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What is Widefield Microscopy?

Widefield microscopy refers to a basic sample illumination principle in microscopy. Since widefield microscopy permanently illuminates the whole sample, it can be distinguished from confocal microscopy where only one single focal spot is illuminated and recorded at a time. Typically, widefield microscopy utilizes light sources such as halogen, metal halide lamps or LED for sample illumination. Detection is performed through oculars or with the help of a digital camera. Several contrast methods increase widefield microscopy capabilities, starting from Phase Contrast through to Differential Interference Contrast (DIC) or fluorescence, to name but a few. Computer based deconvolution can be applied to increase widefield fluorescence image quality and enable 3D image reconstruction. Moreover, TIRF and GSDIM super-resolution microscopy can be assigned to widefield microscopy.

  • mTORC1 Promotes Proliferation of Immature Schwann Cells and Myelin Growth of Differentiated Schwann Cells

    The myelination of axons is essential for neuronal wiring and normal nervous system functions. In the peripheral nervous system, Schwann cells (SCs) form myelin sheaths around axons during nerve development. Such myelination is compromised in a number of diseases. Hence, identification and understanding of the key pathways regulating SC development and myelinogenesis are essential for therapeutic progress. Here we uncover two separate roles of the cellular signaling node mTORC1 (mechanistic target of rapamycin complex 1) for regulating the development of SCs and subsequently the growth of myelin sheaths. Moreover, we demonstrate that defective SCs possess a remarkable plasticity to remyelinate axons via mTORC1. Thus, manipulating mTORC1 activity in diseased SCs could be therapeutically beneficial.
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  • Effect of curcumin analogs onα-synuclein aggregation and cytotoxicity

    Alpha-synuclein (α-Syn) aggregation into oligomers and fibrils is associated with dopaminergic neuron loss occurring in Parkinson’s disease (PD) pathogenesis. Compounds that modulate α-Syn aggregation and interact with preformed fibrils/oligomers and convert them to less toxic species could have promising applications in the drug development efforts against PD. Curcumin is one of the Asian food ingredient which showed promising role as therapeutic agent against many neurological disorders including PD.
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  • Expression Analysis of Platelet‐derived Growth Factor Receptor Alpha and its Ligands in the Developing Mouse Lung

    Activation of the platelet‐derived growth factor receptor‐α (PDGFR α) signaling pathway is critically important during lung alveogenesis, the process in lung development during which alveoli are formed from the terminal alveolar sacs. Several studies have aimed to characterize the expression patterns of PDGFR α and its two ligands (PDGF‐A and ‐C) in the lung, but published analyses have been limited to embryonic and/or perinatal time points, and no attempts have been made to characterize both receptor and ligand expression simultaneously. In this study, we present a detailed map of the expression patterns of PDGFR α, PDGF‐A and PDGF‐C during the entire period of lung development, that is, from early embryogenesis until adulthood.
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  • 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.
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  • Adipose Stem Cells Display Higher Regenerative Capacities and More Adaptable Electro-Kinetic Properties Compared to Bone Marrow-Derived Mesenchymal Stromal Cells

    Adipose stem cells (ASCs) have recently emerged as a more viable source for clinical applications, compared to bone-marrow mesenchymal stromal cells (BM-MSCs) because of their abundance and easy access. In this study we evaluated the regenerative potency of ASCs compared to BM-MSCs. Furthermore, we compared the dielectric and electro-kinetic properties of both types of cells using a novel Dielectrophoresis (DEP) microfluidic platform based on a printed circuit board (PCB) technology. Our data show that ASCs were more effective than BM-MSCs in promoting neovascularization in an animal model of hind-limb ischemia.
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  • Real Time Observation of Neutrophil White Blood Cell Recruitment to Bacterial Infection In Vivo

    The zebrafish (Danio rerio) is an emerging vertebrate model organism to study infection. The transparent larva comprises a fully functional innate immune system and enables live imaging of fluorescent immune cells in transgenic animals. Zebrafish infection models have been developed for both the human bacterial pathogen Shigella flexneri and the natural fish bacterial pathogen Mycobacterium marinum. Importantly, whilst S. flexneri causes acute infection and is typically used as an inflammatory paradigm, M. marinum causes a chronic disease similar to tuberculosis in humans. Here, we use real time fluorescence microscopy to image transgenic zebrafish larvae with neutrophils (granulocyte white blood cells) expressing the green fluorescent protein eGFP.
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  • Introduction to Widefield Microscopy

    One of the most basic microscopy techniques is known as ‘Widefield Microscopy’. It is fundamentally any technique in which the entire specimen of interest is exposed to the light source with the resulting image being viewed either by the observer or a camera (which can also be attached to a computer monitor).
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  • 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.
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  • Definitions of Basic Technical Terms for Digital Microscope Cameras and Image Analysis

    Most microscopes today are operated with a camera. The characteristics of the camera often decide whether the acquired image will reveal what a researcher wants to see. But when diving into camera terminology, the technical terms can be overwhelming. We have compiled the most important terms with a concise explanation to provide orientation.
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  • 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.
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  • Infinity Optical Systems

    “Infinity Optics” refers to the concept of a beam path with parallel rays between the objective and the tube lens of a microscope. Flat optical components can be brought into this “Infinity Space” without influencing image formation, which is critical for the utilization of contrast methods such as DIC or fluorescence. Modern microscopy techniques require the addition of multiple optical instruments, such as light sources or laser devices, into the infinite light path. Different approaches to fulfill this need have emerged and are described here.
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  • Introduction to Digital Camera Technology

    A significant majority of modern optical microscopy techniques require the use of a digital camera. By working with digital devices researchers can observe specimens on a screen in real time or acquire and store images and quantifiable data. Here we introduce the basic principles behind digital camera technologies commonly encountered in scientific imaging.
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  • Color Infidelity: Why Using a Light Source Incorrectly is Cheating on your Data

    There are many influences on color in the imaging process including lighting, optics, sensor, and monitor, and ultimately print. The first, and generally most important, is lighting. There are plenty of options for light sources, Halogen, LED, and arc lamps are among the most popular for microscopes. Each light source has its own advantages and disadvantages and it is up to the user to learn which is best for the sample and application.
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  • 3D Localization Microscopy With Ground State Depletion (GSD)

    With the latest development of a GSD 3D super-resolution platform, it is now possible to achieve a lateral resolution of down to 20 nm and an axial resolution of 70 nm. The technology is based on an astigmatism approach using a manipulated PSF to localize the molecule in z. This following tutorial describes the basic principles of the 3D GSD technology.
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  • Step by Step Guide to Fluorescence Microscopy

    Fluorescence Microscopy is a special form of light microscopy. It uses fluorescence to highlight structures in fixed and living biological specimens instead of using absorption, phase or interference effects. The fluorescence is delivered either by inorganic dyes, proteins, synthetic beads or by autofluorescent structures within a sample. In this tutorial the principles of fluorescence microscopy will be explained.
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  • Abstracts of the 3rd European Super-Resolution User-Club Meeting

    The 3rd meeting of the Leica Super-Resolution User Club was held from June 17th to 19th, 2013 in collaboration with Alberto Diaspro and the Italian Institute of Technology (IIT) in Genoa. Confocal and widefield super-resolution users from ten European countries took three days’ out to deepen their knowledge on super-resolution techniques and applications and make use of an opportunity for full exchange of experiences.
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  • The Principles of Polarization Contrast

    Polarization contrast microscopy is a convenient way to make birefringent crystalline structures like starch grains or cellulose visible without staining. This tutorial will explain the optical elements in the light path and the operating mode of polarization contrast taking the example of an inverted and motorized high-end research light microscope which can be used for transmitted light contrasting methods and fluorescence microscopy.
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  • 50 Years of Image Analysis

    Modern image analysis systems perform highly sophisticated image processing functions on images from an automated microscope and digital camera. 50 years ago, the first image analysis system was analogue, based on a video camera and the area measurements could be read from a meter. Nevertheless, it marked the beginning of automation in this field.
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  • Abstracts of the 2nd European Super-Resolution User-Club Meeting

    The 2nd meeting of the Leica Super-resolution User club was held from September 25 to 27, 2012 in collaboration with the Science for Life Laboratory at the Karolinska Institute, Stockholm, Sweden. With a mixture of engaging talks by key experts in the field of super-resolution microscopy and stimulating discussion sessions, the meeting proved as popular as last year’s event, attracting a wide range of scientists interested in both confocal and widefield super-resolution and sample preparation techniques.
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  • Digital Camera Technologies for Scientific Bio-Imaging

    This four-part series of articles published in Microscopy and Analysis covers the factors to consider in choosing a camera among CCD, EMCCD, and scientific-grade CMOS camera technologies for biological imaging applications. The differences among the sensor architectures and the impact of parameters such as pixel size, noise, and QE on signal-to-noise performance, image quality, and Nyquist sampling are considered.
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  • Widefield Super-Resolution with GSDIM

    Great advancements in biology have been possible by using fluorescence microscopy. So far, the resolution of the images was limited due to physical constraints. In the past couple of years, new methods evolved circumventing these limitations and bringing fluorescence microscopy to a new level of resolution, boosting the possibilities in science with fluorescence microscopes.
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  • Deconvolution

    Fluorescence microscopy is a modern and steadily evolving tool to bring light to current cell biological questions. With the help of fluorescent proteins or dyes it is possible to make discrete cellular components visible in a highly specific manner. A prerequisite for these kinds of investigations is a powerful fluorescence microscope. One special aim is the three-dimensional illustration of a structure to get an impression of full plasticity. This poses a certain problem for the experimenter using a classical light microscope.
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  • The Principles of Modulation Contrast

    Modulation contrast creates 3D-like images of unstained specimens, rendering it the contrasting method of choice for applications like e.g. in vitro fertilization, where DIC is not possible (due to the usage of plastics) or phase contrast does not deliver satisfying results. This tutorial explains the optical elements in the light path and the operating mode of the modulation contrast.
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  • Fluorescent Dyes

    A basic principle in fluorescence microscopy is the highly specific visualization of cellular components with the help of a fluorescing agent. This can be a fluorescing protein – for example GFP – genetically linked to the protein of interest. If cloning is impossible – for instance in histologic samples – it is required to use other techniques like immunofluorescence staining to visualize the protein of interest.
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  • The Principles of Phase Contrast

    In this tutorial the principle of phase contrast imaging is described taking the example of an inverted research microscope. Additionally, the alignment of the components needed for phase contrast is shown in the interactive part of the tutorial.
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  • Fluorescent Proteins – Introduction and Photo Spectral Characteristics

    The prospects of fluorescence microscopy changed dramatically with the discovery of fluorescent proteins in the 1950s. The starting point was the detection of the jellyfish Aequorea victoria green fluorescent protein (GFP) by Osamo Shimomura. Hundreds of GFP mutants later, the range of fluorescent proteins reaches from the blue to the red spectrum.
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  • Total Internal Reflection Fluorescence (TIRF) Microscopy

    Total internal reflection fluorescence (TIRF) is a special technique in fluorescence microscopy developed by Daniel Axelrod at the University of Michigan, Ann Arbor in the early 1980s. TIRF microscopy delivers images with an outstandingly high axial resolution below 100 nm. This allows the observation of membrane-associated processes.
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  • Applications of TIRF Microscopy in Life Science Research

    The special feature of TIRF microscopy is the employment of an evanescent field for fluorophore excitation. Unlike standard widefield fluorescence illumination procedures with arc lamps, LEDs or lasers, the evanescent field only penetrates the specimen by about 100 nm starting from the coverslip/medium interface.
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  • Differential Interference Contrast

    The examination of live unstained biological specimens often suffers from poor contrast and therefore bad visibility of the specimen. Thick specimens in particular, such as brain slices, show up as nothing more than light grey structures instead of single cells. This tutorial explains the optical elements in the light path and the operating mode of DIC (differential interference contrast) on the example of an inverted and motorized high-end research light microscope which can be used for transmitted light contrasting methods and fluorescence microscopy.
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Useful Links

Communities and Web Sources

www.researchgate.net/Social network for scientists

www.lsoft.com/scripts/wl.exe?SL1=CONFOCALMICROSCOPY&H=LISTS.UMN.EDUConfocal Microscopy Mailing List, University of Minnesota

www.ibiology.org/Teaching tools, video lectures on biology and microscopy

www.ibiology.org/Teaching tools, video lectures on biology and microscopy

bitesizebio.comOnline magazine and community for molecular and cell biology researchers

www.somersault1824.comResource for high-end scientific illustrations, images and animations

Search Engines and Data Bases

www.cellimagelibrary.orgPublic resource database of images, videos, and animations of cells

harvester.fzk.de/harvesterBioinformatic meta search engine for genes and proteins

www.gopubmed.comSearch interface for pubmed

en.wikipedia.org/wiki/List_of_academic_databases_and_search_enginesList of academic databases and search engines

scholar.google.comBeta of Google's search engine for scientific article abstracts

Journals

www.doaj.org/Directory of open access journals

emboj.embopress.org/The EMBO Journal

www.lifescied.orgCBE-Life Sciences Education – an ASCB online journal

www.sciencemag.org/Science

www.nature.com/Nature

www.cell.com/Biweekly publication of exceptional research articles

jcs.biologists.org/Journal of Cell Science

dev.biologists.org/Development

jeb.biologists.org/The Journal of Experimental Biology

dmm.biologists.org/DMM Disease Models & Mechanisms

www.biotechniques.com/International Journal of Life Science Methods

www.opticsinfobase.org/Collection of Journals and Proceedings in Optics and Photonics

spie.org/x576.xmlSPIE - peer-reviewed journals on applied research in optics and photonics

onlinelibrary.wiley.com/journal/10.1002/(ISSN)1864-0648Journal of Biophotonics

www.plosone.org/home.actionInternational, peer-reviewed, open-access, online publication

rspb.royalsocietypublishing.org/Proceedings B - the Royal Society's biological research journal

www.microscopy-analysis.com/International Journal for microscopists

Organizations

www.microscopy.org/Microscopy Society of America

www.eurmicsoc.org/European Microscopy Society

www.rms.org.uk/Royal Microscopical Society

www.ascb.org/ASCB American Society of Cell Biology

www.biologists.com/cob_activities.htmlthe company of biologists

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