imaging Protocols

Category: Molecular Imaging: Light Microscopy

Ahmanson Center for Advanced Electron Microscopy and Imaging. Use of Semi-Thin Cryosections for Light Microscopy, Immunolabeling of Cryosections on Glass Slides, Problems with Autofluorescence. House Ear Institute - [Read Light Microscopy Techniques]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition and processing of confocal fluorescent and bright field images of live cells, expressing cyan fluorescent protein(CFP) and/or yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope. This procedure is used to help determine if N- or Cterminal tagging of signaling molecules alters the steady state localization pattern of the signaling protein in question. - [Read Live Cell Spinning Disk Confocal Fluore Imaging of Cells- Colocalization of Fluorescent Protein Tags]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition and processing of confocal fluorescent and bright field images of live cells expressing yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope when three planes along the z-axis of the cell are acquired. Protocol includes: Description of Microscope and Imaging Setup; Description of Acquisition Parameters; Image Processing. - [Read Live Cell Spinning Disk Confocal Fluorescence Imaging of Cells- YFP & Bright Field—Three Z Axis]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition and processing of confocal fluorescent and bright field images of live cells expressing yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope. Protocol includes: Description of Microscope and Imaging Setup; Description of Acquisition Parameters; Image Processing. - [Read Live Cell Spinning Disk Confocal Fluorescence Imaging of Cells- YFP and Bright Field Images]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition and processing of confocal fluorescent images of live cells expressing yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope. Protocol includes: Description of Microscope and Imaging Setup; Description of Acquisition Parameters; Movie Processing. - [Read Live Cell Spinning Disk Confocal Fluorescence Imaging of Cells- YFP Time Series for Markers]

Category: C. elegans Protocols

To image early cleavages and chromatin dynamics, it is convenient to use histone H2B fused to GFP or lamin::GFP. Time-lapse movies can be obtained using conventional confocal microscope systems and their included software. Early embryos dissected from transgenic hermaphrodites are placed with egg salts on agar pads. - [Read Live Imaging of Caenorhabditis elegans: Examples]

Category: Microscopy Protocols: In Vivo Imaging Protocols

Live nematodes can be studied at all levels of microscopic resolution. Data collection includes information on: Photographic Records, Video Microscopy,Multiple-Focal-Plane Time-Lapse Recording Systems and Digital Imaging. - [Read Live Imaging of Caenorhabditis elegans: Observation of Nematodes and Data Collection]

Category: C. elegans Protocols

At low magnification, dissection microscopes are useful, whereas at higher magnification and resolution, a wide range of optical methods can be used, including differential interference contrast (DIC) optics, phase contrast, fluorescence, polarized light, confocal, and dark field. - [Read Live Imaging of Caenorhabditis elegans: Observation of Nematodes and Data Collection Protocol]

Category: Microscopy Protocols: In Vivo Imaging Protocols

Caenorhabditis elegans, a small (adults are ~1 mm long), free-living soil nematode that feeds on bacteria, is an ideal organism for applying various live microscopy techniques. This protocol describes useful techniques for preparing C. elegans for live microscopic analysis. Details of sample preparation depend on the developmental stage of the worm to be studied. - [Read Live Imaging of Caenorhabditis elegans: Preparation of Samples]

Category: C. elegans Protocols

Protocol describes useful techniques for preparing C. elegans for live microscopic analysis. Details of sample preparation depend on the developmental stage of the worm to be studied. - [Read Live Imaging of Caenorhabditis elegans: Preparation of Samples]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

GFP serves as a molecular marker that can be imaged dynamically in living cells, both in its native form & as a fusion to other proteins. For GFP imaging, plants present the challenge of autofluorescence from chlorophyll, lignified cell walls, vacuolar contents, and other cell materials, all of which can obscure the GFP signal. Maximizing the signal-to-noise ratio is a major concern, and careful consideration should be given to the choice of tissue imaged, GFP expression level, etc. - [Read Live-Cell Imaging of GFP in Plants]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Confocal laser scanning microscopy (CLSM) is a relatively new light microscopical imaging technique which has found wide applications in the biological sciences. The primary value of the CLSM to the biologist is its ability to produce optical sections through a 3-D specimen-e.g., an entire cell or a piece of tissue - that, to a good approximation, contain information from only one focal plane. Article includes principle and applications of confocal laser scanning microscope. - [Read Looking Inside Cells and Tissues by Optical Sectioning with a Confocal Laser Scanning Microscope]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Live-cell imaging techniques provide a critical insight into the fundamental nature of cellular and tissue function, especially due to the rapid advances that are currently being witnessed in fluorescent protein and synthetic fluorophore technology. Because of these advances, live-cell imaging has become a requisite analytical tool in most cell biology laboratories. - [Read Maintaining Live Cells on the Microscope Stage]

Category: Microscopy Protocols: Optical Microscopy Protocols

Diffraction-limited optical microscopy requires that the spatial resolution of an image is limited by the wavelength of the incident light & by the numerical apertures of the condenser & objective lens systems.The development of near-field scanning optical microscopy (scanning near-field optical microscopy) has allowed for a imaging technique that retains the various contrast mechanisms afforded by optical microscopy methods while attaining spatial resolution beyond the optical diffraction limit - [Read Near-Field Scanning Optical Microscopy]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition of confocal fluorescent and bright field images of live cells, expressing cyan fluorescent protein (CFP) and/or yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope. Protocol includes: Description of Microscope and Imaging Setup; Description of Acquisition Parameters; Image Processing; Movie Processing. - [Read Protocol for Live Cell Spinning Disk Confocal Fluorescence Imaging of Cells on a Zeiss]

Category: Microscopy Protocols: In Vivo Imaging Protocols

To image early cleavages and chromatin dynamics, it is convenient to use histone H2B fused to GFP or lamin::GFP. Time-lapse movies can be obtained using conventional confocal microscope systems and their included software. Early embryos dissected from transgenic hermaphrodites are placed with egg salts on agar pads. Chromatin dynamics can be followed easily, and wild-type embryonic cells can be compared with mutants or RNAi-treated embryos. - [Read Protocol Live Imaging of Caenorhabditis Elegans]

Category: Microscopy Protocols

Reflected light microscopy is often referred to as incident light, epi-illumination, or metallurgical microscopy, and is the method of choice for fluorescence and for imaging specimens that remain opaque even when ground to a thickness of 30 microns. - [Read Reflected Light Microscopy]

Category: Microscopy Protocols: In Vivo Imaging Protocols

In the past two decades, there have been many revolutions in light microscopy techniques made possible by improvements in optics, detector technology, and computers. Furthermore, there is no indication that the rate of development of new equipment is slowing down. Here we attempt to provide an overview of available options and important considerations applicable to imaging Drosophila cells and tissues. - [Read Selection of Appropriate Imaging Equipment and Methodology for Live Cell Imaging in Drosophila]

Category: Pharmacology and Toxicology Protocols: Drug Discovery

Protocol for selection of appropriate imaging equipment and methodology for live cell imaging in Drosophila. - [Read Selection of Appropriate Imaging Equipment and Methodology for Live Cell Imaging in Drosophila]

Category: Primary Cell Isolation and Culture Protocols

This protocol describes a method for static culture of early postimplantation mouse embryos on a microscope stage. Embryos between 6.5 and 9.5 days post coitum (dpc) can be cultured and imaged for 24 hours, with very little growth retardation. - [Read Static Culture of Postimplantation Embryos for Imaging Protocol]

Category: Mouse Protocols: Mouse Cell Culture Protocols

Protocol describes a method for static culture of early postimplantation mouse embryos on a microscope stage. Embryos between 6.5 and 9.5 days post coitum (dpc) can be cultured and imaged for 24 hours, with very little growth retardation. - [Read Static Culture of Postimplantation Embryos for Imaging Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Cycle Analysis Protocols

Protocol describes methods for maintaining healthy, dividing mammalian cells in culture and during imaging, when mitosis can be examined. Rose chambers are preferable for observation and microinjection of living mitotic cells, but slide/coverslip preparations are easy to make and do not require any special equipment. - [Read Studying Mitosis in Cultured Mammalian Cells Protocol]

Category: Cytogenetics Protocols

Protocol describes methods for maintaining healthy, dividing mammalian cells in culture and during imaging, when mitosis can be examined. Rose chambers are preferable for observation and microinjection of living mitotic cells, but slide/coverslip preparations are easy to make and do not require any special equipment. Another inexpensive and easy-to-use alternative is to grow cells in a culture dish with a glass bottom. Such dishes are suitable for microinjection experiments. - [Read Studying Mitosis in Cultured Mammalian Cells Protocol]

Category: Cell Biology and Cell Culture: Cell Cycle Protocols: Mitosis Protocols

Protocol describes methods for maintaining healthy, dividing mammalian cells in culture and during imaging, when mitosis can be examined. Rose chambers are preferable for observation and microinjection of living mitotic cells, but slide/coverslip preparations are easy to make and do not require any special equipment. Another inexpensive and easy-to-use alternative is to grow cells in a culture dish with a glass bottom. Such dishes are suitable for microinjection experiments. - [Read Studying Mitosis in Cultured Mammalian Cells Prtocol]

Category: Microscopy Protocols: In Vivo Imaging Protocols

Sophisticated fluorescence microscopy methods & equipment, now allow cellular events to be studied at high resolution in living material. The studying of living fly tissues presents unique difficulties in keeping the cells alive, introducing fluorescent probes, & imaging through thick hazy cytoplasm. This protocol outlines the preparation of major tissue types amenable to study by time-lapse cinematography and different methods for keeping them alive. - [Read Time-Lapse Cinematography in Living Drosophila Tissues: Preparation of Material]