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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.

  • 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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  • How Widefield Super-Resolution GSDIM Images are Created

    The localization microscopy technique GSDIM is a proven technology to achieve super-resolution images with a resolution of up to 20 nm. In the following tutorial we will describe the basic principles and features of GSDIM.
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  • Optical Contrast Methods

    Optical contrast methods give the potential to easily examine living and colorless specimens. Different microscopic techniques aim to change phase shifts caused by the interaction of light with the specimen into amplitude shifts that are visible to the human eye as differences in brightness.
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  • Phase Contrast

    Phase contrast is an optical contrast technique for making unstained phase objects (e.g. flat cells) visible under the light microscope. Cells that appear inconspicuous and transparent in brightfield can be viewed in high contrast and rich detail using phase contrast microscopy.
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  • Differential Interference Contrast (DIC)

    Differential interference contrast (DIC) microscopy is a good alternative to brightfield microscopy for gaining proper images of unstained specimens that often only provide a weak image in brightfield.
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  • Integrated Modulation Contrast (IMC)

    Hoffman modulation contrast has established itself as a standard for the observation of unstained, low-contrast biological specimens. The integration of the modulator in the beam path of themodern inverted microscopes allows a wide range of brightfield or phase objectives to be used, rather than a small selection of special objectives.
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Communities and Web Sources

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Social network for scientists

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Confocal Microscopy Mailing List, University of Minnesota

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Teaching tools, video lectures on biology and microscopy

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Teaching tools, video lectures on biology and microscopy

bitesizebio.com
Online magazine and community for molecular and cell biology researchers

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Resource for high-end scientific illustrations, images and animations

Search Engines and Data Bases

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

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

www.gopubmed.com
Search interface for pubmed

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

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Beta of Google's search engine for scientific article abstracts

Journals

www.doaj.org/
Directory of open access journals

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The EMBO Journal

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CBE-Life Sciences Education – an ASCB online journal

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Science

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Nature

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Biweekly publication of exceptional research articles

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Journal of Cell Science

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Development

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The Journal of Experimental Biology

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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.xml
SPIE - peer-reviewed journals on applied research in optics and photonics

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

www.plosone.org/home.action
International, 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

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European Microscopy Society

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Royal Microscopical Society

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ASCB American Society of Cell Biology

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