relevance-for-functional-imaging/ Wed, 28 Aug 2019 10:13:00 +0000 Professor Ammasi Periasamy, Dr. Giulia Ossato https://www.leica-microsystems.com/25143 Quantitative Imaging How FRET/FLIM Biosensors and Lifetime Imaging Helped Identify the Importance of Zinc for Human B-Cell Development In her latest studies, Dr. Mukta Deobagkar-Lele investigated the role of ZIP7, a zinc transporter protein, in modulating B cell activity and its effects on human immunity. During an interview with Dr. Luis Alvarez from Leica Microsystems, she describes the key findings of these studies published in her recent article in Nature Immunology. She also discusses her introduction to lifetime imaging with the SP8 FALCON and how this modality helps her explore B-cell biology and related immunodeficiencies. https://www.leica-microsystems.com/science-lab/how-fretflim-biosensors-and-lifetime-imaging-helped-identify-the-importance-of-zinc-for-human-b-cell-development/ Tue, 30 Jul 2019 09:10:00 +0000 PhD Mukta Deobagkar, Dr. Luis Alvarez https://www.leica-microsystems.com/25094 Quantitative Imaging Single-residue bioorthogonal labeling of G protein-coupled receptors (GPCRs) in live cells for quantitative analysis To perform quantitative studies of membrane bound G protein-coupled receptors (GPCRs) with high-performance microscopy, the proteins must first be labeled with bright and photostable dyes. The labeling method should be minimally invasive to avoid perturbing the GPCR function and result in quantitative yields to allow stoichiometric data analysis. For the precise quantification of dye yields, a method based on fluorescence fluctuation microscopy (molecular brightness) has been developed. The method can extract the number of labeling sites at the single-cell level. Because the reported technique is performed using a commercially available microscope, the approach can be used for the general study of membrane proteins with fluorescence microscopy. https://www.leica-microsystems.com/science-lab/single-residue-bioorthogonal-labeling-of-g-protein-coupled-receptors-gpcrs-in-live-cells-for-quantitative-analysis/ Wed, 26 Jun 2019 10:03:00 +0000 PhD James DeRose, PhD Paolo Annibale https://www.leica-microsystems.com/24942 Quantitative Imaging Phasor Analysis for FLIM (Fluorescence Lifetime Imaging Microscopy) The Phasor analysis approach to analyze fluorescence lifetime does not require any fitting. Phasor FLIM (fluorescence lifetime imaging microscopy) provides a 2D graphical view of lifetime distributions. This graphical view enables any observer to distinguish and separate different lifetime populations within a FLIM image rapidly. The interpretation of phasor FLIM distributions is straightforward. Multiple molecular species are resolved within a single pixel, because every species has a specific phasor. https://www.leica-microsystems.com/science-lab/phasor-analysis-for-flim-fluorescence-lifetime-imaging-microscopy/ Tue, 07 May 2019 22:00:00 +0000 Dr. Giulia Ossato https://www.leica-microsystems.com/24692 Quantitative Imaging Researchers use Functional Imaging to Elucidate the Role of Cellular Zn²⁺ in Human Immunity An international consortium of researchers has found a new mutation in a gene harboring a reticulum-to-cytoplasm zinc transporter. The gene is called ZIP7 and it is responsible for the early onset of infections in patients. These findings led them to uncover the role of ZIP7 in B cell development. To elucidate the cellular function of ZIP7, researchers used CRISPR-Cas9 mutagenesis to recreate the observed mutations in mice. They then studied primary cells with functional imaging and zinc biosensors to corroborate the mutations and see how cells handle zinc. https://www.leica-microsystems.com/science-lab/researchers-use-functional-imaging-to-elucidate-the-role-of-cellular-zn2-in-human-immunity/ Wed, 27 Feb 2019 23:00:00 +0000 Dr. Luis Alvarez https://www.leica-microsystems.com/20689 Multiphoton Microscopy SP8 DIVE Multiphoton Microscope Image Gallery Today’s life science research focusses on complex biological processes, such as the causes of cancer and other human diseases. A deep look into tissues and living specimens is vital to understanding the conditions and mechanisms in cells and finding answers to crucial questions challenging the life sciences. https://www.leica-microsystems.com/science-lab/galleries/image-gallery-tunable-multicolor-deep-in-vivo-imaging/ Thu, 01 Nov 2018 23:00:00 +0000 https://www.leica-microsystems.com/20359 Quantitative Imaging Precise and lightning-fast spectral Fluorescence Lifetime Imaging at video rate integrated in a high-end confocal microscope Förster (Fluorescence) Resonance Energy Transfer (FRET) has become a powerful tool to study protein-protein interactions and signal transduction in living cells. FRET is commonly read out either by detecting the ratio of the donor and acceptor intensities (sensitized emission) or by detecting the excited state lifetime of the donor, which decreases with increasing FRET (Fluorescence Lifetime IMaging or FLIM). https://www.leica-microsystems.com/science-lab/precise-and-lightning-fast-spectral-fluorescence-lifetime-imaging-at-video-rate-integrated-in-a-high-end-confocal-microscope/ Tue, 07 Aug 2018 22:00:00 +0000 PhD Kees Jalink https://www.leica-microsystems.com/20297 Confocal Microscopy FLIM Image gallery SP8 FALCON (FAst Lifetime CONtrast) is the fast, and fully integrated platform for fluorescence lifetime imaging (FLIM) and analysis. It enables the observation of fast dynamic cellular events with lifetime contrasts between fluorescent molecules down to sub-second time scales. https://www.leica-microsystems.com/science-lab/image-gallery-flim-for-high-speed-investigation-of-molecular-interactions/ Wed, 04 Jul 2018 22:00:00 +0000 Dr. Johanna Berndt https://www.leica-microsystems.com/20208 Quantitative Imaging 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 https://www.leica-microsystems.com/20109 Quantitative Imaging 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. https://www.leica-microsystems.com/science-lab/lifetime-a-proper-alternative/ Wed, 28 Mar 2018 22:00:00 +0000 https://www.leica-microsystems.com/20133 Quantitative Imaging FCS - Fluorescence Correlation Spectroscopy FCS is a fluorescence-based measurement method. Fluorescent molecules passing through a strongly focused, fixed laser beam are excited for fluorescence emission. After passing a confocal pinhole, the emitted photons are registered using very sensitive detectors. https://www.leica-microsystems.com/science-lab/fcs-fluorescence-correlation-spectroscopy/ Wed, 28 Mar 2018 22:00:00 +0000 Dr. Lioba Kuschel https://www.leica-microsystems.com/20141 Quantitative Imaging FLIM FRET - Förster Resonance Energy Transfer 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. The SP8 FALCON system together with the integrated FRET analyzer provides FRET-efficiency and binding maps. https://www.leica-microsystems.com/science-lab/flim-fret-fluorescence-resonance-energy-transfer/ Wed, 28 Mar 2018 22:00:00 +0000 Dr. Lioba Kuschel https://www.leica-microsystems.com/20129 Quantitative Imaging What is FLIM - Fluorescence Lifetime Imaging? The fluorescence lifetime is a measure of how long a fluorophore remains on average in its excited state before returning to the ground state by emitting a fluorescence photon. https://www.leica-microsystems.com/science-lab/what-is-flim-fluorescence-lifetime-imaging/ Sun, 25 Mar 2018 22:00:00 +0000 Dr. Lioba Kuschel https://www.leica-microsystems.com/20130 Quantitative Imaging SP FLIM - Spectral Fluorescence Lifetime Imaging The SP8 FALCON is the ideal tool for spectral FLIM detection. No emission filters in front of the FLIM detectors are necessary. This grants a much higher flexibility to the experimental design. https://www.leica-microsystems.com/science-lab/sp-flim-spectral-fluorescence-lifetime-imaging/ Sun, 25 Mar 2018 22:00:00 +0000 Dr. Lioba Kuschel https://www.leica-microsystems.com/20132 Quantitative Imaging FCCS - Fluorescence Cross-Correlation Spectroscopy FCCS (Fluorescence Cross-Correlation Spectroscopy) can be measured using the Leica TCS SP8 FCS system. Similar to FCS , it analyzes fluorescence intensity fluctuations derived from a small observation volume. https://www.leica-microsystems.com/science-lab/fccs-fluorescence-cross-correlation-spectroscopy/ Sun, 25 Mar 2018 22:00:00 +0000 Dr. Lioba Kuschel https://www.leica-microsystems.com/19107 Confocal Microscopy Fluorescence Microscopy Testing the Münch Hypothesis of Long Distance Phloem Transport in Plants Long distance transport in plants occurs in sieve tubes of the phloem. The pressure flow hypothesis introduced by Ernst Münch in 1930 describes a mechanism of osmotically generated pressure differentials that are supposed to drive the movement of sugars and other solutes in the phloem, but this hypothesis has long faced major challenges. The key issue is whether the conductance of sieve tubes, including sieve plate pores, is sufficient to allow pressure flow. We show that with increasing distance between source and sink, sieve tube conductivity and turgor increases dramatically in Ipomoea nil. Our results provide strong support for the Münch hypothesis, while providing new tools for the investigation of one of the least understood plant tissues. https://www.leica-microsystems.com/science-lab/testing-the-muench-hypothesis-of-long-distance-phloem-transport-in-plants/ Tue, 14 Feb 2017 09:08:00 +0000 https://www.leica-microsystems.com/13942 Fluorescence Microscopy Quantitative Imaging 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/18431 Quantitative Imaging Live-Cell Imaging 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. https://www.leica-microsystems.com/science-lab/individual-macromolecule-motion-in-a-crowded-living-cell/ Mon, 27 Jun 2016 04:57:00 +0000 https://www.leica-microsystems.com/13262 Quantitative Imaging 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. https://www.leica-microsystems.com/science-lab/frap-with-tcs-sp8-resonant-scanner/ Tue, 26 Jan 2016 15:39:00 +0000 Dr. Jan Schröder https://www.leica-microsystems.com/16458 Confocal Microscopy Light Sheet Microscopy Super-Resolution "The Leica Digital Light Sheet Module – a Clever Example of Thinking Out of the Box" Bram van den Broek is a postdoctoral fellow at the Netherlands cancer institute in Amsterdam where he supports the advanced microscopy techniques in the laboratory of Kees Jalink. Working with Leica Microsystems as a collaboration partner for beta-testing of microscopes he enjoys very much. https://www.leica-microsystems.com/science-lab/the-leica-digital-light-sheet-module-a-clever-example-of-thinking-out-of-the-box/ Tue, 29 Sep 2015 14:39:00 +0000 Bram van den Broek, Dipl. oec.-troph. Anja Schué, PhD Isabelle Köster https://www.leica-microsystems.com/16176 Super-Resolution Quantitative Imaging 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. https://www.leica-microsystems.com/science-lab/a-straightforward-approach-for-gated-sted-fcs-to-investigate-lipid-membrane-dynamics/ Mon, 03 Aug 2015 10:29:00 +0000 https://www.leica-microsystems.com/16174 Super-Resolution Quantitative Imaging Live-Cell Imaging 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. https://www.leica-microsystems.com/science-lab/cortical-actin-networks-induce-spatio-temporal-confinement-of-phospholipids-in-the-plasma-membrane-a-minimally-invasive-investigation-by-sted-fcs/ Fri, 24 Jul 2015 13:02:00 +0000 Débora Machado Andrade https://www.leica-microsystems.com/15239 Super-Resolution Quantitative Imaging 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. https://www.leica-microsystems.com/science-lab/a-lipid-bound-actin-meshwork-organizes-liquid-phase-separation-in-model-membranes/ Wed, 15 Jul 2015 13:49:00 +0000 https://www.leica-microsystems.com/15511 Super-Resolution Encoding and Decoding Spatio-Temporal Information for Super-Resolution Microscopy The challenge of increasing the spatial resolution of an optical microscope beyond the diffraction limit can be reduced to a spectroscopy task by proper manipulation of the molecular states. The nanoscale spatial distribution of the molecules inside the detection volume of a scanning microscope can be encoded within the fluorescence dynamics and decoded by resolving the signal into its dynamics components. https://www.leica-microsystems.com/science-lab/encoding-and-decoding-spatio-temporal-information-for-super-resolution-microscopy/ Fri, 17 Apr 2015 10:35:00 +0000 https://www.leica-microsystems.com/15406 Confocal Microscopy Super-Resolution Live-Cell Imaging Multiphoton Microscopy Fluorescence Microscopy Quantitative Imaging 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. https://www.leica-microsystems.com/science-lab/interview-with-dr-gertrude-bunt-and-prof-fred-s-wouters-on-the-fom-2015/ Fri, 27 Mar 2015 11:36:00 +0000 Dr. Gertrude Bunt, Dr. Fred S. Wouters, Dipl. oec.-troph. Anja Schué https://www.leica-microsystems.com/14656 Confocal Microscopy Super-Resolution CLEM Live-Cell Imaging Quantitative Imaging 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. https://www.leica-microsystems.com/science-lab/icln-a-new-regulator-of-non-erythroid-41r-localisation-and-function/ Mon, 03 Nov 2014 18:02:00 +0000 https://www.leica-microsystems.com/12287 Fluorescence Microscopy Quantitative Imaging Live-Cell Imaging 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. https://www.leica-microsystems.com/science-lab/novel-fluorescent-carbonic-nanomaterials-for-sensing-and-imaging/ Thu, 16 Jan 2014 11:53:00 +0000 https://www.leica-microsystems.com/10111 Confocal Microscopy Multiple Microscopy Modes in a Single Sweep with Supercontinuum White Light Lasers have been critical to the advancement on confocal microscopy, and the white light laser (WLL) offers particular advantages. Finessing WLL output for bioimaging is a complex task, though, and traditional approaches retain key limitations. But acousto-optical beamsplitting enables smoother operation, leading to enhanced microscopy capabilities. https://www.leica-microsystems.com/science-lab/multiple-microscopy-modes-in-a-single-sweep-with-supercontinuum-white-light/ Fri, 14 Jun 2013 14:09:00 +0000 Dr. Lioba Kuschel https://www.leica-microsystems.com/9759 Quantitative Imaging 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. The resulting information can be used to determine kinetic properties, like the diffusion coefficient, mobile fraction, and transport rate of the fluorescently labeled molecules. FRAP employs irradiation of a fluorophore with a short laser pulse. State of the art laser scanning microscopes, like the TCS SP8 confocal system, have the advantage of using a high intensity laser for photobleaching and a low intensity laser for image recording. With the LAS AF application wizard you can choose between different ways to carry out a FRAP experiment. https://www.leica-microsystems.com/science-lab/step-by-step-guide-for-frap-experiments/ Fri, 17 May 2013 13:50:00 +0000 Dr. Jan Schröder https://www.leica-microsystems.com/9492 Live-Cell Imaging Fluorescence Microscopy Quantitative Imaging 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. https://www.leica-microsystems.com/science-lab/imaging-enzymes-at-work/ Fri, 05 Apr 2013 14:27:00 +0000 https://www.leica-microsystems.com/6656 Live-Cell Imaging Quantitative Imaging 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. https://www.leica-microsystems.com/science-lab/widefield-calcium-imaging-with-calcium-indicator-fura2/ Sun, 13 Jan 2013 23:00:00 +0000 Gabriele Burger, Simone Diehl https://www.leica-microsystems.com/7855 Quantitative Imaging Confocal Microscopy Live-Cell Imaging Fluorescence Microscopy 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. https://www.leica-microsystems.com/science-lab/integrative-open-source-software-for-image-analysis-in-biology/ Tue, 27 Nov 2012 23:00:00 +0000 Christian Dietz, Martin Horn, Prof. Dr. Michael Berthold https://www.leica-microsystems.com/6600 Confocal Microscopy Live-Cell Imaging Quantitative Imaging 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. https://www.leica-microsystems.com/science-lab/label-free-flim/ Thu, 09 Aug 2012 22:00:00 +0000 Dr. Constantin Kappel https://www.leica-microsystems.com/6602 Confocal Microscopy Live-Cell Imaging Quantitative Imaging 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. https://www.leica-microsystems.com/science-lab/fret-with-flim/ Tue, 07 Aug 2012 22:00:00 +0000 Dr. Constantin Kappel https://www.leica-microsystems.com/6609 Confocal Microscopy Quantitative Imaging 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. https://www.leica-microsystems.com/science-lab/fluorescence-correlation-spectroscopy/ Thu, 02 Aug 2012 22:00:00 +0000 Dr. Constantin Kappel https://www.leica-microsystems.com/5899 Quantitative Imaging 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. https://www.leica-microsystems.com/science-lab/quantitative-fluorescence-1/ Wed, 18 Apr 2012 22:00:00 +0000 https://www.leica-microsystems.com/5108 Confocal Microscopy Live-Cell Imaging Live-cell Imaging Techniques The understanding of complex and/or fast cellular dynamics is an important step for exploring biological processes. Therefore, today’s life science research is increasingly focusing on dynamic processes like cell migration, morphological changes of cells, organs or whole animals and physiological (e.g. changes of intracellular ion composition) events in living specimens in real time. https://www.leica-microsystems.com/science-lab/live-cell-imaging-techniques/ Thu, 23 Feb 2012 23:00:00 +0000 Dr. Thomas Veitinger, Dr. Zhongxiang Jiang https://www.leica-microsystems.com/5217 Confocal Microscopy Fluorescence Microscopy Live-Cell Imaging Neuroscience TIRF Microscopy Quantitative Imaging 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. https://www.leica-microsystems.com/science-lab/the-new-repository-on-the-block/ Mon, 06 Feb 2012 23:00:00 +0000 M.B.A. David N. Orloff, Ph.D. Janet Iwasa, Ph.D. Caroline Kane https://www.leica-microsystems.com/5082 Confocal Microscopy Quantitative Imaging 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. https://www.leica-microsystems.com/science-lab/flcs-advances-in-fluorescence-correlation-spectroscopy/ Wed, 14 Dec 2011 23:00:00 +0000 Dr. Andreas Bülter, Dr. Andrea Bleckmann, Uwe Ortmann https://www.leica-microsystems.com/4811 Confocal Microscopy Quantitative Imaging 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. https://www.leica-microsystems.com/science-lab/fluorescence-recovery-after-photobleaching-frap-and-its-offspring/ Wed, 23 Nov 2011 07:19:37 +0000 Dr. Jan Schröder https://www.leica-microsystems.com/4356 Confocal Microscopy Quantitative Imaging 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. https://www.leica-microsystems.com/science-lab/foerster-resonance-energy-transfer-fret/ Wed, 09 Nov 2011 09:45:00 +0000 Gabriele Burger https://www.leica-microsystems.com/10321 Quantitative Imaging 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 Imaging 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 Imaging ATP Changes the Fluorescence Lifetime of Cyan Fluorescent Protein via an Interaction with His148 This study employed fluorescence lifetime spectroscopy to investigate the influence of ATP (adenosine triphosphate) on the fluorescence intensities of yellow and cyan fluorescent proteins (YFP/CFP) which are used as the basis for FRET (Förster resonance energy transfer) sensors. This work further elucidates the complex and heterogeneous fluorescence properties of CFP and its mutant H148D, as well as the sensitivity of CFP fluorescence to changes in solute conditions. 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 Eugenia Petoukhov, PhD James DeRose https://www.leica-microsystems.com/8082 Quantitative Imaging Widefield Microscopy FRET Sensitized Emission Wizard Widefield Förster Resonance Energy Transfer (FRET) is a technique, which allows insight into the interactions between proteins or molecules in proximities beyond light microscopic resolution. An excited fluorophore, called the donor, transfers its excited state energy to a light absorbing molecule which is called the acceptor. This transfer of energy is non-radiative. Sensitized Emission is one established method for the evaluation of FRET efficiencies. It can be applied to live cells as well as to fixed samples. https://www.leica-microsystems.com/science-lab/fret-sensitized-emission-wizard-widefield/ Tue, 14 Apr 2009 22:00:00 +0000 Markus Schechter, Gabriele Burger https://www.leica-microsystems.com/10190 Quantitative Imaging Multiphoton Microscopy A New FRAP/FRAPa Method for Three-Dimensional Diffusion Measurements Based on Multiphoton Excitation Microscopy Quantitative measurement method based on FRAP and FRAPa using multiphoton microscopy. We present a new convenient method for quantitative three-dimensionally resolved diffusion measurements based on the photobleaching (FRAP) or photoactivation (FRAPa) of a disk-shaped area by the scanning laser beam of a multiphoton microscope. Contrary to previously reported spot-photobleaching protocols, this method has the advantage of full scalability of the size of the photobleached area and thus the range of diffusion coefficients, which can be measured conveniently. https://www.leica-microsystems.com/science-lab/a-new-frapfrapa-method-for-three-dimensional-diffusion-measurements-based-on-multiphoton-excitation-microscopy/ Fri, 11 Jul 2008 15:06:00 +0000 Davide Mazza https://www.leica-microsystems.com/8084 Confocal Microscopy Quantitative Imaging FRET Sensitized Emission Wizard Confocal Förster Resonance Energy Transfer (FRET) is a technique, which allows insight into the interactions between proteins or molecules in proximities beyond light microscopic resolution. https://www.leica-microsystems.com/science-lab/fret-sensitized-emission-wizard-confocal/ Thu, 14 Sep 2006 22:00:00 +0000 Markus Schechter, Gabriele Burger https://www.leica-microsystems.com/10023 Quantitative Imaging Fluorescence Recovery after Photobleaching with the Leica TCS SP2 Among all photobleaching experiments which have been described, fluorescence recovery after photobleaching (FRAP) is the most popular. It employs irradiation of a fluorophore in a living sample with a short laser pulse to degrade it and thereby abolish fluorescence followed by time-resolved image recording of the sample. https://www.leica-microsystems.com/science-lab/fluorescence-recovery-after-photobleaching-with-the-leica-tcs-sp2/ Sun, 15 Aug 2004 19:15:00 +0000 Dr. Constantin Kappel, Professor Dr. Roland Eils