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Imaging In Depth: 3-Dimensional Microscopy Featured In Cold Spring Harbor Protocols

November 1, 2010

Imaging has rapidly become a defining tool of the current era in biological research. But finding the right method and optimizing it for data collection can be a daunting process, even for an established imaging laboratory. Cold Spring Harbor Protocols is one of the world’s leading sources for detailed technical instruction for implementation of imaging methods(http://cshprotocols.cshlp.org/cgi/collection/imaging_microscopy_general), and the November issue (http://cshprotocols.cshlp.org/TOCs/toc11_10.dtl) features articles detailing standard and cutting-edge laboratory techniques.

The confocal microscope is a workhorse of the modern life science laboratory. Its popularity stems from its ability to permit volume objects to be imaged and rendered in three dimensions. But the confocal microscope itself does not produce three-dimensional images; in fact, it only images very thin sections of a specimen that lie within its focal region. To produce a three-dimensional image, a series of thin optical sections are collected, and computer processing is used to combine them into a volumetric rendering. In “Spinning-Disk Microscopy Systems,” Oxford University’s Tony Wilson (http://acara.eng.ox.ac.uk/som/SOMG_home.html) reviews the many methods for producing optical sections, of which the confocal optical system is just one. He also describes a number of convenient methods of implementation that can lead to, among other things, real-time image formation. The article is freely available on the journal’s website (http://cshprotocols.cshlp.org/cgi/content/full/2010/11/pdb.top88).

While confocal microscopy relies on optical sectioning, array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of tissue are cut using an ultramicrotome, and attached in order to a glass coverslip. These coverslips are then stained as desired and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The thin sections allow for rapid staining and imaging and the array format allows much of the process to be automated. Stephen J. Smith and colleagues from Stanford University (http://smithlab.stanford.edu/Smithlab/Home.html) present “Array Tomography: High-Resolution Three-Dimensional Immunofluorescence,” a guide to this technique that allows for visualizing previously inaccessible features of tissue structure and molecular architecture. The article is freely available on the journal’s website (http://cshprotocols.cshlp.org/cgi/content/full/2010/11/pdb.top89).

Both articles are adapted from the forthcoming Imaging: A Laboratory Manual (http://www.cshlpress.com/link/imagingp.htm). Available later this month, the collection is the cornerstone of a new laboratory manual series, designed as an essential guide for investigators who need these visualization techniques.

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