[Autophagy 4:2, 257-260; 16 February 2008]; 2008 Landes Bioscience

Views and Commentaries

To be or not to be?

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Examples of incorrect identification of autophagic compartments in conventional transmission electron

microscopy of mammalian cells
Eeva-Liisa Eskelinen

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Department of Biological and Environmental Sciences; University of Helsinki; Helsinki, Finland

Abbreviations: LC3, light chain 3; TEM, transmission electron microscopy

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Further, quantitative TEM can also be used to determine the lifetime

of autophagic compartments.9,10 In addition to the great sensitivity,
the advantage of this method is that it does not depend on the avail
ability of specific antibodies or probes. However, the drawbacks are
that the method requires an electron microscopy laboratory, takes
more time than most other methods, and that the correct interpreta
tion of electron microscopy pictures requires special expertise that
can only be gained by experience.
Review articles have been published by the author and others on
the detailed morphology of autophagosomes at different maturation
stages.8,1114 These aspects are, therefore, described only shortly.
The main purpose of this commentary is to point out a few most
common mistakes in the identification of autophagic compartments
in conventional TEM images of mammalian cells. Conventional
TEM includes aldehyde fixation, osmium tetroxide postfixation,
dehydration in alcohol and propylene oxide or acetone, and embed
ding in resin that is polymerized by heat.
By definition, autophagosomes, amphisomes and autolysosomes
are membranebound compartments that contain cytoplasmic mate
rial and/or organelles. In conventional TEM, autophagosomes have a
double or sometimes multiple limiting membrane, and the cytoplasm
inside has the same morphology and electron density as the cyto
plasm outside the autophagosome. In conventional TEM samples,
the number and contrast of autophagosome limiting membranes
may vary, probably due to limitations in the preservation of lipids
during sample preparation. Sometimes autophagosomes seem to
have only one electrondense membrane, sometimes the limiting
membrane may seem to consist of two, and sometimes of several,
separate membranes. Further, sometimes the limiting membrane
may not have contrast at all, probably due to lipid extraction during
sample preparation. The same applies to the ultrastructure of phago
phores (also called isolation membranes or segregating membranes),
membrane structures that seem to form new autophagosomes.12,13,15
The ultrastructure of the phagophore and autophagosome limiting
membranes may depend on the fixation and sample preparation
method. More sophisticated methods providing better preserva
tion of lipids should be used if the exact morphology of limiting
membranes is to be investigated.16,17 Thus, the presence of a double
limiting membrane should not be used as a sole criterion for the
identification of autophagosomes in conventional TEM.

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Transmission electron microscopy is one of the most sensitive

methods to detect autophagic compartments in mammalian cells.
The number of scientists that are experts in this technique has
declined during recent years, probably because the method was
developed over forty years ago and is not in fashion today. This has
lead to a situation where, too many times, authors and reviewers
alike are not able to interpret electron microscopy pictures correctly.
In this commentary I present some common mistakes in identification of autophagic compartments in conventional transmission
electron microscopy of mammalian cells.

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Key words: autophagy, transmission electron microscopy, aldehyde fixation, plastic embedding, mammalian cells

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Autophagy was originally described using transmission electron

microscopy.14 For decades, electron microscopy remained the only
morphological tool to detect the presence of autophagic compart
ments in cells. Today, a specific marker protein for autophagosomes
called LC3 is known.5 LC3 can be used in light and electron micros
copy as a marker for autophagic compartments. The validity of LC3
as a marker in light microscopy has, however, been challenged. LC3
has been shown to incorporate into protein aggregates independent
of autophagy.6 Further, microscopy quality antibodies against LC3
are not freely available at present. Expression of epitopetagged
LC3 can be used to overcome the lack of antiLC3, but especially
when tissue samples are used this is not always possible. Further,
stable expression of tagged LC3 is recommended because transiently
expressed LC3 tends to form aggregates.7 Biochemical methods have
also been developed to measure autophagic activity,7 but microscopy
methods are often needed to complement, strengthen, or replace,
biochemistry. Thus, conventional transmission electron microscopy
(TEM) is still needed. Especially when the results are quantified,
TEM still remains one of the most sensitive methods to detect the
accumulation of autophagic compartments in mammalian cells.8
*Correspondence to: Eeva-Liisa Eskelinen; Department of Biological and Environmental
Sciences; Division of Biochemistry; PO Box 56; University of Helsinki; Helsinki 00014
Finland; Tel.: +358919159566; Fax: +358919159068; Email eeva-liisa.eskelinen@
helsinki.fi
Submitted: 08/08/07; Revised: 10/16/07; Accepted: 10/17/07
Previously published online as an Autophagy E-publication:
www.landesbioscience.com/journals/autophagy/article/5179
www.landesbioscience.com

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Figure 2. Rough endoplasmic reticulum often surrounds organelles such as

mitochondria (indicated by ?). Sometimes these structures are mistakenly
interpreted as autophagosomes. The insert shows the presence of ribosomes
on the endoplasmic reticulum membrane. Autophagosome limiting membranes do not have ribosomes. Two autophagosomes are also visible, indicated by 1. The autophagosome on the left contains a ringshaped cistern of
rough endoplasmic reticulum. The limiting membrane of this autophagosome
is indicated by arrowheads. Note that the two limiting membranes of this
autophagosome are very close to each other, while the two limiting membranes of the autophagosome on the right are located further away from
each other. The picture was taken from an isolated mouse hepatocyte.

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Figure 1. Autophagic compartments in a mouse fibroblast (1, 2). Note the

presence of normallooking ribosomes inside the autophagosomelike compartment indicated by 1. The two limiting membranes are also visible. In the
compartment indicated by 2, the ribosomes are more electron dense and
form dark granular/amorphous masses. Ly, dense lysosomes.

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Identification of autophagic compartments in electron microscopy

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When autophagosomes grow older, their cytoplasmic contents

start to look partially degraded in TEM samples. In most cases this
means increased electron density. Partially degraded ribosomes form
dark granular/amorphous clumps inside these compartments. In
many cases, the electron density of the cytoplasmic contents increases
before the complete disappearance of the inner limiting membrane
(Figs. 1 and 3). Autophagosomes mature by fusing with endosomes,
forming amphisomes, and with lysosomes, forming autolysosomes.
When analyzing conventional TEM images one cannot absolutely
define certain structures as amphisomes or autolysosomes without
specific markers, which is not always practical or even possible.
That is, it is not clear whether an autophagosome has fused with
an endosome, a lysosome or both. Therefore, it has been helpful
to have additional terms where such ambiguity exists. This is the
reason for the origin of the terms initial autophagic vacuole (AVi)
and degradative autophagic vacuole (AVd).1820 The use of the word
vacuole may cause confusion with the organelle vacuole, which is
involved in autophagy in yeast and plants. Therefore I do not use
the terms AVi and AVd but instead speak of autophagosomes (that
contain morphologically intact cytoplasmic material) and autophagic
compartments (that contain partially degraded cytoplasmic material).
The cytoplasmic contents of autophagosomes start to disintegrate
and turn electron dense before the inner limiting membrane disap
pears. This is why autophagic compartments with electrondense
contents may still be bound by a double limiting membrane. Figure
1 shows typical examples of autophagic compartments in mouse
fibroblasts. In tissues rich in mitochondria such as liver and muscle,
autophagosomes frequently contain these organelles. This helps the
identification of the cytoplasmic content in autophagic compart
ments at different stages of maturation.
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Rough endoplasmic reticulum often surrounds organelles such

as mitochondria (Fig. 2). This close localization is thought to play a
role in calcium transport from the endoplasmic reticulum to mito
chondria.21 These endoplasmic reticulum wrapped organelles should
not be confused with autophagosomes, because autophagosome
limiting membranes do not have ribosomes.18,22 Another confu
sion may arise from the morphology of the endoplasmic reticulum
itself. Depending on the plane and orientation of the thin section,
rough endoplasmic reticulum cisternae can sometimes form cup or
ringshaped structures in the section (Fig. 3). Again, the presence
of ribosomes on the membranes distinguishes these structures from
autophagosomes.
Like autophagosomes, mitochondria also have two limiting
membranes. If mitochondria are swollen or contain precipitates,
they can be misinterpreted as autophagosomes (Fig. 4). The presence
of cristae (infoldings of the inner limiting membrane) distinguishes
these from autophagosomes, where the inner limiting membrane
does not form such infoldings. In addition, in mitochondria the
distance between the two limiting membranes is shorter and more
uniform than in autophagosomes.
Electron lucent or empty vacuoles (Fig. 5) are also sometimes
incorrectly called autophagic vacuoles. However, because these
vacuoles have no contents, it is not possible to say whether they are

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2008; Vol. 4 Issue 2

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Identification of autophagic compartments in electron microscopy

Figure 5. Electronlucent or empty vacuoles (indicated by ?) should not be

classified as autophagic compartments. The neighboring cell contains an
autophagosome (1). The plasma membranes of the two cells are indicated
by PM. The picture was taken from mouse fibroblasts.

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Figure 4. Swollen mitochondria (indicated by ?) are sometimes incorrectly

identified as autophagosomes, probably because they also have a double
limiting membrane. Mitochondria can be identified by the presence of cristae, which are shown at higher magnification in the insert (arrowheads).
Nu, nucleus. The picture was taken from a Chinese hamster ovary cell.

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Figure 3. Rough endoplasmic reticulum can form cup or ringshaped

structures in thin sections (indicated by ?). The presence of ribosomes
identifies the membranes as rough endoplasmic reticulum. Depending on the
orientation of the section, polysomes can occasionally be identified on the
membrane (arrowhead). An autophagosome (1) and two later autophagic
compartments (2) are also present. The picture was taken from a mouse
fibroblast.

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autophagic compartments or some other kind of vacuoles. The elec

tron lucent vacuole in Figure 5 is most likely either an endosome, or
a cross section of an invagination of the plasma membrane.
These examples show that there are several pitfalls for an inex
perienced electron microscopist when trying to identify autophagic
compartments. Therefore it is recommendable to contact a colleague
who has more experience in electron microscopy, if possible in
connection with autophagy. Review articles also exist that a beginner
may find helpful.8,1114 Even after identification of several autophagy
proteins, the possibilities to reliably detect autophagic activity
remain limited.7 Therefore, electron microscopy is still needed and
it is important that the knowledge and skills of this method do not
disappear because of the invasion of more fashionable techniques
into life sciences.
www.landesbioscience.com

Acknowledgements

I thank Antti Arstila, Pirkko Hirsimki and Hilkka Reunanen

for the first introduction to the secrets of electron microscopy,
ultrastructure of mammalian cells, and autophagy. Work in my labo
ratory is supported by the Academy of Finland, Helsinki University
Foundations, Biocentrum Helsinki, and the Ehrnrooth Foundation.
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