Using X-Ray Microscopy to Increase Targeting Accuracy in Serial Block-Face Scanning Electron Microscopy

  • Eric A. BushongEmail author
  • Sébastien Phan
  • Mark H. Ellisman
Part of the Neuromethods book series (NM, volume 155)


In this chapter, we describe the use of X-ray microscopy (XRM) as a method for improving the accuracy and efficiency of volume electron microscopy (volume EM). By providing a means of nondestructively imaging EM specimens prior to performing volume EM, XRM allows the investigator to pinpoint specific regions of interest (ROIs) for imaging. In addition, given the excellent contrast and resolution that can be achieved with XRM when specimens are stained with protocols compatible with volume EM, it can also dramatically enhance the value of volume EM data, either by revealing how the EM data fits into a larger context and/or by improving the ability to perform correlated light microscopy (LM) and EM imaging. This chapter will focus on the combined use of XRM with diamond knife-based serial block-face scanning electron microscopy (SBEM). We also briefly describe software we have developed to ease tracking of ROIs across imaging modalities and allow direct targeting of ROIs in an SEM as guided by XRM volumes.

Key words

X-Ray microtomography MicroCT Serial block-face scanning electron microscopy Confocal microscopy Correlated microscopy CLEM 



The National Center for Microscopy and Image Research is supported by a grant to Mark Ellisman from the National Institutes of General Medical Sciences (P41 GM103412). We would like to acknowledge Chih-Ying Su (R01 DC015519), Angela Tsang, Katerina Akassoglou (R35 NS097976), and Victoria Rafalsky for providing some of the specimens used as examples in figures.

Supplementary material

Supplemental Movie S1

This movie shows the coregistration of LM, XRM, and SBEM volumes in taken from an area of GFP-expressing mouse cortex. The movie begins with a low resolution image of the vibratome slices taken with a stereoscope and then proceeds to display confocal and microCT volumes of the ROI taken at increasing resolution. Once the target cell is identified, a volume showing GFP-signal from this cell warped to be coregistered with the targeted SBEM volume collected of the cell (MP4 129,432 kb)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Eric A. Bushong
    • 1
    Email author
  • Sébastien Phan
    • 1
  • Mark H. Ellisman
    • 1
  1. 1.National Center for Microscopy and Imaging Research, Center for Research in Biological SystemsUniversity of California – San DiegoLa JollaUSA

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