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Medical Molecular Morphology
https://doi.org/10.1007/s00795-018-0194-y
REVIEW
Methods for array tomography with correlative light and electron
microscopy
Taro Koike
1
· Hisao Yamada
1
Received: 23 April 2018 / Accepted: 27 May 2018
© The Japanese Society for Clinical Molecular Morphology 2018
Abstract
The three-dimensional ultra-structure is the comprehensive structure that cannot be observed from a two-dimensional electron
micrograph. Array tomography is one method for three-dimensional electron microscopy. In this method, to obtain consecu-
tive cross sections of tissue, connected consecutive sections of a resin block are mounted on a flat substrate, and these are
observed with scanning electron microscopy. Although array tomography requires some bothersome manual procedures to
prepare specimens, a recent study has introduced some techniques to ease specimen preparation. In addition, array tomog-
raphy has some advantages compared with other three-dimensional electron microscopy techniques. For example, sections
on the substrate are stored semi-eternally, so they can be observed at different magnifications. Furthermore, various stain-
ing methods, including post-embedding immunocytochemistry, can be adopted. In the present review, the preparation of
specimens for array tomography, including ribbon collection and the staining method, and the adaptability for correlative
light and electron microscopy are discussed.
Keywords Three-dimensional electron microscopy · Array tomography · Correlative light and electron microscopy · Pre-
embedding staining · Post-embedding staining
Introduction
The three-dimensional ultra-structure has been determined
with transmission electron microscopy (TEM) for a long
time. Serial section TEM (ssTEM) is a technique for three-
dimensional microscopy with TEM, where consecutive
ultra-thin sections are mounted on TEM grids, and these
sections are observed with TEM [1]. These procedures
include preparative difficulties as described below. How-
ever, recently, back scattered electron imaging with scanning
electron microscopy (SEM) has been employed to obtain
the three-dimensional ultra-structure. There are mainly two
methods to obtain serial cross sections of resin-embedded
tissue: “automated method” and “array tomography”. Auto-
mated methods require repeating 2 step procedures in the
SEM chamber; capturing of the block face followed by
cutting the block face with a diamond knife or focused ion
beam, and these methods are called serial block face SEM
(SBF/SEM) or focused ion beam SEM (FIB/SEM), respec-
tively [2, 3].
Array tomography is an imaging technique that was pro-
posed by Micheva and Smith [4]. In this technique, con-
nected consecutive sections, called ribbons, are mounted on
a solid substrate, and the sections are observed with fluo-
rescent microscopy and/or SEM. Unlike an automated tech-
nique (SBF/SEM and FIB/SEM), array tomography requires
some manual procedures to mount the ribbons on the sub-
strate, and to adjust axis of serial image. This manual pro-
cess needs more time to obtain data set of three-dimensional
EM, comparison with automated technique. However, this
technique is cost-effective, because specialized expensive
machines are not involved. In addition, several studies to
improve specimen preparation have been performed. There
is also free software to help axis adjustment of serial image
[5, 6]. Only array tomography enables post-embedding
immunocytochemistry (ICC), because the ribbons for array
tomography are mounted on a solid substrate, while, the sec-
tions do not remain after capturing in automated techniques.
In the present review, ribbon collection methods and staining
methods for array tomography are discussed.
* Taro Koike
koiket@hirakata.kmu.ac.jp
1
Department of Anatomy and Cell Science, Kansai Medical
University, Shinmachi 2-5-1, Hirakata, Osaka, Japan
@Phil_Robichaud
@deepthiw
@JoseServera