NIEF manager, Dr. Otero, participated in the Quantitative Fluorescence Microscopy course held at the MDI Biological Laboratory in Bar Harbor, ME. This international course in fluorescence microscopy and imaging provided the opportunity to train in several high-end microscopes and imaging analysis software and take lectures that cover the theory, mechanics, and applications of this biomedical technology.. Dr. Otero received a competitive scholarship from MDI Biological Laboratory Education and won the Chroma Cube Award for providing an outstanding individual scientific presentation at the course.. #gallery-1{margin:auto}#gallery-1 .gallery-item{float:left;margin-top:10px;text-align:center;width:33%}#gallery-1 img{border:2px solid #cfcfcf}#gallery-1 .gallery-caption{margin-left:0} ...
Total Internal Reflection Microscopy (TIRM) is a sensitive non-invasive technique to measure the interaction potentials between a colloidal particle and a wall with femtonewton resolution. The equilibrium distribution of the particle-wall separation distance z is sampled monitoring the intensity I scattered by the Brownian particle under evanescent illumination. Central to the data analysis is the knowledge of the relation between I and the corresponding z, which typically must be known a priori. This poses considerable constraints to the experimental conditions where TIRM can be applied (short penetration depth of the evanescent wave, transparent surfaces). Here, we introduce a method to experimentally determine I(z) by relying only on the distance-dependent particle-wall hydrodynamic interactions. We demonstrate that this method largely extends the range of conditions accessible with TIRM, and even allows measurements on highly reflecting gold surfaces where multiple reflections lead to a ...
Total internal reflection microscopy (TIRM) is a sensitive non-invasive technique to measure the interaction potentials between a colloidal particle and a wall with femtonewton resolution. Our work has largely extended the range of conditions accessible with TIRM, e.g. gold coated surfaces or large penetration depths.
Imaging single-channel Ca21 signals by total internal reflection fluorescence microscopy. (A) Schematic of the TIRFM imaging system. The 488-nm beam from an argon ion laser (50 mW) passes through a 53 beam expander (BE) and is focused by a lens (FL; f ¼ 150 mm) via a dichroic mirror (DM)to a spot at the back focal plane of the microscope objective lens (Olympus TIRFM 603, oil immersion, NA ¼ 1.45). The focusing lens is mounted on a micrometer-driven translation stage, so that the laser beam can be adjusted to enter the periphery of the objective aperture so as to achieve total internal reflection at the interface between the cover glass and the aqueous bathing medium. An adjustable rectangular knife-blade aperture (A) located at a conjugate image plane defines the field of excitation. Fluorescence excited in the specimen by the evanescent wave is collected by the same objective, passes through the dichroic mirror and a barrier filter (BF) blocking the laser wavelength, and is imaged by an ...
Total internal reflection fluorescence microscopy (TIRFM) is an elegant optical technique utilized to observe single molecule fluorescence at surfaces and interfaces. This section is an index to our discussions, references, and interactive Java tutorials that describe TIRFM.
The regulated trafficking or exocytosis of cargo-containing vesicles to the cell surface is fundamental to all cells. By coupling the technology of fluorescently tagged fusion proteins with total internal reflection fluorescence microscopy (TIRFM), it is possible to achieve the high spatio-temporal resolution required to study the dynamics of sub-plasma membrane vesicle trafficking and exocytosis. TIRFM has been used in a number of cell types to visualize and dissect the various steps of exocytosis revealing how molecules identified via genetic and/or biochemical approaches are involved in the regulation of this process. Here, we summarize the contribution of TIRFM to our understanding of the mechanism of exocytosis and discuss the novel methods of analysis that are required to exploit the large volumes of data that can be produced using this technique.
We report the use of a high-refractive-index aplanatic solid immersion lens (ASIL) in total internal reflection fluorescence (TIRF) microscopy. This new solid immersion total internal reflection fluorescence (SITIRF) microscopy allows highly confined surface imaging with a significantly reduced imaging depth compared with conventional TIRF microscopy. We explore the application of a high refractive index, low optical dispersion material zirconium dioxide in the SITIRF microscope and also introduce a novel system design which enables the SITIRF microscope to work either in the epi-fluorescence or TIRF modes with variable illumination angles. We use both synthetic and biological samples to demonstrate that the imaging depth in the SITIRF microscope can be confined to a few tens of nanometers. SITIRF microscopy has the advantages of performing highly selective imaging and high-resolution high-contrast imaging. Potential applications in biological imaging and future developments of SITIRF microscopy ...
TY - JOUR. T1 - Real time imaging of single fluorophores on moving actin with an epifluorescence microscope. AU - Sase, I.. AU - Miyata, H.. AU - Corrie, J. E T. AU - Craik, J. S.. AU - Kinosita, K.. PY - 1995. Y1 - 1995. N2 - Relatively simple modifications of an ordinary epifluorescence microscope have greatly reduced its background luminescence, allowing continuous and real time imaging of single fluorophores in an aqueous medium. Main modifications were changing the excitation light path and setting an aperture stop so that stray light does not scatter inside the microscope. A simple and accurate method using actin filaments is presented to establish the singularity of the observed fluorophores. It was possible, at the video rate of 30 frames/s, to image individual tetramethylrhodamine fluorophores bound to actin filaments sliding over heavy meromyosin. The successful imaging of moving fluorophores demonstrates that conventional microscopes may become a routine tool for studying dynamic ...
Anytime: Self-serve breakfast in the dining hall. 09:00-09:30 Introduction Welcome, Introductions, Define Course Goals (Piston and St. Croix). 09:30-10:30 The Microscope 1 Advances in Modern Imaging for Biological Problems (Kenworthy). 10:30-11:00 Break at Maren Conference Center. 11:00-12:00 The Microscope 2 Transmitted Light Contrast in Microscopy (Piston). 12:00-13:00 Lunch. 13:00-14:45 LAB 1 TRANSMITTED LIGHT MICROSCOPY. 15:00-15:45 The Microscope 3 Fluorescence Microscopy and Quantitation (Piston). 15:45-16:45 Live Cell Imaging 1 Green-Fluorescent Protein (Kremers). 16:45-17:00 Break. 17:00-18:00 Digital Imaging 1 Digital Imaging, Resolution, and Detectors (Watkins). 18:00-19:00 Dinner. 19:00-20:00 Digital Imaging 2 Optical Filters and Brightness Demonstration (Watkins et al.). 20:30-23:00 LAB 2 STANDARD FLUORESCENCE MICROSCOPY. ...
Friday, May 16, 2014. 16:00 Arrival on campus, check-in to housing, get keys at Maren Conference Center. 18:00 Dinner and Social at Co-Op. 19:30 Reception at Maren Conference Center and conference material pickup. Late arrival keys placed in Dining Hall keybox for pick-up. Saturday, May 17, 2014. anytime self-serve breakfast in the dining hall. 09:00-09:30 Introduction Welcome, Introductions, Define Course Goals (Piston and St. Croix). 09:30-10:30 The Microscope 1 Advances in Modern Imaging for Biological Problems (Kenworthy). 10:30-11:00 Break at Maren Conference Center. 11:00-12:00 The Microscope 2 Transmitted Light Contrast in Microscopy (Piston). 12:00-13:00 Lunch. 13:00-14:45 LAB 1 TRANSMITTED LIGHT MICROSCOPY. 15:00-15:45 The Microscope 3 Fluorescence Microscopy and Quantitation (Piston). 15:45-16:45 Live Cell Imaging 1 Green-Fluorescent Protein (Kremers). 16:45-17:00 Break. 17:00-18:00 Digital Imaging 1 Digital Imaging, Resolution, and Detectors (Watkins). 18:00-19:00 Dinner. 19:00-20:00 ...
In 2008, the first international Theodor Förster lecture series took place at the University of Cambridge http://laser.ceb.cam.ac.uk/foerster. Throughout the year, leading researchers were invited from all over the world to hold talks and engage with the life science community at Cambridge. The focus was on developments in quantitative optical microscopy techniques, which are revolutionizing research in the biological sciences today.. The name of the series commemorates the brilliant contributions of Theodor Förster, who, 60 years ago, published his seminal paper on the quantitative theory of electronic energy transfer (Förster 1948). The process, now known as Förster resonance energy transfer (FRET), takes place frequently in nature and refers to the non-radiative transport of energy from a donor to an acceptor molecule. Förster recognized that through a sequence of such interactions, energy can migrate over distances much larger than the molecular scale and in one sweep he had resolved ...
In electromagnetics, an evanescent field, or evanescent wave, is an oscillating electric and/or magnetic field that does not propagate as an electromagnetic wave but whose energy is spatially concentrated in the vicinity of the source (oscillating charges and currents). Even when there in fact is an electromagnetic wave produced (e.g., by a transmitting antenna) one can still identify as an evanescent field the component of the electric or magnetic field that cannot be attributed to the propagating wave observed at a distance of many wavelengths (such as the far field of a transmitting antenna). A hallmark of an evanescent field is that there is no net energy flow in that region. Since the net flow of electromagnetic energy is given by the average Poynting vector, that means that the Poynting vector in these regions, as averaged over a complete oscillation cycle, is zero. In many cases one cannot simply say that a field is or is not evanescent. For instance, in the above illustration energy is ...
Topology of cell adhesion on a substrate is measured with nanometre precision by variable-angle total internal reflection fluorescence...
Wide-field fluorescence microscopy is a widely applied imaging technique used to examine cells and investigate their internal structures.
We describe a method of obtaining optical sectioning with a standard wide-field fluorescence microscope. The method involves acquiring two images, one with nonuniform illumination (in our case, speckle) and another with uniform illumination (in our case, randomized speckle). An evaluation of the loc …
Principal Investigator:FUNATSU Takashi, Project Period (FY):2009-07-23 - 2014-03-31, Research Category:Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Project Area:Integrative understanding of biological processes mediated by transient macromolecular complexes; New technology for visualizing physiologically metastable states.
A wide range of optical designs for TIRFM have been employed with both inverted and upright microscope configurations. This discussion focuses on TIRFM configurations that utilize a prism to direct light toward the TIR interface.
Basic molecular mechanisms of cell surface receptors that mediate transmembrane signals can be elucidated by integrating information from multiple interdisciplinary approaches. Our studies focus on the receptor (FceRI) for immunoglobulin E (IgE) that plays a central role in the allergic response and serves as a model for other types of immune receptors. Binding and cross-linking of IgE-FceRI complexes by antigen initiates signal transduction resulting in cell activation and release of chemical mediators.. We measure kinetics and thermodynamics of binding and cross-linking between cell-bound IgE and structurally defined ligands with fluorescence methods and analyze with realistic theoretical models to determine features that are critical for signaling. We employ quantitative fluorescence microscopy, including confocal imaging and total internal reflection fluorescence (TIRF) microscopy, to monitor changes in the distribution and dynamics of the receptor and signaling components (and genetically ...
Single molecule fluorescence measurements are used to probe the effects of GM1 in DPPC monolayers. Langmuir-Blodgett films of GM1 and DPPC were doped with ~10−8 mol% of the fluorescent lipid probe, BODIPY-PC, and transferred onto glass substrates at 23 mN/m. As shown previously, the individual orientation of each BODIPY-PC probe in the membrane can be measured using defocused polarized total internal reflection fluorescence microscopy, revealing changes in film properties at the molecular level. Here, BODIPY-PC tilt angle histograms are used to characterize the effects of GM1 in DPPC films from 0.05 mol% to 100 mol% GM1. At high GM1 levels (,5 mol% GM1), trends in the single molecule measurements agree with previous bulk measurements showing the turnover from condensing to expanding influence of GM1 at ~20 mol%, thus validating the single molecule approach. At biologically relevant, low concentrations of GM1 (,5 mol% GM1), where bulk fluorescence measurements are less informative, the single ...
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BILLERICA, Mass. -- Bruker today announced that it has acquired Vutara Incorporated, a technology leader in high-speed, three-dimensional (3D), ...
As a high-resolution wide-field near-surface microscopy, total internal reflection fluorescence microscopy (TIRFM) has been widely applied for the study of biomolecules. Unlike those costly, sample consuming and time consuming traditional detection assays, the application of TIRFM enable the direct quantification of biomolecules in a sample pretreatment and enrichment free fashion. Taking advantages of the TIRFM imaging system, in this thesis we have applied the TIRFM imaging system to directly quantify the content of different cancer associated biomarkers. Four different detection approaches for direct cancer biomarkers quantification with the aid of TIRFM were herein presented respectively. In Chapter 2, a direct quantification of nasopharyngeal carcinoma associated miRNAs was described. In the assay, five different miRNAs were chosen as the target analytes, which hybridized with the synthetic complementary LNA, probe in solution. The duplex was labeled with intercalating fluorescence dye YOYO-1 and
Total internal reflection fluorescence (TIRF) microscopy is a powerful technique. It provides extremely thin axial sectioning with excellent signal-to
Total internal reflection fluorescence (TIRF) microscopy is a high‐contrast imaging technique suitable for observing biological events that occur on or near the cell membrane
TY - JOUR. T1 - Threshold-free evaluation of near-surface diffusion and adsorption-dominated motion from single-molecule tracking data of single-stranded DNA through total internal reflection fluorescence microscopy. AU - Hanasaki, Itsuo. AU - Uehara, Satoshi. AU - Arai, Yoshiyuki. AU - Nagai, Takeharu. AU - Kawano, Satoyuki. PY - 2015/12. Y1 - 2015/12. N2 - Total internal reflection fluorescence (TIRF) microscopy enables the single-molecule observation in liquid near substrate surface. However, the evaluation of the diffusion from their individually-tracked positions entails the difficulty in the treatment of molecular adsorption on the substrate. We propose a novel technique to evaluate them, and two types of near-surface Brownian motion were determined for DNA. One is the diffusion near glass surface, and the other is the adsorption-dominated motion, which is also found to be diffusive rather than anchored to the substrate. Our technique does not require the threshold values for the ...
Fluorescence microscopy images of cells double stained with phalloidin for actin filaments (red) and DAPI for nuclei (blue) on Ti (A) and USP-Ti (B) samples.Abb
Video articles in JoVE about tirf include A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors, SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy, Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy, A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins, Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy, Single Molecule Fluorescence Microscopy on Planar Supported Bilayers, Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking, Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair, Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes, Counting Proteins in Single Cells with Addressable
Dr. Jorge Bernardino de la Serna (JbdlS) is a Senior Lecturer at the National Heart and Lung Institute, Imperial College London, and Visiting Scientist at the United Kingdom Research and Innovation. JBdlS has a markedly multidisciplinary research track record; he has ~25 publications in the last 5 years. JBdlSs research has been highlighted on 6 journal covers for his work on protein-lipid localisation and dynamics; Tcell lipid-protein interactions in atopic dermatitis; advanced quantitative fluorescence microscopy and spectroscopy; and peptide/peptoid-protein interactions with biological films. All these publications had in common the employment of highly-advanced microscopy techniques. JBdlS last publication in ACSnano were he bserved the theragnostic effect in Gram Negative Bacteria of a fluorescent molecule at the nanoscale. This article has been highlighted in The Telegraph and BBC Sheffield. JBdlS has a track record of microscopy method development and customisation to understand ...
Dr. Jorge Bernardino de la Serna (JbdlS) is a Senior Lecturer at the National Heart and Lung Institute, Imperial College London, and Visiting Scientist at the United Kingdom Research and Innovation. JBdlS has a markedly multidisciplinary research track record; he has ~25 publications in the last 5 years. JBdlSs research has been highlighted on 6 journal covers for his work on protein-lipid localisation and dynamics; Tcell lipid-protein interactions in atopic dermatitis; advanced quantitative fluorescence microscopy and spectroscopy; and peptide/peptoid-protein interactions with biological films. All these publications had in common the employment of highly-advanced microscopy techniques. JBdlS last publication in ACSnano were he bserved the theragnostic effect in Gram Negative Bacteria of a fluorescent molecule at the nanoscale. This article has been highlighted in The Telegraph and BBC Sheffield. JBdlS has a track record of microscopy method development and customisation to understand ...
In this study we show that as a single agent in Hct116 cells in vitro, SN-38 induces cell cycle arrest without cell death. This correlates to the absence of CRs observed in vivo with CPT-11 alone. The addition of flavopiridol to SN-38-treated Hct116 cells caused cell death in vitro, and in vivo this translated into greater tumor regression as well as CRs. With clonogenic assays, we observed that SN-38-treated cells remained attached to the plate as viable single cells. These cells did not form colonies because cell cycle arrest by SN-38, and this resulted in the inhibition of colony formation. However, SN-38 alone did not induce apoptosis; therefore, we did not observe PARP cleavage or caspase-3 activation. In contrast, flavopiridol added to SN-38-treated Hct116 cells induced significant apoptosis as indicated by quantitative fluorescence microscopy assays, PARP cleavage, and caspase-3 activation. We observed neither single viable cells nor colonies by clonogenic assays. In essence, flavopiridol ...
Today is exciting because I get to do two things. 1: Post the first successful images of West Nile virus infectined cells that I took on my own and 2: Give a basic explanation one of the most visually impressive techniques at our disposal: immunofluorescent microscopy. For those of you not familiar with fluorescent microscopy…
Endocytosis/recycling and exocytosis are mechanisms conserved through evolution allowing cells to communicate with their external medium. In order to study these dynamic processes, the present work proposes a patch-based method for detecting recycling or exocytotic events at the Plasma membrane in fast TIRF microscopy combined with the computation of normalized temporal representations of those events. Evaluation, performed on TIRF sequences showing Transferrin receptor (TfR) recycling, validates a high detection rate fully compatible with an automatic data extraction and analysis of the plasma membrane recycling process.
TY - CONF. T1 - Chirality in evanescent fields: conditions, limits and guidelines (Conference Presentation). AU - Raziman, T. V.. AU - Godiksen, Rasmus H.. AU - Müller, Moos A.. AU - Curto, Alberto G.. PY - 2020/4/1. Y1 - 2020/4/1. KW - optical chirality. KW - circular dichroism. KW - evanescent waves. KW - spin. U2 - 10.1117/12.2555726. DO - 10.1117/12.2555726. M3 - Paper. SP - 24. T2 - Metamaterials XII. Y2 - 6 April 2020 through 10 April 2020. ER - ...
We study the molecular mechanisms underlying bacterial multiplication. Bacteria are renowned for their fast multiplication. During their short cell cycles, bacteria grow, replicate and segregate their chromosomes and divide. They do all of this with sometimes blazing speed and with remarkable temporal and spatial accuracy, which explains their success. Despite the medical, agricultural and ecological importance of bacteria, little is known about the molecular mechanisms involved in growth, DNA segregation, cell division and cell morphogenesis. Similarly, the temporal and spatial mechanisms regulating these essential processes remain largely elusive. We address these fundamental questions using genetics, biochemistry, quantitative fluorescence microscopy and modeling.. ...
Dragonfly is a multi-dimensional imaging platform offering three key imaging modalities. At its core is a multi-point confocal for high-speed and high-sensitivity image capture. With speed at least 10x faster than conventional confocal technology, this mode is the optimal solution for live cell imaging, avoiding sensitivities to phototoxicity and photobleaching.. A second mode is laser-illuminated widefield epifluorescence. This mode is ideal for applications that do not benefit from confocal imaging, such as very thin samples or applications that require high laser power density, such as single molecule localisation. This mode benefits from Borealis illumination and is complemented by GPU accelerated deconvolution.. The third mode is total internal reflectance microscopy (TIRF) which is available as a factory-installed option. This is the tool of choice for imaging protein dynamics at or proximal to the cell membrane.. The Dragonfly platform is controlled by Andors Fusion software, which ...
The nature of light limits the size of the spot to which light can be focused. According to the diffraction limit a focused light distribution cannot be made smaller than approximately half of the wavelength of the used light. Uncovered in the 19th century by Ernst Abbe this has been a barrier of the achievable resolution of fluorescence light microscopes for a long time. While resolution is denoted by the ability to discern different objects of the same kind, localizing or tracking of single particles have been performed with a precision much below the diffraction limit. Several improvements in microscopy techniques have been invented in the 20th century and have resulted in increased resolution and contrast to some extent. In 1978 first theoretical ideas have been developed to break this barrier by using a 4Pi microscope as a confocal laser scanning fluorescence microscope where the light is focused ideally from all sides to a common focus which is used to scan the object by point-by-point ...
Understanding a proteins subcellular localization is critical to understanding a proteins role in the cell. The physical location of a protein limits its possible interaction partners and suggests possible biological functions for the protein [1]. The subcellular localization of a protein may be readily assessed by covalently binding it to a fluorescent protein, such as green fluorescent protein (GFP), and viewing the resulting fluorescence by microscopy. The observed pattern and intensity of fluorescence indicate the location and relative quantity of the protein in the cell.. Protein-protein interactions may be assessed via fluorescence microscopy by observing the relative subcellular localization of separate proteins simultaneously tagged with different fluorescent proteins. Proteins with strongly overlapping patterns of subcellular localization are said to colocalize; and this colocalization may indicate similar biological function or possible protein-protein interaction. Interactions may ...
Total internal reflection microscopy excites only to a depth of 100 nm, which is ideally suited to visualize fluorescent membrane proteins or proteins that operate adjacent to a membrane. Our 2 setups allow the use of UV, blue, green, yellow and red excitation, and the combination with Fluorescence Revovery after Photobleaching (FRAP). This technique uses bleaching of a small area within cells and determines protein mobility by measuring regain of fluorescence within the bleached area over time. Differential FRAP can determine association constants of protein complexes within live cells, which is done in the Flow Cytometry & Imaging Facility of the MPI Micter http://www.mpi-marburg.mpg.de/facs. Our TIRF microscopes are based on Zeiss Axioobservers, feature fast EM-CCD cameras and can also be used for regular wide field microscopy, including 3D reconstruction and time lapse microscopy.. ...
As changes in cytosolic free Ca2+ play key roles in coupling responses in neutrophils, it is important to locate and identify Ca2+ storage sites within these cells. Here, recent data is presented which highlights the functional link between microanatomical structure and cell signalling function. Fluorescent optical probes for cytosolic free Ca2+ have been used, together with organelle specific markers. We present evidence from conventional fluorescence microscopy, together with ratiometric and confocal laser scanning fluorescence microscopy, which pin-points two cellular locations for Ca2+ within the neutrophil; one within the nuclear lobes, and the other towards the cell periphery. Knowledge of these two locations provides a clear insight into how signalling in this cell type is regulated and provides a framework for explaining how specific stimuli act to produce specific responses. ...
Skeletal muscle. Rotating fluorescence light microscopy footage of skeletal muscle tissue from a tongue. Fluorescent dyes have been used to highlight tissues, cellular structures and proteins. Proteins highlighted include actin (green). - Stock Video Clip K004/6259
Blood vessel. Rotating fluorescence light microscopy footage of a blood vessel (pink) traversing skeletal muscle in tissue from the tongue. Fluorescent dyes have been used to highlight tissues, cellular structures and proteins. - Stock Video Clip K004/6256
Fluorescence microscopy applications are rapidly expanding in many medical and biological research laboratories. The mainstream of fluorescence microscopy has undergone an almost total shift from utilizing transmitted light to incident light, accompanied by the introduction of many new and different fluorochromes. Fluorescence microscopy applications are rapidly expanding in many medical and biological research laboratories. The mainstream of fluorescence microscopy has undergone an almost total shift from utilizing transmitted light to incident light, accompanied by the introduction of many new and different fluorochromes.
Fluorescence is one of the most commonly used physical phenomena in biological and analytical microscopy, mainly because of its high sensitivity and high specificity. Fluorescence is a form of luminescence. Fluorescence microscopy even allows users to determine the distribution of a single molecule species, its amount and its localization inside a cell. Colocalization and interaction studies can be performed, and ion concentrations, as well as intra- and intercellular processes like endocytosis and exocytosis, can be observed. With the help of super-resolution fluorescence microscopy, it is even possible to image sub-resolution structures.. ...
Bayesian localization microscopy reveals nanoscale podosome dynamics. Susan Cox, Edward Rosten, James Monypenny, Tijana Jovanovic-Talisman, Dylan T Burnette, Jennifer Lippincott-Schwartz, Gareth E Jones & Rainer Heintzmann. Nature Methods. 2012, volume 9: 195-200. doi: 10.1038/nmeth.1812. http://f1000.com/13978956 An analytical approach based on the concept of fluorophore localisation provides dynamic super-resolution data of xFP- labelled live cells using a common arc lamp based wide-field fluorescence microscope. One method of achieving fluorescence super-resolution is based around finding the positions of fluorescent molecules that label the cellular structure of interest. In this approach, positions can be determined precisely and accurately using fluorescent probes that can be photoactivated, photoconverted or photoswitched to generate single images with emitter densities of only about one active fluorophore per diffraction-limited area. Many images each containing subsets of active ...
Bayesian localization microscopy reveals nanoscale podosome dynamics. Susan Cox, Edward Rosten, James Monypenny, Tijana Jovanovic-Talisman, Dylan T Burnette, Jennifer Lippincott-Schwartz, Gareth E Jones & Rainer Heintzmann. Nature Methods. 2012, volume 9: 195-200. doi: 10.1038/nmeth.1812. http://f1000.com/13978956 An analytical approach based on the concept of fluorophore localisation provides dynamic super-resolution data of xFP- labelled live cells using a common arc lamp based wide-field fluorescence microscope. One method of achieving fluorescence super-resolution is based around finding the positions of fluorescent molecules that label the cellular structure of interest. In this approach, positions can be determined precisely and accurately using fluorescent probes that can be photoactivated, photoconverted or photoswitched to generate single images with emitter densities of only about one active fluorophore per diffraction-limited area. Many images each containing subsets of active ...
Emissive base analogs are powerful tools for probing nucleic acids at the molecular level. Herein we describe the development and thorough characterization of pentacyclic adenine (pA), a versatile base analog with exceptional fluorescence properties. When incorporated into DNA, pA pairs selectively with thymine without perturbing the B-form structure and is among the brightest nucleobase analogs reported so far. Together with the recently established base analog acceptor qAnitro, pA allows accurate distance and orientation determination via Förster resonance energy transfer (FRET) measurements. The high brightness at emission wavelengths above 400 nm also makes it suitable for fluorescence microscopy, as demonstrated by imaging of single liposomal constructs coated with cholesterol-anchored pA-dsDNA, using total internal reflection fluorescence microscopy. Finally, pA is also highly promising for two-photon excitation at 780 nm, with a brightness (5.3 GM) that is unprecedented for a base analog.
Define wide-field fluorescence microscopy: Wide-field fluorescence microscopy uses either naturally occurring structures or staining with fluorescent ...
Light Microscopy and Resolution -- Super-Resolution Microscopy: Principles, Techniques, and Applications -- Foundations of STED Microscopy -- Application of Real-Time STED Imaging to Synaptic Vesicle Motion -- Photoactivated Localization Microscopy for Cellular Imaging -- Data Analysis for Single-Molecule Localization Microscopy -- Super-Resolution Fluorescence Microscopy Using Structured Illumination -- Application of Three-Dimensional Structured Illumination Microscopy in Cell Biology: Pitfalls and Practical Considerations -- Scanning Near-Field Optical Microscopy for Investigations of Bio-Matter -- Atomic Force Microscopy of Living Cells -- X-Ray Microscopy for Neuroscience: Novel Opportunities by Coherent Optics -- Nonlinear Optics Approaches Towards Sub-Diffraction Resolution in CARS Imaging -- Photo-Oxidation Microscopy: Bridging the Gap Between Fluorescence and Electron Microscopy -- Requirements for Samples in Super-Resolution Fluorescence Microscopy -- Probing Biological Samples in ...
Fingerprint Dive into the research topics of A fluorescence microscopy study of quantum dots as fluorescent probes for brain tumor diagnosis. Together they form a unique fingerprint. ...
This dissertation describes the application of fluorescence microscopy techniques to investigations of mass transport phenomena in self-assembled nanomaterials. The microscopic morphologies of the materials and the mass-transport dynamics of probe molecules dispersed within them were assessed with high temporal and spatial resolution by single molecule imaging and spectroscopic methods. Three distinct sets of experiments were performed in completing the work for this dissertation. In the first study, single molecule imaging was employed to explore the interactions and field-induced migration of double-stranded DNA (ds-DNA) molecules with nanostructured Pluronic F127 gels. While DNA interactions with nanostructured gels have been explored in the past, none had apparently looked at these interactions in gels comprising hexagonally ordered arrays of cylindrical micelles. Therefore, these studies focused on materials DNA dispersed in flow aligned hexagonal F127. DNA molecules were found to be ...
Super-resolution microscopy has tremendously progressed our understanding of cellular biophysics and biochemistry. Specifically, 4pi fluorescence microscopy technique stands out because of its axial super-resolution capability. All types of 4pi-microscopy techniques work well in conjugation with deconvolution techniques to get rid of artifacts due to side-lobes. In this regard, we propose a technique based on spatial filter in a 4pi-type-C confocal setup to get rid of these artifacts. Using a special spatial filter, we have reduced the depth-of-focus. Interference of two similar depth-of-focus beams in a 4 pi geometry result in substantial reduction of side-lobes. Studies show a reduction of side-lobes by 46% and 76% for single and two photon variant compared to 4pi - type - C confocal system. This is incredible considering the resolving capability of the existing 4pi - type - C confocal microscopy. Moreover, the main lobe is found to be 150 nm for the proposed spatial filtering technique as ...
Total Internal Reflection Fluorescence Microscopy (TIRFM) is a common method to study structural and dynamic processes at the single-molecule level. The principle is based on an optical phenomenon where electromagnetic radiation is not refracted but totally reflected on the boundary surface of two different media. An evanescent field is generated at the boundary surface and penetrates the medium with lower optical density where fluorescent molecules can be excited. This evanescent wave attains its maximum intensity at the surface and then exponentially decays with increasing penetration depth. Therefore, only molecules located near the surface are excited, as opposed to molecules situated at distances exceeding approx. 100 nm, and thus TIRF efficiently decreases background signals of the bulk solution and allows sensitive detection down to single molecules ...
Part of the prestigious Max Planck Society based in Germany, MPFI is the first and only institute of its kind in North America. Situated in the new biosciences cluster in scenic Palm Beach County in South Florida, MPFI provides a vibrant, collaborative environment where scientists are provided generous ongoing support to conduct high impact research at the cutting edge.. ...
Box 1. Determining the quality of a TIRF set up. The test samples described below can be used to check the quality of any TIRF set up. They should always be prepared using a cover slip with the correct thickness and refractive index for the objective.. Test samples. Fluorescent microbeads. These can be purchased from many sources, including Invitrogen (Carlsbad, CA) and Bangs Laboratories (Fishers, IN). The beads should be of subresolution size (100 nm diameter or less), and selected to have excitation and emission spectra that match typical experimental conditions. The beads should be diluted in water and applied to the cover slip. PBS can be added to increase the number of beads that adhere to the surface.. DiI. A convenient, uniform, fluorescent film can be easily made on a cover slip surface with the lipophilic fluorophore DiI (Invitrogen, Carlsbad, CA). Dissolve the DiI at 0.5 mg/ml in ethanol and place a single droplet of the solution on a glass cover slip. Then, before the solution dries, ...
Super-resolution fluorescence imaging methods based on reversible photoswitching of fluorophores with subsequent localization currently develop to promising tools for cellular imaging. Since most of these methods rely on the transfer of the majority of fluorophores to a non-fluorescent dark state and precise localization of separated fluorescent fluorophores, the photophysical properties of photoswitchable fluorophores have to be carefully controlled. The achievable resolution and herewith the ability to resolve a structural feature depends not only on the brightness of the fluorophores, but also on the labeling density and on the stability or lifetime of the non-fluorescent dark state. Here, we discuss how the ratio of off- and on-switching of a fluorophore affects resolution. We compare experimental data with theoretical simulations and present a strategy to customize photoswitching characteristics to achieve optimal optical resolution. (C) 2010 Elsevier B.V. All rights reserved ...
The ASU Library acknowledges the twenty-two Native Nations that have inhabited this land for centuries. Arizona State Universitys four campuses are located in the Salt River Valley on ancestral territories of Indigenous peoples, including the Akimel Oodham (Pima) and Pee Posh (Maricopa) Indian Communities, whose care and keeping of these lands allows us to be here today. ASU Library acknowledges the sovereignty of these nations and seeks to foster an environment of success and possibility for Native American students and patrons. We are advocates for the incorporation of Indigenous knowledge systems and research methodologies within contemporary library practice. ASU Library welcomes members of the Akimel Oodham and Pee Posh, and all Native nations to the Library.. ...
A super-resolution platform for correlative live single-molecule imaging and STED microscopy. Super-resolution microscopy offers tremendous opportunities to unravel the complex and dynamic architecture of living cells. However, current super-resolution microscopes are well suited for revealing protein distributions or cell morphology, but not both. We present a super-resolution platform that permits correlative single-molecule imaging and stimulated emission depletion microscopy in live cells. It gives nanoscale access to the positions and movements of synaptic proteins within the morphological context of growth cones and dendritic spines.. ...
0050] This species of surgical fluorescence stereomicroscopes requires specific properties known to one skilled in the art, and specific constituents of the microscope that make said properties possible. Those pertinent to conventional fluorescence microscopy (which already existed long before the creation of surgical fluorescence stereomicroscopes) are: [0051] an excitation light source or excitation illumination device (formerly, for example, often a mercury vapor lamp), [0052] an excitation filter to improve the quality of the excitation light (spectrally filtering out those light wavelength regions that do not contribute particularly well, or at all, to fluorescence excitation), and a blocking filter or observation filter in the observation beam path of the surgical fluorescence stereomicroscope. The latter serves in turn to filter out the excitation light to a greater or lesser extent, since little or none of it is after all intended in principle to be seen, but the emission of the ...
When Eric Betzig and colleagues first described their new microscopy method, PALM, they chose to highlight its power by comparing it to an ultra-high-resolution approach: transmission electron microscopy (TEM). PALM, or photoactivated localization microscopy, is a super-resolution fluorescence technique allowing users to circumvent the 200 nm diffraction limit that constrains optical microscopy, mapping fluorophores to within…
Day 1. Cell growth. MCF7, LLC-PK1 cells. Grow MCF7 or LLC-PK1 cells at 37˚C in high glucose DMEM, supplemented with 8% FBS, sodium pyruvate and antibiotics [15, 16]. 24 hours prior to the experiment, trypsinize cells and culture overnight on glass cover slips coated with poly-L-lysine.. Superior cervical ganglion (SCG) neurons. Prepare SCG neurons from postnatal day 1 to 7 as described [11]. Culture cells on glass coverslips (coated with laminin) in modified 35 mm dishes (Corning) at 37˚C for 7-10 days; change medium twice per week.. Day 2. Treatment with MitoTracker®. Remove medium and treat cells with fresh DMEM supplemented with MitoTracker® (diluted 1:5,000 in DMEM). Incubate for 30 minutes at 37˚C in a cell culture incubator (5% CO2).. Fixation, permeabilization, block of non-specific binding sites * MCF7 and LLC-PK1 cells*. After 30 min incubation with MitoTracker®, remove the tissue culture medium, wash cells once with pre-warmed PBS. Fix samples in 3.7 % formaldehyde/PBS for 15 min ...
Structural characterization by super-resolution microscopy has become increasingly widespread, particularly in the biological community. The technique is powerful because it can produce real-space images with resolutions of tens of nanometers, while sample preparation is relatively non-invasive. Previous studies have applied these techniques to important scientific problems in the life sciences, but relatively little work has explored the attainable limit of resolution using samples of known structure. In this work, we apply photo-activated localization microscopy (PALM) to polymer films that have been nanopatterned using electron-beam lithography. Trace amounts of a rhodamine spiroamide dye are dispersed into nanostructured poly(methyl methacrylate), and UV-induced switching of the fluorophores enables nanoscale localization of single molecules to generate a final composite super-resolution image. Features as small as 25 nm half-pitch are clearly resolvable.. We also explore the effect of ...
Fluorescence filters help to isolate specific wavelengths of fluoresced light - which might seem light years away from anything to do with jellyfish evolution.. But in fact, many modern scientific processes are derived from naturally occurring biochemical capabilities, such as the green fluorescent proteins (GFPs) found off the coast of North America in the jellyfish species Aequorea victoria.. These green proteins can be used as fluorescent markers for biological phenomena, by attaching them as genetic tags and then using fluorescence filters to see where they go.. Some GFPs can do more though - including changing colour to become red - and these are especially valuable for super-resolution fluorescence microscopy.. A team at Arizona State University have now discovered a hinge migration mechanism that they say is the key for evolution of a green-to-red photoconvertible phenotype in a GFP.. It took eight years to unlock this process, which is described in a paper published in the academic ...
Visualizing 3D structure and dynamics at the molecular scale is a current and critical need in biomedical research. Many sub-cellular features, for example the morphology of many organelles or the 3D organization of chromatin, cannot be resolved by standard light microscopy. Improving the resolution of light microscopy has therefore been an urgent need of biological research for many decades. Today, several methods achieve sub-100 nm resolution by taking advantage of reversible or irreversible photo-physical switching properties of fluorescent markers. Our research group in the Department of Cell Biology at Yale University School of Medicine is developing new fluorescence microscopy techniques with spatial and/or temporal resolutions exceeding far beyond current technology and also applying them to a diverse set of biological questions.. Specialized Terms: Super-resolution fluorescence ...
-A key step of adaptive immune responses is the T lymphocyte capacity to detect the presence of foreign antigens on specialized cells with high speed and specificity during contacts lasting a few minutes. Much evidence suggests that there is a deep link between the lifetime of molecular interactions between T cell receptors and ligands and T cell activation, but the precise mechanisms of bond formation and dissociation remain incompletely understood. Previous experiments done with interference reflection microscopy/reflection interference contrast microscopy disclosed transverse motions with several nanometer average amplitude of micrometer size membrane zones. More recently, total internal reflection fluorescence microscopy was used to show that the initial interaction between primary T lymphocytes and model surfaces involved the tip of microvilli (typically 0.2 lm 2 area) generating apparent contacts of a few seconds that allowed cells to detect ligands of their membrane receptors. Here we show that
The study of protein dynamics is essential for understanding the multi-molecular complexes at subcellular levels. Fluorescent Protein (XFP)-tagging and time-lapse fluorescence microscopy enable to observe molecular dynamics and interactions in live cells, unraveling the live states of the matter. Original image analysis methods are then required to process challenging 2D or 3D image sequences. Recently, tracking methods that estimate the whole trajectories of moving objects have been successfully developed. In this paper, we address rather the detection of meaningful events in spatio-temporal fluorescence image sequences, such as apparent stable stocking areas involved in membrane transport. We propose an original patch-based Markov modeling to detect spatial irregularities in fluorescence images with low false alarm rates. This approach has been developed for real image sequences of cells expressing XFP-tagged Rab proteins, known to regulate membrane trafficking.
Our other large Zeiss epifluorescent microscope is the Zeiss Axiophot. In addition to fluorescence microscopy, this microscope is also used for phase contrast and differential interference contrast (DIC) microscopy. Though it is an older scope, it was recently outfitted with a modern digital Zeiss AxioCam MR camera and utilizes the same Zen software as the new Axio Imager 2. Phase contrast and DIC microscopy allow for observation of unstained, live cells and structures. For fluorescent microscopy, this microscope utilizes a Zeiss XBO 75 (75W xenon arc lamp) and HBO 100 (100W mercury arc lamp).. For additional information including manuals, manufacturer links, protocols and publications, see our resources page. To see images captured by this microscope, see the gallery.. ...
Find Molecular Probes® fluorescent labels for multiplexed super-resolution microscopy (SRM) applications, including STORM, STED, SIM, and two-photon microscopy.
phdthesis{f9c27a48-2eb3-4555-b7c6-9d9c5fb019ce, abstract = {In the research area of cleaning and process hygiene it is important to understand the underlying mechanisms behind the deposition of particles and macromolecules onto surfaces. The deposition process is described as the transport of particles to the surface followed by attachment. If the hydrodynamic forces are strong enough, the particles can be re-entrained.,br/,,br, ,br/,,br, In this work particle deposition was studied <i>in situ</i>. In order to obtain a well-defined mass transfer to a glass surface covered with indium-tin oxide onto which deposition was to occur, an experimental setup consisting of a wall-jet cell was employed. Model solutions consisting of polystyrene latex particles of two particle radii, 0.23 um and 0.38 um, were used in the deposition experiments. The deposited particles were visualized by total internal reflection microscopy. The construction of the cell made it possible to radially scan the ...
2. What did the paper show that normal microscopy could not show. In this paper Schermelleh et al. noted that although normal fluorescence light microscopy permits the cellular components to be visualised in various colours, it is limited in providing adequate resolution. They overcame this problem by applying the use of 3D-SIM. In this paper they use it to study the mammalian nucleus. In particular, with the use of 3D-SIM they were able to resolve single NPCs[1], differentially localize distinct NPC components[2] and detect double-layered invaginations of the nuclear envelope[3], all of which would not be possible with the use of conventional microscopy. Schermelleh et al. notes that the use 3D-SIM will allow for a greater exploration of the subcellular structures beyond the diffraction limit of the emitted light[4] Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. --Z3254598 00:52, 22 March 2012 (EST) ...
This course will cover the theory and practical application of current super-resolution microscopy techniques to biological questions.
We introduce a method for correlative in-resin super-resolution fluorescence and electron microscopy (EM) of biological structures in mammalian culture cells. Cryo-fixed resin embedded samples offer superior structural preservation, performing in-resin super-resolution, however, remains a challenge. We identified key aspects of the sample preparation procedure of high pressure freezing, freeze substitution and resin embedding that are critical for preserving fluorescence and photo-switching of standard fluorescent proteins, such as mGFP, mVenus and mRuby2. This enabled us to combine single molecule localization microscopy with transmission electron microscopy imaging of standard fluorescent proteins in cryo-fixed resin embedded cells. We achieved a structural resolution of 40-50 nm (~17 nm average single molecule localization accuracy) in the fluorescence images without the use of chemical fixation or special fluorophores. Using this approach enabled the correlation of fluorescently labeled ...
The cytoskeleton is involved in many cellular processes. Over the last decade, super-resolution microscopy has become widely available to image cytoskeletal structures, such as microtubules and actin, with great detail. For example, Single-Molecule Localization Microscopy (SMLM) achieves resolutions of 5-50 nm through repetitive sparse labeling of samples, followed by Point-Spread-Function ... read more analysis of individual fluorophores. Whereas initially this approach depended on the controlled photoswitching of fluorophores targeted to the structure of interest, alternative techniques now depend on the transient binding of fluorescently labeled probes, such as the small polypeptide lifeAct that can transiently interact with polymerized actin. These techniques allow for simple multicolor imaging and are no longer limited by a fluorophores blinking properties. Here we describe a detailed step-by-step protocol to purify, label, and utilize the lifeAct fragment for SMLM. This purification and ...
RhoGTPases are important regulators of the cell cytoskeleton, controlling cell shape, migration and proliferation. Previously we showed that ARHGAP18 in endothelial cells is important in cell junctions. Here we show, using structured illumination microscopy (SIM), ground-state depletion (GSD), and total internal reflection fluorescence microscopy (TIRF) that a proportion of ARHGAP18 localizes to microtubules in endothelial cells, as well as in nonendothelial cells, an association confirmed biochemically. In endothelial cells, some ARHGAP18 puncta also colocalized to Weibel-Palade bodies on the microtubules. Depletion of ARHGAP18 by small interfering RNA or analysis of endothelial cells isolated from ARHGAP18-knockout mice showed microtubule destabilization, as evidenced by altered morphology and decreased acetylated α-tubulin and glu-tubulin. The destabilization was rescued by inhibition of ROCK and histone deacetylase 6 but not by a GAP-mutant form of ARHGAP18. Depletion of ARHGAP18 resulted ...
Project Description: The Kner and De La Fuente Labs are interested in using a novel 3D superresolution fluorescence microscopy approach to study nucleosome organization and the role of specific proteins in chromatin organization. The project involves the engineering of a new superresolution microscope, imaging of chromosomes, and biological questions that are important for in reproduction.. REU Student Role and Responsibility: The REU student will assist a graduate student in developing the microscope and in imaging experiments on samples form the De La Fuente lab. The student will assist in developing software to run the microscope and process the images, will help run the microscope to collect data, and will help prepare the samples for imaging.. Expected Outcome for REU student: The student will be trained in cutting-edge microscopy techniques and exposed to important questions in modern biology. Superresolution imaging of the nucleus is a hot topic. This project is expected to result in at ...
Endocrinology. 2003 Aug;144(8):3532-40. Ovine placental lactogen-induced heterodimerization of ovine growth hormone and prolactin receptors in living cells is demonstrated by fluorescence resonance energy transfer microscopy and leads to prolonged phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3. Biener E, Martin C, Daniel N, Frank SJ, Centonze VE, Herman B, Djiane J, Gertler A ...
Researchers from UCL, the National Physical Laboratory and the Royal Free Hospital have differentiated between patients with a rare bleeding disorder and healthy volunteers using super-resolution microscopy, providing an alternative method for accurately and cost-effectively diagnosing rare platelet diseases.
Get an introduction to the technologies, unique benefits, practical examples, and future developments of super-resolution microscopy.
Repulsive guidance molecules (RGMs) control crucial processes including cell motility, adhesion, immune-cell regulation and systemic iron metabolism. RGMs signal via the neogenin (NEO1) and the bone morphogenetic protein (BMP) pathways. Here, we report crystal structures of the N-terminal domains of all human RGM family members in complex with the BMP ligand BMP2, revealing a new protein fold and a conserved BMP-binding mode. Our structural and functional data suggest a pH-linked mechanism for RGM-activated BMP signaling and offer a rationale for RGM mutations causing juvenile hemochromatosis. We also determined the crystal structure of the ternary BMP2-RGM-NEO1 complex, which, along with solution scattering and live-cell super-resolution fluorescence microscopy, indicates BMP-induced clustering of the RGM-NEO1 complex. Our results show how RGM acts as the central hub that links BMP and NEO1 and physically connects these fundamental signaling pathways. Repulsive guidance molecule is a structural ...
Extraordinary transmission based axial imaging (EOT-AIM) for cell microscopy is reported. EOT-AIM uses linear arrays of nanoapertures, each of which samples target fluorescence up to a preset axial distance from surface, in combination with wide-field microscopy for acquisition of lateral images. Current design of nanoapertures provides EOT-AIM with axial super-resolution that is as small as 20 nm for a depth range of 500 nm. Experiments were performed for the measurement of the axial distribution of ganglioside in mouse macrophage (RAW264.7) cells using FITC-conjugated cholera toxin subunit B. The results were successfully confirmed with conventional confocal and total internal reflection fluorescence microscopy. ...
Definition of Evanescent wave in the Legal Dictionary - by Free online English dictionary and encyclopedia. What is Evanescent wave? Meaning of Evanescent wave as a legal term. What does Evanescent wave mean in law?
Recently implemented fluorescence imaging techniques, such as total internal reflection fluorescence microscopy and two-photon laser scanning micro-scopy, have made possible multiscale analysis of the immune response from single molecules in an interface to cells moving in lymphoid tissues and tumors. In this review, we consider components of T cell sensitivity: the immunological synapse, the coordination of migration, and antigen recognition in vivo. Potency, dose, and detection threshold for peptide-MHC determine T cell sensitivity. The immunological synapse incorporates T cell receptor microclusters that initiate and sustain signaling, and it also determines the positional stability of the T cells through symmetry and symmetry breaking. In vivo decisions by T cells on stopping or migration are based of antigen stop signals and environmental go signals that can sometimes prevent arrest of T cells altogether, and thus can change the outcome of antigen encounters. © 2010 Springer-Verlag Berlin
Recently implemented fluorescence imaging techniques, such as total internal reflection fluorescence microscopy and two-photon laser scanning micro-scopy, have made possible multiscale analysis of the immune response from single molecules in an interface to cells moving in lymphoid tissues and tumors. In this review, we consider components of T cell sensitivity: the immunological synapse, the coordination of migration, and antigen recognition in vivo. Potency, dose, and detection threshold for peptide-MHC determine T cell sensitivity. The immunological synapse incorporates T cell receptor microclusters that initiate and sustain signaling, and it also determines the positional stability of the T cells through symmetry and symmetry breaking. In vivo decisions by T cells on stopping or migration are based of antigen stop signals and environmental go signals that can sometimes prevent arrest of T cells altogether, and thus can change the outcome of antigen encounters. © 2010 Springer-Verlag Berlin
BILLERICA -- Bruker Corp. recently announced that it has acquired Vutara Inc., a Salt Lake City-based provider of super-resolution fluorescence microscopy for life-science applications.
Scattering-Assisted Localization Microscopy (SALM) is a super-resolution microscopy tool where high resolution is obtained based on the size of the grain in the speckle pattern on the sample.
Researchers measured times from cytokinesis to budding (G1) and from budding to cytokinesis in haploids, diploids or tetraploids (mothers and daughters), using time-lapse fluorescence microscopy of strains expressing Myo1 tagged with green fluorescent protein (Myo1-GFP). Standard methods were used throughout strain and plasmid constructions. All strains are W303-congenic. All integrated constructs were characterized by Southern blot analysis. Cells were prepared for time-lapse microscopy as described (Bean et al. 2006 PMID 16387649). Researchers observed growth of microcolonies with fluorescence time-lapse microscopy at 30?°C using a Leica DMIRE2 inverted microscope with a Ludl motorized XY stage. Images were acquired every 3?min for cells grown in glucose and every 6?min for cells grown in glycerol/ethanol with a Hamamatsu Orca-ER camera. They used custom Visual Basic software integrated with ImagePro Plus to automate image acquisition and microscope control ...
Impaired insulin secretion in type 2 diabetes (T2D) is linked to reduced insulin granule docking, disorganization of the exocytotic site, and impaired glucose-dependent facilitation of insulin exocytosis. We show in β cells from 80 human donors that the glucose-dependent amplification of exocytosis is disrupted in T2D. Spatial analyses of granule fusion events, visualized by total internal reflection fluorescence microscopy in 24 of these donors, demonstrated that these events are nonrandom across the surface of β cells from donors with no diabetes. The compartmentalization of events occurs within regions defined by concurrent or recent membrane-resident secretory granules. This organization, and the number of membrane-associated granules, is glucose dependent and notably impaired in T2D β cells. Mechanistically, multichannel Kv2.1 clusters contribute to maintaining the density of membrane-resident granules and the number of fusion hotspots, while SUMOylation sites at the channel N- (K145) ...
Impaired insulin secretion in type 2 diabetes (T2D) is linked to reduced insulin granule docking, disorganization of the exocytotic site, and impaired glucose-dependent facilitation of insulin exocytosis. We show in β cells from 80 human donors that the glucose-dependent amplification of exocytosis is disrupted in T2D. Spatial analyses of granule fusion events, visualized by total internal reflection fluorescence microscopy in 24 of these donors, demonstrated that these events are nonrandom across the surface of β cells from donors with no diabetes. The compartmentalization of events occurs within regions defined by concurrent or recent membrane-resident secretory granules. This organization, and the number of membrane-associated granules, is glucose dependent and notably impaired in T2D β cells. Mechanistically, multichannel Kv2.1 clusters contribute to maintaining the density of membrane-resident granules and the number of fusion hotspots, while SUMOylation sites at the channel N- (K145) ...
Fluorescence Microscope for sale, new Infinitive Plan Phase contrast Microscope Inverted Fluorescence Microscope CE A16.1023 of Opto-Edu (Beijing) Co., Ltd. from China.
We are a little late to the party (it has been a busy month at Protocols HQ!), but Nature Protocols would like to extend their warmest congratulations to all this years winners of the Nobel Prize for Chemistry: Eric Betzig, Stefan Hell and William E. Moerner for their contributions to the development of super-resolution microscopy.. We are very pleased to have published a Nature Protocol from the Betzig lab earlier this year on Bessel beam plane illumination microscopy. And we are equally pleased that the Protocol Exchange can claim a Nobel Laureate amongst its authors; the Moerner lab has published a guide to using Easy-DHPSF to measure the precise localisations of molecules in images acquired using a wide-field DH epifluorescence microscope. I would also encourage you to visit Moerners very informative lab website, if only to find out about the guacamole!. ...
If you are looking for a Distributor of Olympus IX83 Microscope in India then you need to look no further than DSS Image. The fully motorized and automated inverted microscope system IX83 is the most advanced in the IX3 series of inverted imaging systems
Scientific-grade fluorescence microscope MF43 MF43 is a research-grade fluorescence microscope latest developed by Mshot with excellent performance . The mi
By surpassing Abbes diffraction limit, super-resolution microscopy (SRM) has opened the door to the visualization and the understanding of complex structures in the 1-100 nm range. SRM combines typical features of optical microscopy (low invasiveness, high penetration depth, high sensitivity, chemical specificity, straightforward sample preparation) with nanometer spatial resolution. Despite its widespread in cell-biology and bio-physics, SRM is still in its infancy for in-depth visualization of mesoscale features in man-made materials. At the VoetsLab, we use SRM techniques such as PAINT (point accumulation for imaging in nanoscale topography), PALM (photo-activatable localization microscopy) and STORM (stochastic optical reconstruction microscopy) to visualize in-situ structural composition, stability, exchange dynamics and growth direction of self-assembled architectures ranging from one-dimensional fibers to multi-dimensional colloids. By doing so, we aim for a more rational and ...
The assembly process of the human immunodeficiency virus 1 (HIV-1) is driven by the viral polyprotein Gag. Fluorescence imaging of Gag protein fusions is widely performed and has revealed important information on viral assembly. Gag fusion proteins are commonly co-transfected with an unlabeled form of Gag to prevent labeling artifacts such as morphological defects and decreased infectivity. Although viral assembly is widely studied on individual cells, the efficiency of the co-transfection rescue has never been tested at the single cell level. Here, we first develop a methodology to quantify levels of unlabeled to labeled Gag in single cells using a fluorescent reporter protein for unlabeled Gag and fluorescence correlation spectroscopy. Using super-resolution imaging based on photoactivated localization microscopy (PALM) combined with molecular counting we then study the nanoscale morphology of Gag clusters as a function of unlabeled to labeled Gag ratios in single cells. We show that for a ...