Leica Science Lab - Tag : Live Imaging https://www.leica-microsystems.com//science-lab/tag/tags/live-imaging/show/Tag/ Article tagged with Live Imaging en-US https://www.leica-microsystems.com/4421 Coherent Raman Scattering (CRS) Coherent Raman Scattering Microscopy Publication List CRS (Coherent Raman Scattering) microscopy is an umbrella term for label-free methods that image biological structures by exploiting the characteristic, intrinsic vibrational contrast of their molecules. The two most important CRS techniques are Coherent Anti-Stokes Raman Scattering (CARS) and Stimulated Raman Scattering (SRS). The biochemical image contrast of CRS is in many ways complementary to the molecular contrast obtained in fluorescence microscopy. A second crucial advantage of these methods is that they preserve the specimen/sample in a near pristine state. This reference list presents current and basic papers on CRS microscopy. https://www.leica-microsystems.com//science-lab/cars-publication-list/ Sun, 26 May 2019 22:00:00 +0000 Dr. Volker Schweikhard https://www.leica-microsystems.com/24669 Super-Resolution Extending Nanoscopy Possibilities with STED and exchangeable fluorophores When it comes to STED Nanoscopy, keeping high signal-to-noise is key to achieve the best possible resolution in fixed and living cells. This can be challenging in the case of experiments in 3D and/or with time series, where the sample undergoes many rounds of image acquisition and photobleaching becomes an issue. If fluorophores were completely immune to photobleaching, it should be possible to perform STED indefinitely using the same molecules over and over. In practice, one performs STED with the best available fluorophores in terms of brightness and photostability (Grimm, Muthusamy et al. 2017), and at high labeling densities. However, there is a clever alternative to come closer to the ideal situation: if “fresh” fluorophores replenish the sample in each round of STED, imaging will take place with intact fluorophores every time. https://www.leica-microsystems.com//science-lab/extending-nanoscopy-possibilities-with-sted-and-exchangeable-fluorophores/ Tue, 12 Feb 2019 23:00:00 +0000 Dr. Julia Roberti https://www.leica-microsystems.com/24562 Confocal Microscopy Kaggle Competition for Multi-label Classification of Cell Organelles in Proteome Scale Human Protein Atlas Data The Cell Atlas, a part of the Human Protein Atlas, was created by the group of Prof. Emma Lundberg at the SciLifeLab, KTH Royal Institute of Technology, in Stockholm, Sweden. Currently, she is a visiting professor at Stanford University through the support of the Chan Zuckerberg Initiative. The Cell Atlas was created, in large part, using data acquired with Leica confocal instruments. In the scope of the Kaggle competition regarding the Human Protein Atlas Image Classification Prof. Lundberg gave an interview to Dr. Constantin Kappel from Leica Microsystems. https://www.leica-microsystems.com//science-lab/kaggle-competition-for-multi-label-classification-of-cell-organelles-in-proteome-scale-human-protein-atlas-data/ Sun, 09 Dec 2018 23:00:00 +0000 Dr. Constantin Kappel https://www.leica-microsystems.com/20653 Laser Microdissection Webinar: Live Cell Isolation by Laser Microdissection Laser microdissection is a tool for the isolation of homogenous cell populations from their native niches in tissues to downstream molecular assays. Beside its routine use for fixed tissue sections, laser microdissection may be applied for live cell isolation. Unlike other well-established and widely used techniques for live cell isolation and single cell cloning—such as FACS, MACS, cloning by limited dilution, and so on—laser microdissection allows for capturing live cells and cell colonies without their detachment from the carrier. https://www.leica-microsystems.com//science-lab/webinar-live-cell-isolation-by-laser-microdissection/ Tue, 16 Oct 2018 22:00:00 +0000 https://www.leica-microsystems.com/20061 Live-Cell Imaging Solvent immersion imprint lithography We expand upon our recent, fundamental report on solvent immersion imprint lithography (SIIL) and describe a semi-automated and high-performance procedure for prototyping polymer microfluidics and optofluidics. The SIIL procedure minimizes manual intervention through a cost-effective (∼$200) and easy-to-assemble apparatus. We analyze the procedure's performance specifically for Poly (methyl methacrylate) microsystems and report repeatable polymer imprinting, bonding, and 3D functionalization in less than 5 min, down to 8 μm resolutions and 1:1 aspect ratios. In comparison to commercial approaches, the modified SIIL procedure enables substantial cost reductions, a 100-fold reduction in imprinting force requirements, as well as a more than 10-fold increase in bonding strength. https://www.leica-microsystems.com//science-lab/solvent-immersion-imprint-lithography/ Wed, 21 Mar 2018 23:00:00 +0000 https://www.leica-microsystems.com/19922 Fluorescence Microscopy Widefield Microscopy Acute Transcriptional Up-regulation Specific to Osteoblasts/Osteoclasts in Medaka Fish Immediately after Exposure to Microgravity Bone loss is a serious problem in spaceflight; however, the initial action of microgravity has not been identified. To examine this action, we performed live-imaging of animals during a space mission followed by transcriptome analysis using medaka transgenic lines expressing osteoblast and osteoclastspecific promoter-driven GFP and DsRed. In live-imaging for osteoblasts, the intensity of osterix- or osteocalcin-DsRed fluorescence in pharyngeal bones was significantly enhanced 1 day after launch; and this enhancement continued for 8 or 5 days. https://www.leica-microsystems.com//science-lab/acute-transcriptional-up-regulation-specific-to-osteoblastsosteoclasts-in-medaka-fish-immediately-after-exposure-to-microgravity/ Tue, 13 Feb 2018 23:00:00 +0000 https://www.leica-microsystems.com/19686 Live-Cell Imaging Introduction to Mammalian Cell Culture Mammalian cell culture is one of the basic pillars of life sciences. Without the ability to grow cells in the lab, the fast progress in disciplines like cell biology, immunology, or cancer research would be unthinkable. This article gives an overview of mammalian cell culture systems. Mainly, they can be categorized according to their morphology, as well as cell type and organization. Moreover, you can find basic information about the correct growth conditions and what kind of microscope you need to watch your cells. https://www.leica-microsystems.com//science-lab/introduction-to-mammalian-cell-culture/ Thu, 10 Aug 2017 10:54:00 +0000 Dr. Christoph Greb https://www.leica-microsystems.com/19342 Super-Resolution Axial Tubule Junctions Control Rapid Calcium Signaling in Atria The canonical atrial myocyte (AM) is characterized by sparse transverse tubule (TT) invaginations and slow intracellular Ca2+ propagation but exhibits rapid contractile activation that is susceptible to loss of function during hypertrophic remodeling. Here, we have identified a membrane structure and Ca2+-signaling complex that may enhance the speed of atrial contraction independently of phospholamban regulation. This axial couplon was observed in human and mouse atria and is composed of voluminous axial tubules (ATs) with extensive junctions to the sarcoplasmic reticulum (SR) that include ryanodine receptor 2 (RyR2) clusters. In mouse AM, AT structures triggered Ca2+ release from the SR approximately 2 times faster at the AM center than at the surface. https://www.leica-microsystems.com//science-lab/axial-tubule-junctions-control-rapid-calcium-signaling-in-atria/ Wed, 31 May 2017 09:21:00 +0000 https://www.leica-microsystems.com/18994 Fluorescence Microscopy Live-Cell Imaging Widefield Microscopy Image Restoration and Deconvolution Chronic Inflammation Under the Microscope In the course of chronic inflammation certain body areas are recurrently inflamed. This goes along with many human diseases. With the help of widefield light microscopy, the underlying processes can be examined from a cellular level to whole organisms. This article presents several widefield microscopy applications such as immunofluorescence, live-cell imaging, histology, and ratiometric analysis to get insight into the development of chronic inflammation, the related diseases, and their treatment. https://www.leica-microsystems.com//science-lab/chronic-inflammation-under-the-microscope/ Mon, 09 Jan 2017 17:42:00 +0000 M.Sc. Jan Neumann, M.Sc. Anne Scherhag, Susanne Otten, Ph.D. Fangxia Shen, Anna Lena Leifke, Dr. Udo Birk, Dr. Christoph Greb, Dr. Kurt Lucas, Prof. Dr. Dr. Christoph Cremer https://www.leica-microsystems.com/18278 Super-Resolution Live-Cell Imaging Super-Resolution Optical Microscopy of Lipid Plasma Membrane Dynamics Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS, and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS, the STED-FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid–protein interactions and the traditional lipid ‘raft’ theory. https://www.leica-microsystems.com//science-lab/super-resolution-optical-microscopy-of-lipid-plasma-membrane-dynamics/ Fri, 23 Dec 2016 14:16:00 +0000 Prof. Christian Eggeling https://www.leica-microsystems.com/18952 Super-Resolution Live-Cell Imaging Image Restoration and Deconvolution Discovery of Novel Peptides Targeting Pro-Atherogenic Endothelium in Disturbed Flow Regions -Targeted siRNA Delivery to Pro-Atherogenic Endothelium in vivo Application example of HyVolution Super-Resolution - Atherosclerosis occurs preferentially in arterial regions exposed to disturbed blood flow. Targeting these pro-atherogenic regions is a potential anti-atherogenic therapeutic approach, but it has been extremely challenging. Here, using in vivo phage display approach and the partial carotid ligation model of flow-induced atherosclerosis in mouse, we identified novel peptides that specifically bind to endothelial cells (ECs) exposed to disturbed flow condition in pro-atherogenic regions. https://www.leica-microsystems.com//science-lab/discovery-of-novel-peptides-targeting-pro-atherogenic-endothelium-in-disturbed-flow-regions-targeted-sirna-delivery-to-pro-atherogenic-endothelium-in-vivo/ Thu, 10 Nov 2016 15:28:00 +0000 https://www.leica-microsystems.com/18730 Confocal Microscopy Live-Cell Imaging Adeno-associated Viral Vectors do not Efficiently Target Muscle Satellite Cells Adeno-associated viral (AAV) vectors are becoming an important tool for gene therapy of numerous genetic and other disorders. Several recombinant AAV vectors (rAAV) have the ability to transduce striated muscles in a variety of animals following intramuscular and intravascular administration, and have attracted widespread interest for therapy of muscle disorders such as the muscular dystrophies. Here we examined the relative ability of rAAV vectors derived from AAV6 to target myoblasts, myocytes, and myotubes in culture and satellite cells and myofibers in vivo. AAV vectors are able to transduce proliferating myoblasts in culture, albeit with reduced efficiency relative to postmitotic myocytes and myotubes. In contrast, quiescent satellite cells are refractory to transduction in adult mice. https://www.leica-microsystems.com//science-lab/adeno-associated-viral-vectors-do-not-efficiently-target-muscle-satellite-cells/ Mon, 05 Sep 2016 05:58:00 +0000 https://www.leica-microsystems.com/15428 Confocal Microscopy Live-Cell Imaging Highly Selective Fluorescent and Colorimetric Probe for Live-cell Monitoring of Sulphide Based on Bioorthogonal Reaction H2S is the third endogenously generated gaseous signaling compound and has also been known to involve a variety of physiological processes. To better understand its physiological and pathological functions, efficient methods for monitoring of H2S are desired. Azide fluorogenic probes are popular because they can take place bioorthogonal reactions. In this work, by employing a fluorescein derivative as the fluorophore and an azide group as the recognition unit, we reported a new probe 5-azidofluorescein for H2S with improved sensitivity and selectivety. https://www.leica-microsystems.com//science-lab/highly-selective-fluorescent-and-colorimetric-probe-for-live-cell-monitoring-of-sulphide-based-on-bioorthogonal-reaction/ Fri, 29 Jul 2016 11:41:00 +0000 https://www.leica-microsystems.com/15645 Confocal Microscopy Live-Cell Imaging Label-free in vivo Imaging of Myelinated Axons in Health and Disease with Spectral Confocal Reflectance Microscopy We report a new technique for high-resolution in vivo imaging of myelinated axons in the brain, spinal cord and peripheral nerve that requires no fluorescent labeling. This method, based on spectral confocal reflectance microscopy (SCoRe), uses a conventional laser scanning confocal system to generate images by merging the simultaneously reflected signals from multiple lasers of different wavelengths. https://www.leica-microsystems.com//science-lab/label-free-in-vivo-imaging-of-myelinated-axons-in-health-and-disease-with-spectral-confocal-reflectance-microscopy/ Fri, 01 Jul 2016 09:29:00 +0000 https://www.leica-microsystems.com/18431 Quantitative Fluorescence 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/17977 Confocal Microscopy Live-Cell Imaging The Bimodally Expressed MicroRNA miR‐142 Gates Exit from Pluripotency A stem cell's decision to self‐renew or differentiate is thought to critically depend on signaling cues provided by its environment. It is unclear whether stem cells have the intrinsic capacity to control their responsiveness to environmental signals that can be fluctuating and noisy. Using a novel single‐cell microRNA activity reporter, we show that miR‐142 is bimodally expressed in embryonic stem cells, creating two states indistinguishable by pluripotency markers. https://www.leica-microsystems.com//science-lab/the-bimodally-expressed-microrna-mir-142-gates-exit-from-pluripotency/ Fri, 13 May 2016 13:47:00 +0000 https://www.leica-microsystems.com/17931 Camera Technology Live-Cell Imaging What Makes sCMOS Microscope Cameras so Popular? sCMOS cameras are more sensitive and are capable of much higher acquisition speed than cameras with other sensor types. Even though CCD cameras are widely used in live cell imaging and time-lapse recordings, researchers are often concerned that their camera does not detect faint signals. In this interview, Dr. Karin Schwab, Product Manager at Leica Microsystems, talks about the characteristics of sCMOS cameras and how researchers benefit from the latest camera sensor technology. https://www.leica-microsystems.com//science-lab/what-makes-scmos-microscope-cameras-so-popular/ Mon, 11 Apr 2016 10:22:00 +0000 Claudia Müller, Dr. Karin Schwab https://www.leica-microsystems.com/17667 Live-Cell Imaging Widefield Microscopy How to do a Proper Cell Culture Quick Check In order to successfully work with mammalian cell lines, they must be grown under controlled conditions and require their own specific growth medium. In addition, to guarantee consistency their growth must be monitored at regular intervals. This article describes a typical workflow for subculturing an adherent cell line with detailed illustrations of all of the necessary steps. https://www.leica-microsystems.com//science-lab/how-to-do-a-proper-cell-culture-quick-check/ Thu, 24 Mar 2016 09:18:00 +0000 PhD Tamara Straube, Claudia Müller https://www.leica-microsystems.com/17669 Confocal Microscopy Super-Resolution Image Restoration and Deconvolution HyVolution – the Smart Path to Confocal Super-Resolution Super-resolution refers to any device or method that can resolve better than the classical Abbe limit. Apart from infinite super-resolution techniques such as STED (stimulated emission depletion) and SMLM (single-molecule localization methods) that can theoretically resolve to any detail, there are also methods for limited super-resolution. Here we present HyVolution by Leica, which merges optical super-resolution and computational super-resolution. The optical part is provided by confocal microscopy, and the computational part by deconvolution. Lateral resolution of 140 nm is demonstrated. HyVolution offers multiple fluorescence recording in truly simultaneous mode. https://www.leica-microsystems.com//science-lab/hyvolution-the-smart-path-to-confocal-super-resolution/ Fri, 18 Mar 2016 14:49:00 +0000 Dr. Rolf T. Borlinghaus, Dr. Constantin Kappel https://www.leica-microsystems.com/17611 Super-Resolution Multiphoton Microscopy Live-Cell Imaging 4Pi-RESOLFT Nanoscopy Here we apply the 4Pi scheme to RESOLFT nanoscopy using two-photon absorption for the on-switching of fluorescent proteins. We show that in this combination, the lobes are so low that low-light level, 3D nanoscale imaging of living cells becomes possible. Our method thus offers robust access to densely packed, axially extended cellular regions that have been notoriously difficult to super-resolve. Our approach also entails a fluorescence read-out scheme that translates molecular sensitivity to local off-switching rates into improved signal-to-noise ratio and resolution. https://www.leica-microsystems.com//science-lab/4pi-resolft-nanoscopy/ Fri, 26 Feb 2016 12:41:00 +0000 https://www.leica-microsystems.com/13262 Quantitative Fluorescence 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/16587 Light Sheet Microscopy Live-Cell Imaging Confocal Microscopy Light Sheet Microscopy Turned Vertically Living cells and organisms often suffer from the high light intensities used for fluorescent imaging. Light sheet microscopy reduces phototoxic effects and bleaching by illuminating a specimen in only a single plane at a time. A new light sheet microscope combines light sheet and confocal microscopy in one system without compromising either functionality and allows the combination of the two methods, e.g. confocal photomanipulation with subsequent light sheet acquisition, for new applications. https://www.leica-microsystems.com//science-lab/light-sheet-microscopy-turned-vertically/ Fri, 02 Oct 2015 13:34:00 +0000 PhD Isabelle Köster, Dr. Petra Haas https://www.leica-microsystems.com/16521 Super-Resolution Live-Cell Imaging Fluorescence Microscopy Probes that FIT RNA We have been developing new tools based on fluorogenic forced intercalation (FIT) probes for RNA detection quantification and interference in biological samples. Upon duplex formation with target nucleic acids, the base surrogates TO dye increases its quantum yield and brightness substantially (>10 fold). https://www.leica-microsystems.com//science-lab/probes-that-fit-rna/ Tue, 22 Sep 2015 17:25:00 +0000 PhD Imre Gaspar https://www.leica-microsystems.com/16174 Super-Resolution Quantitative Fluorescence 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/15700 Super-Resolution Live-Cell Imaging CRISPR-Cas9 CRISPR/Cas9-mediated Endogenous Protein Tagging for RESOLFT Super-Resolution Microscopy of Living Human Cells Overexpression is a notorious concern in conventional and especially in super-resolution fluorescence light microscopy studies because it may cause numerous artifacts including ectopic sub-cellular localizations, erroneous formation of protein complexes, and others. Nonetheless, current live cell super-resolution microscopy studies generally rely on the overexpression of a host protein fused to a fluorescent protein. https://www.leica-microsystems.com//science-lab/crisprcas9-mediated-endogenous-protein-tagging-for-resolft-super-resolution-microscopy-of-living-human-cells/ Wed, 22 Jul 2015 16:24:00 +0000 https://www.leica-microsystems.com/15241 Super-Resolution Live-Cell Imaging Multi-protein Assemblies Underlie the Mesoscale Organization of the Plasma Membrane Most proteins have uneven distributions in the plasma membrane. Broadly speaking, this may be caused by mechanisms specific to each protein, or may be a consequence of a general pattern that affects the distribution of all membrane proteins. https://www.leica-microsystems.com//science-lab/multi-protein-assemblies-underlie-the-mesoscale-organization-of-the-plasma-membrane/ Thu, 09 Jul 2015 13:44:00 +0000 https://www.leica-microsystems.com/13642 CLEM EM Sample Preparation Correlative Light and Electron Microscopy – Get Your Free CLEM e-Book for Download The urge to go deeper into the microscopic world has led researchers to combine the versatility of the light microscopy (LM) with the resolution power of the electron microscope (EM) to produce Correlative Light and Electron Microscopy (CLEM). CLEM’s most significant and powerful characteristic for cell biology research is an ability to study the same cell using two different microscopy platforms. The Essential Knowledge Briefing describes the basics of CLEM and also reviews potential pitfalls and problems as well as tricks for solving them. https://www.leica-microsystems.com//science-lab/correlative-light-and-electron-microscopy-get-your-free-clem-e-book-for-download/ Thu, 28 May 2015 16:34:00 +0000 Dr. Paul Verkade https://www.leica-microsystems.com/15430 Multiphoton Microscopy Confocal Microscopy Live-Cell Imaging Third Harmonic Generation Microscopy – Label-Free 3D-Tissue Imaging and Blood Flow Characterization THG microscopy as special variants of multiphoton microscopy. Third Harmonic Generation (THG) microscopy is a non-fluorescent multi-photon technique that combines the advantages of label-free imaging with restriction of signal generation to the focal spot of the scanning laser. It allows three-dimensional imaging of refraction index mismatches and of hemoglobin. https://www.leica-microsystems.com//science-lab/third-harmonic-generation-microscopy-label-free-3d-tissue-imaging-and-blood-flow-characterization/ Sat, 11 Apr 2015 17:34:00 +0000 Dr. Steffen Dietzel https://www.leica-microsystems.com/15111 Super-Resolution Universal PAINT – Dynamic Super-Resolution Microscopy Super-resolution microscopy techniques have revolutionized biology for the last ten years. With their help cellular components can now be visualized at the size of a protein. Nevertheless, imaging living cells is a challenge for most of the super-resolution principles. https://www.leica-microsystems.com//science-lab/universal-paint-dynamic-super-resolution-microscopy/ Mon, 02 Mar 2015 08:24:00 +0000 Ph.D. Eric Hosy, Dr. Christoph Greb https://www.leica-microsystems.com/15203 Confocal Microscopy Live-Cell Imaging Video: High Speed Scanning – With two Scanners in one System High speed scanning is necessary to image rapidly changing biological processes. With traditional scanning techniques, imaging speed is limited by the number of fluorophores in a specimen. And, rapid acquisition often comes at the cost of image resolution. https://www.leica-microsystems.com//science-lab/video-high-speed-scanning-with-two-scanners-in-one-system/ Tue, 24 Feb 2015 15:15:00 +0000 PhD Christopher Vega