Leica Science Lab - Topic : Quantitative Imaging https://www.leica-microsystems.com//science-lab/topics/quantitative-fluorescence/?tx_leicaacademy_pi2%5Baction%5D=listPagesForTopic&tx_leicaacademy_pi2%5Bcontroller%5D=Topic&tx_leicaacademy_pi2%5Btopic%5D=5&cHash=a2f34a529537892079727b12ece71eb1 Article tagged with Quantitative Imaging en-US https://www.leica-microsystems.com/75169 Live-Cell Imaging Quantitative Imaging Fluorescence Microscopy Digital Microscopy Learning the Cellular Architecture from its Optical Properties In the last 3 years, microscopists have started to use "AI based" solutions for a wide range of applications, including image acquisition optimization (smart microscopy), object classification, image classification, segmentation, restoration, super resolution and virtual staining. The symposium highlighted scientific breakthroughs which are enabled by machine learning or deep learning technologies. The 6th edition of the AI Microscopy Symposium offered a unique forum for presenting and discussing the latest AI-based technologies and tools in the field of microscopy and biomedical imaging. https://www.leica-microsystems.com/science-lab/learning-the-cellular-architecture-from-its-optical-properties-1/ Thu, 11 Nov 2021 10:34:00 +0000 PhD Shalin Mehta, Corporate Communications https://www.leica-microsystems.com/73002 Quantitative Imaging Confocal Microscopy Phasor FLIM Detects Metabolic Differences The maintenance of normal blood glucose levels is defective in diabetes caused by dysfunction of the alpha and beta cells in islets. To understand these regularities, we used multiphoton phasor-FLIM NADH autofluorescence imaging to detect metabolic changes of living islet cells before and after glucose stimulation. Multiphoton phasor FLIM NADH autofluorescence imaging provides a straightforward detection and analysis to monitor metabolic states in living organisms. https://www.leica-microsystems.com/science-lab/phasor-flim-detects-metabolic-differences/ Mon, 28 Jun 2021 08:19:00 +0000 PhD Peiyu Wang 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 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/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 Ph.D. 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/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/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 Dr. Rolf T. Borlinghaus 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 Dr. Rolf T. Borlinghaus 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/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