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 H. Wong, Nicholas R. Smith, Lynn Y. Sakai, William A. Horton

Research output: Chapter in Book/Report/Conference proceedingChapter

11 Scopus citations

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)
Title of host publicationMethods in Cell Biology
PublisherAcademic Press Inc.
Pages391-417
Number of pages27
DOIs
StatePublished - Jan 1 2014

Publication series

NameMethods in Cell Biology
Volume124
ISSN (Print)0091-679X

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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

Keene, D. R., Tufa, S. F., Wong, M. H., Smith, N. R., Sakai, L. Y., & Horton, W. A. (2014). 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. In Methods in Cell Biology (pp. 391-417). (Methods in Cell Biology; Vol. 124). Academic Press Inc.. https://doi.org/10.1016/B978-0-12-801075-4.00018-5