Received: 15 July 2019 Revised: 12 August 2019 Accepted: 13 August 2019 Uncorrected manuscript published: 16 August 2019 Published

Published on: 3 October 2019

DOI: 10.1111/tra.12684

COMMENTARY

Twenty years of traffic: A 2020 vision of cellular electron

microscopy

Robert G. Parton

Institute for Molecular Bioscience and Centre for

Microscopy and Microanalysis, The University of Abstract
Queensland, Brisbane, Queensland, Australia The last 20 years have seen incredible advances in electron microscopy (EM). Cryo-
Correspondence electron microscopy can now resolve protein structures to a previously unimaginable
Robert G. Parton, Institute for Molecular resolution but the advances in cellular EM are just as significant. I will take this
Bioscience, The University of Queensland, St
Lucia, Brisbane, Queensland 4072, Australia. opportunity to briefly summarize some of the new developments in cellular EM.
Email: r.parton@imb.uq.edu.au
KEYWORDS
Funding information
Apex, CLEM, cryoEM, electron microscopy, electron tomography, lipid, localization, nanobody,
Australian Research Council, Grant/Award
Number: Centre of Excellence in Convergent stereology
BioNano Science; National Health and Medical
Research Council, Grant/Award Numbers:
APP1140064, APP1150083, APP1156489

Peer Review
The peer review history for this article is
available at https://publons.com/publon/
10.1111/tra.12684/

1 | I N T RO D UC T I O N approaches. In the past 20 years, these methods have been comple-

The range of electron microscopy (EM) methods available to the cell
biologist today is truly revolutionary. 20 years ago, digital images were
still a relatively new development. Now we have gigabyte 3D datasets
from cells and tissues, we can routinely correlate light and EM, and
we have genetic tags for EM to rival the use of green fluorescent pro-
mented by scanning EM methods allowing automated 3D sectioning
within the electron microscope and generation of entire 3D datasets
from cells, tissues and even whole organisms (Figure 1).1 Serial
blockface scanning electron microscopy (SEM) and focussed ion beam
(FIB) SEM both rely on removal of a defined slice of tissue and imaging
of the exposed blockface and are providing unprecedented new views

tein (GFP) in light microscopy. Cryoelectron microscopy is making cel- of complex architecture, including huge volumes of brain tissue to

lular structural biology possible, and we can even explore EM data in
virtual reality. Here I discuss some of the exciting new EM advances
that are of relevance to trafficking studies.
2 | TH E M OV E TO 3 D
The use of transmission EM to study cells and tissues has generally
involved thin plastic sections. 3D views were usually reliant on manual
serial sectioning or, more recently, electron tomography. Com-
plementing this approach, proteins could be identified using antibody-
based methods on sections. The use of thawed frozen sections and
Lowicryl resins allowed the preservation of cellular architecture and
the localization of antigens of interest in on-section labeling
reveal neuronal connections,2 a tumor cell within the brain as it
migrates across the blood brain barrier,3 or entire early embryos.4
As with any new approach there are challenges but step-by-step
these are being overcome. Charging of the sample can contribute to
suboptimal imaging but is being minimized by specialized sample
preparation methods, technological advances such as focal charge
compensation5 and conductive resins. The size of the blockface that
can be imaged is being increased through new technological
advances and automated stitching of the images. And deconvolution,
taking advantage of the different penetration depths of electrons at
different accelerating voltages, is adding extra resolution.6 The qual-
ity of modern SEM detectors is such that SEM may even replace
transmission electron microscopy for cellular EM. Combining SEM
with serial sections and light microscopy has been elegantly used in

© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

156 wileyonlinelibrary.com/journal/tra Traffic. 2020;21:156–161.

PARTON
F I G U R E 1 3D electron microscopy. Serial blockface electron
microscopy of zebrafish embryonic muscle. Segmentation of
T-tubules (red) and caveolae (orange) in part of a muscle fiber.
N. Ariotti and R. Parton
array tomography, a complementary technique in which serial sec-
tions can be analyzed by EM and, in parallel, labeled with specific
fluorescent markers for light microscopic analysis.7
2.1 | Genetic tags: Efficient 3D EM localization
Protein localization has traditionally involved labeling of sections with
antibodies. Immunolabeling of thawed frozen sections, the most
widely-used “on section” approach, continues to be an important
method for localization, particularly with the ability to preserve the
fluorescence of tags such as GFP for correlative approaches.8,9 How-
ever, with the increasing availability of 3D EM approaches, the need
to localize proteins within the depths of the tissue, rather than just
the surface of a section, also becomes more pressing. A number of
genetic tags have been introduced to the scientific community includ-
ing ReAsh and, more recently, APEX (Figure 2). Of these, APEX, a
modified soybean ascorbate peroxidase, 10,11
is perhaps the simplest
to use as detection relies on diaminobenzidine (DAB) oxidation, a
method long used to detect horseradish peroxidase (HRP) (Figure 2).
APEX uses haem as a cofactor in its active site but this does not
appear to limit its applicability in a range of cellular and organismal
systems. APEX can be directly fused to the protein of interest or GFP-
or mCherry-tagged proteins can be co-expressed with nanobodies
against the fluorescent proteins tagged with APEX.12-14
A few features of APEX-based methods are worth emphasizing.
First, the resolution is surprisingly high. Despite the insoluble DAB pre-
cipitate being produced through an enzymatic reaction, it remains in
close proximity to the tagged protein.13 This resolution is shared with
other DAB-based methods as illustrated in recent elegant studies to
15
determine 3D chromatin structure. Secondly, the method is so simple,
157
F I G U R E 2 Use of APEX in cellular electron microscopy. Expression
of a GFP-tagged lipid binding probe together with an anti-GFP-
nanobody linked to APEX2. The electron dense DAB-reaction product is
associated with the cytoplasmic face of early endosomes. Insets show
the quantitation of APEX-DAB reaction in the two boxed regions; region
A (red box; endosomal membrane) shows specific DAB reaction product
as illustrated in the linescan (higher signal on the y axis indicates higher
electron density as the image was inverted before surface scan analysis).
Region B (plasma membrane) shows no significant enrichment of APEX
using the same analysis conditions. Bar, 2 μm
involving only transfection, fixation, DAB visualization and conventional
processing, that it can be used alongside IF to localize multiple proteins
as a first step in protein characterization, or in high throughput screens
or to compare multiple conditions in one experiment. This can change
the way that EM experiments are performed. Third, the methods are
becoming applicable to serial blockface SEM, FIB SEM and to cryo-
approaches.16 Finally, it is worth noting that as for fluorescence micros-
copy, the APEX reaction product can be quantified, for example by lin-
escan analysis of electron density over areas of interest (Figure 2).
Modifications of the basic APEX method are bringing additional
applications. A split-APEX allows the detection of interacting proteins
when the two segments of the active enzyme are brought together by
the interaction of their fusion proteins.17 GFP or mCherry-tagged pro-
teins can be detected using nanobodies fused to APEX that are co-
expressed in the cells allowing use of existing tagged constructs and
lines and high throughput localization.12,13 Conditionally-stabilized ver-
sions of these nanobody-APEX constructs are proteasomally degraded
unless they bind their target proteins allowing detection of low abun-
dance antigens and their use to detect protein complexes using a split
GFP approach.14 All these approaches remove the dependence on high
158 PARTON

quality antibodies but they do require heterologous expression of the
tagged proteins, either transiently or as stable lines, and independent
validation of their functionality, as for GFP-tagged proteins. However,
genome-editing methods are now available to replace genes with the
tagged genes of interest allowing expression at endogenous levels.
applications to cells and tissues are yielding hugely exciting results. Thin
regions of cells cultured on grids and then fast frozen can be analyzed
in situ while FIB milling of cryofixed material to produce cryolamellae
for cryoelectron tomography is now allowing high resolution analysis of
cells and tissue samples in their native state.29-32 While in its infancy,
structural EM in cells is already yielding exciting data,33 allowing corre-

2.2 | CLEM: The power of light and EM combined lation with higher resolution data from isolated proteins or protein
complexes obtained by X-ray crystallography or by cryoEM.34
Light microscopy provides a dynamic view of cellular processes that EM
cannot provide. Yet the resolution of EM and the sheer quantity of
grayscale information within EM images makes the two methods com- 2.4 | Lipid localization and other challenges
plementary. The last 20 years have seen a surge in correlative light and We should not forget other cellular components such as the hundreds
electron microscopy (CLEM) as researchers have taken advantage of of different lipid species within cells, chromatin structure, post-
the incredible advances in light microscopy and combined them with
translational modifications, small molecules, and all the other events
EM. These approaches are particularly powerful when combined with
allowing a cell to function.
live cell microscopy in which the properties of a dynamic structure can
Lipids are a particular challenge as they are small (many lipid
be analyzed by light microscopy and then the same structure visualized
headgroups could lie under a single colloidal gold particle used for
by EM.8 An extensive summary of the numerous methods now avail-
immunolabeling), can vary in both headgroups and acyl chains, and are
able is beyond the scope of this short perspective and the reader is
not well-fixed by conventional fixatives. Yet a molecular understanding
instead referred to a number of excellent research papers, books and
of lipid distribution would have huge benefits for understanding cellular
technical articles.8,18-22 However, by highlighting a few key studies I will
function and disease. A number of lipid-binding probes, including probes
attempt to give an idea of the range of methods now available.
for specific phospholipids, cholesterol, phosphatidylinositol species,
A number of studies have taken advantage of the retention of GFP
23-25 sphingolipids, or even probes that detect complexes of lipids, such as
fluorescence after embedding in resin and processing for EM. This
sphingomyelin/cholesterol domains,13,35-45 are now available. Many of
can involve high pressure freezing and then rapid freeze substitution and
these can be used “on section” (eg, using the fast freezing/freeze substi-
embedding in plastic resins at low temperature.26 An elegant example of
this method took advantage of yeast as a system and combined the use tution methods also used to retain GFP fluorescence24,35,40,46; Figure 3).

of defined fluorescent marker proteins that associate with different stages

of the lifecycle of a clathrin coated carrier, with 3D tomographic charac-
terization of the ultrastructure of the identified structure, to provide
unprecedented temporal and correlated ultrastructural characterization of
the endocytic process.23 This method has been further developed with
the finding that fluorophores blink when excited within the low vacuum
of the SEM providing a super-resolution CLEM approach.25 In a further
extension of CLEM methods, a novel approach (termed MultiCLEM) has
recently been used in a fluorescent barcoding approach to analyze cellular
ultrastructure in multiple cell populations from a single EM preparation.27
At a different scale, it has now been possible to track single cells
undergoing rare events in whole animals. Cancer cell extravasation is
a critical step in metastatic spread to peripheral tissues. Karreman
et al combined intravital imaging of GFP-tagged cancer cells in living
mice with microCT and 3D EM to give a detailed view of the ultra-
structure of the transmigrating cells.3 These kinds of methods now
allow cell biology to move into whole organisms.28

2.3 | Cellular structural biology

The ideal EM approach would allow us to identify endogenous proteins
and protein complexes in the 3D environment of the cell, tissue or
whole organism under completely native conditions and in relation to
dynamic cellular events. The structures of those proteins would be
explored at atomic resolution in situ. While a single technique cannot
yet achieve this, developments in cryoelectron microscopy and their
F I G U R E 3 High pressure frozen HeLa cells embedded at low
temperature in Lowicryl HM20 resin after freeze substitution.
“On-section” labeling with a probe for the membrane lipid,
phosphatidyethanolamine, followed by antibodies and protein A-gold.
Specific labeling is associated with a number of membranous
compartments including mitochondria (M), the plasma membrane
(PM), late endosomes (L) and the Golgi complex (G). Bar, 1 μm
PARTON
This avoids any cellular perturbation. Alternatively, probes can be
expressed in cells and localized by conventional means using immunogold
labeling on sections or by using APEX either fused directly to probes or in
13
an indirect approach with GFP-labeled probes (see eg, Figure 2).
Localization of expressed lipid probes on plasma membrane sheets gener-
ated from transfected cells has been remarkably effective in providing
quantitative insights into the nanoscale organization of the plasma
membrane, especially when combined with semi-automated quantitation
44,47
techniques. A series of studies have revealed the extent of lipid
colocalization with signaling proteins such as Ras and demonstrated previ-
ously unappreciated complexity in the lipid association of lipid-binding pro-
44
tein domains, both in terms of the lipid headgroups and the acyl chains.
A particularly elegant, but specialized, method for lipid localization involves
depositing a layer of metal onto the exposed surface of a freeze-fractured
cell. This is then used to immobilize membrane lipids while the proteins are
digested using SDS and removed.48 This method can resolve nanoscale
organization within the plane of a single membrane in fast frozen material
37,49
and can distinguish which leaflet is being examined.
Nanobodies combined with APEX can provide additional insights
into complex biological processes. For example, nanobodies specific
50
for particular conformational states of proteins can allow EM visuali-
zation of the spatial organization of signaling pathways and other cel-
lular processes.51 As the library of available nanobodies increases, we
can expect an exciting new wave of studies in this direction.
2.5 | Advances in imaging and visualization
One of the greatest changes to the routine EM of cells in the last
20 years is in the speed and simplicity of imaging. The ease of image
F I G U R E 4 Example of simple quantitative analysis of TEM data as applied to the analysis of liver ultrastructure from WT and KO mice or
analysis of cell populations. A, Sampling (eg, liver 3WT, 3 KO; cell culture 3 control and 3 experimental); EM processing and random sectioning;
B, systematic unbiased image capture (eg, 30 images per tissue sample or cell population); C, image data deposition and blinded stereological
analysis or other quantitative approach (eg, gold density on organelle). Sv, surface density; Vv, volume density
159
capture means that EM experiments can be imaged efficiently in a
systematic unbiased manner to be used for quantitation and uploaded
to accessible storage sites as a resource. In theory, entire EM datasets
could be accessed by groups to address different research questions;
for example, EM images of a liver from a KO animal might be initially
analyzed for endosomal morphology but at a later date assessed by
the same or a different research group for mitochondrial changes
without further experimentation being required (in the same way that
online proteomic datasets can be re-analyzed). Rapid image capture
has also allowed whole datasets comprising systematically captured
images to be provided to collaborators to analyze in a blinded fashion
in their own laboratories. Stereological analysis of the images is a sim-
ple next step in gaining quantitative data from the images, particularly
volumes and surface areas of organelles (an example ideal pipeline is
shown in Figure 4). A recent elegant development is the application of
these stereological methods to quantitative analysis of 3D datasets.52
The analysis of large 3D datasets has necessitated the development
of additional analysis methods including automated segmentation
methods and machine learning allowing automated reconstruction of
structures from huge cell and tissue volumes.53,54
With the emergence of 3D EM methods and the ability to com-
bine structures of molecules and molecular machines with cellular
models, cell biology can be brought to a wider nonspecialist audience.
Virtual reality (VR) representation of real 3D EM data is now possible
allowing a user to move around the cell as a nanoscale explorer.55
Currently, these approaches are extremely popular for demonstrations
and as educational tools. Whether VR will also provide benefits over
existing methods in terms of representing and exploring scientific
data remains to be seen. However, an exciting possibility is that
160
quantitatively accurate cell models could be populated with actual
molecular data of protein density at specific cellular subcompartments,
combined with real-time observations from light microscopy, and then
combined into a manipulable simulation to test key hypotheses.
ACKNOWLEDGMENTS
I am grateful to Nicole Schieber, Charles Ferguson and James Rae for
outstanding technical support, to Nick Ariotti for assistance with
Figure 1, and to Rick Webb and John Drennan for numerous enlight-
ening discussions. This work was supported by the National Health
and Medical Research Council of Australia (grants APP1140064 and
APP1150083 and fellowship APP1156489) and the Australian
Research Council (ARC) Centre of Excellence in Convergent Bio-Nano
Science and Technology. I acknowledge the use of the Microscopy
Australia Research Facility at the Centre for Microscopy and Micro-
analysis at The University of Queensland.
ORCID
Robert G. Parton https://orcid.org/0000-0002-7494-5248
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How to cite this article: Parton RG. Twenty years of traffic: A
2020 vision of cellular electron microscopy. Traffic. 2020;21:
156–161. https://doi.org/10.1111/tra.12684