Correlation of the same fields imaged in the TEM, confocal, LM, and microCT by image registration

From specimen preparation to displaying a final composite image

Douglas R. Keene, Sara F. Tufa, Melissa Wong, Nicholas R. Smith, Lynn Sakai, William A. Horton

    Research output: Contribution to journalArticle

    11 Citations (Scopus)

    Abstract

    Correlated imaging is the process of imaging a specimen with two complementary modalities and then registering and overlaying the fields obtained in each modality to create a composite view. One of the images is made somewhat transparent, allowing detail in the underlying image to be visible and assisting in the registration of the two images. As an example, an image localizing a specific tissue component by fluorescence may be overlaid atop a TEM image of the same field. The resulting composite image would demonstrate specific ultrastructural features in the high-resolution TEM field, which are colorized in the overlay. Other examples include composites from MicroCT or soft X-ray images overlaid atop light microscopy or TEM images. Automated image registration may be facilitated by a variety of sophisticated computer programs utilized by high-throughput laboratories. This chapter is meant for the more occasional user wishing to align images manually. ImageJ is a public domain, image processing program developed at the National Institutes of Health and is available to anyone as a free download. ImageJ performs marvelously well for the purpose of image registration; therefore, step-by-step instructions are included here. Specimen handling, including fixation and choice of embedding media, is not straightforward for correlative imaging. A step-by-step description of the protocols which work in our laboratory is included for simultaneous localization in LM, EM and micro-CT, as well as maintaining GFP emission in tissue embedded for TEM.

    Original languageEnglish (US)
    Pages (from-to)391-416
    Number of pages26
    JournalMethods in Cell Biology
    Volume124
    DOIs
    StatePublished - 2014

    Fingerprint

    X-Ray Microtomography
    Specimen Handling
    Public Sector
    National Institutes of Health (U.S.)
    Microscopy
    Software
    Fluorescence
    X-Rays
    Light

    Keywords

    • CLEM
    • Correlated imaging
    • Correlative microscopy
    • Electron microscopy
    • Fiji
    • Fluorescence microscopy
    • GFP
    • Image overlay
    • Image registration
    • ImageJ
    • Laser scanning confocal microscopy
    • Localization
    • Method
    • Protocol
    • Technique
    • TurboReg

    ASJC Scopus subject areas

    • Cell Biology

    Cite this

    Correlation of the same fields imaged in the TEM, confocal, LM, and microCT by image registration : From specimen preparation to displaying a final composite image. / Keene, Douglas R.; Tufa, Sara F.; Wong, Melissa; Smith, Nicholas R.; Sakai, Lynn; Horton, William A.

    In: Methods in Cell Biology, Vol. 124, 2014, p. 391-416.

    Research output: Contribution to journalArticle

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