To be or not to be? Examples of incorrect identification of autophagic compartments in conventional transmission electron microscopy of mammalian cells
Open accessAbstract
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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The maturation from the phagophore through the autolysosome is a dynamic and continuous process.
“The maturation from the phagophore through the autolysosome is a dynamic and continuous process, 44 and, thus, the classification of compartments into discrete morphological subsets can be problematic”
From Guidelines for the use and interpretation of assays for monitoring autophagy (2012) · cites this paper as [44]
Double-membraned autophagosomes were identified according to well-established criteria.
“double- membraned autophagosomes were identified according to well established criteria”
From Nilotinib restores memory function by preventing dopaminergic neuron degeneration in a mouse model of Alzheimer’s Disease (2021) · cites this paper as Eskelinen, 2008
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“However, the electron microscopy technique might be limited by many factors, such as slice variance-induced variations in membrane structure, interference with other bilayer membrane organelles or the misleading appearance of low-electron-density vacuoles [48].”
BackgroundKrasemann, S., Dittmayer, C., von Stillfried, S. F., Meinhardt, J., Heinrich, F., Hartmann, K., Pfefferle, S., Thies, E., von Manitius, R., Aschman, T., et al. (2022) · EBioMedicine · doi:10.1016/j.ebiom.2022.104193 · Cited by 57 · see citing works
Role of autophagy in environmental neurotoxicity
Pellacani, C., & Costa, L. G. (2018) · Environmental Pollution · doi:10.1016/j.envpol.2017.12.102 · Cited by 53 · see citing works
Lee, J. S., Ha, T. K., Park, J. H., & Lee, G. M. (2013) · Biotechnology and Bioengineering · doi:10.1002/bit.24879 · Cited by 53 · see citing works
“Instead, if the cytoplasmic contents are partially digested or degraded, turning into dark electron dense granular/amorphous masses, the vacuole structures are termed as autophagic compartments (Eskelinen, 2008b).”
The use of electron microscopy for the detection of autophagy in human atherosclerosis
Perrotta, I. D. (2013) · Micron · doi:10.1016/j.micron.2013.03.007 · Cited by 52 · see citing works
St-Pierre, M.-K., Bordeleau, M., & Tremblay, M.-È. (2019) · Methods in Molecular Biology · doi:10.1007/978-1-4939-9658-2_8 · Cited by 53 · see citing works
Autophagy—A Story of Bacteria Interfering with the Host Cell Degradation Machinery
Riebisch, A. K., Mühlen, S., Beer, Y. Y., & Schmitz, I. (2021) · Pathogens · doi:10.3390/pathogens10020110 · Cited by 46 · see citing works
“NLRX1is the only NOD-like receptor that contains a mitochondrial target sequence and an additional LC3-interacting region (LIR).”
BackgroundInfluentialMyricetin Induces Protective Autophagy by Inhibiting the Phosphorylation of mTOR in HepG2 Cells
Cao, J., Chen, H., Lu, W., Wu, Y., Wu, X., Xia, D., & Zhu, J. (2017) · The Anatomical Record · doi:10.1002/ar.23754 · Cited by 45 · see citing works
“After treatments, autophagosomes were observed using Transmission electron microscopy (TEM) according to standard procedures (Eskelinen, 2008).”
MethodTechniques to Study Autophagy in Plants
Mitou, G., Budak, H., & Gözüaçık, D. (2009) · International Journal of Plant Genomics · doi:10.1155/2009/451357 · Cited by 44 · see citing works
Autophagy appears during the development of the mouse lower first molar
Yang, J., Zhu, L., Yuan, G., Chen, Y., Zhang, L., Zhang, L., & Chen, Z. (2012) · Histochemistry and Cell Biology · doi:10.1007/s00418-012-1016-2 · Cited by 43 · see citing works
“As TEM is one of the most valid and convincing methods to detect autophagic compartments in mammalian cells (Eskelinen 2008), these results were further verified with TEM, which showed the typical ultrastructure of autophagy (autophagic vacuoles), and the colocalization of autophagic vacuoles with apoptotic nuclei in…”
ResultBulk autophagy, but not mitophagy, is increased in cellular model of mitochondrial disease
Morán, M., Delmiro, A., Blázquez, A., Ugalde, C., Arenas, J., & Martín, M. A. (2014) · Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease · doi:10.1016/j.bbadis.2014.03.013 · Cited by 41 · see citing works
“performed as described previously [37-40].”
BackgroundThe versatile electron microscope: An ultrastructural overview of autophagy
Biazik, J. M., Vihinen, H., Anwar, T., Jokitalo, E., & Eskelinen, E. (2014) · Methods · doi:10.1016/j.ymeth.2014.11.013 · Cited by 39 · see citing works
Self-citationThe dynamic interplay between ATP/ADP levels and autophagy sustain neuronal migration in vivo
Bressan, C., Pecora, A., Gagnon, D., Snapyan, M., Labrecque, S., Koninck, P. D., Parent, M., & Saghatelyan, A. (2020) · eLife · doi:10.7554/elife.56006 · Cited by 39 · see citing works
“Labeled cell bodies were then randomly selected, and autophagosomal vesicles were identified and measured based on careful selection criteria (Eskelinen, 2008).”
MethodUchiyama, Y., Koike, M., Shibata, M., & Sasaki, M. (2009) · Methods in Enzymology · doi:10.1016/s0076-6879(08)04003-2 · Cited by 39 · see citing works
Wu, X., Ansari, A. R., Pang, X.-X., Li, H., Zhang, Z.-W., Luo, Y., Arshad, M. I., & Song, H. (2019) · Archivum Immunologiae et Therapiae Experimentalis · doi:10.1007/s00005-019-00544-7 · Cited by 39 · see citing works
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Bogéa, T., Wen, R. H., & Moritz, O. L. (2015) · Investigative Ophthalmology & Visual Science · doi:10.1167/iovs.15-16799 · Cited by 38 · see citing works
“4B, 4D, 4E), consistent with autophagosomes.(28)”
Assessing Autophagy in Mouse Models and Patients with Systemic Autoimmune Diseases
Wang, F., Li, B., Schall, N., Wilhelm, M., & Muller, S. (2017) · Cells · doi:10.3390/cells6030016 · Cited by 39 · see citing works
“5–1 μm in diameter) can be identified as autophagosomes when meeting at least two of the following criteria: double membrane, absence of ribosomes at the cytosolic sides of the vacuole, similar luminal density as that of cytosol, visible organelles or parts of organelles in their lumen [29,79].”
BackgroundHanson, H. H., Reilly, J. E., Lee, R., et al. (2010) · Microscopy and Microanalysis · doi:10.1017/s1431927610094092 · Cited by 39 · see citing works
“The morphology of the material inside the autophagosomes was also suggestive of mitochondrial degradation with apparent cristae visible in some cases, and the dense amorphous material may also be of ribosomal origin as has been noted ~Eskelinen, 2008a, 2008b!.”
BackgroundMethodChloropicrin induces endoplasmic reticulum stress in human retinal pigment epithelial cells
Pesonen, M., Pasanen, M., Loikkanen, J. J., Naukkarinen, A., Hemmilä, M., Seulanto, H., Kuitunen, T., & Vähäkangas, K. (2012) · Toxicology Letters · doi:10.1016/j.toxlet.2012.04.002 · Cited by 38 · see citing works
Exploiting Nanomaterial‐Mediated Autophagy for Cancer Therapy
Wei, W., Rosenkrans, Z. T., Luo, Q., Lan, X., & Cai, W. (2018) · Small Methods · doi:10.1002/smtd.201800365 · Cited by 40 · see citing works
Autophagy: a molecular switch to regulate adipogenesis and lipolysis
Sekar, M., & Thirumurugan, K. (2022) · Molecular and Cellular Biochemistry · doi:10.1007/s11010-021-04324-w · Cited by 38 · see citing works
Michelini, S., Barbero, F., Prinelli, A., Steiner, P., Weiss, R., Verwanger, T., Andosch, A., Lütz‐Meindl, U., Puntes, V., Drobne, D., et al. (2021) · Nanoscale · doi:10.1039/d0nr09153g · Cited by 37 · see citing works
Corral‐Juan, M., Casquero, P., Giraldo-Restrepo, N., et al. (2022) · Brain Communications · doi:10.1093/braincomms/fcac030 · Cited by 37 · see citing works
“Subcellular organelles from 20 fibroblasts sections were classified according to previous 24 publications identifying autophagosomes surrounded by double membrane and autolysosomes filled 25 with undigested lipids.(44,45) 26”
BackgroundCheney, L., Guzik, H. M., Macaluso, F., Macián, F., Cuervo, A. M., & Berman, J. W. (2020) · Cells · doi:10.3390/cells9061426 · Cited by 33 · see citing works
A neurogenic perspective of Sarcopenia: time course study of sciatic nerves from aging mice
Krishnan, V. S., White, Z., McMahon, C. D., Hodgetts, S. I., Fitzgerald, M., Shavlakadze, T., Harvey, A. R., & Grounds, M. D. (2016) · Journal of Neuropathology & Experimental Neurology · doi:10.1093/jnen/nlw019 · Cited by 32 · see citing works
Silva, K. A. S., Ghiarone, T., Schreiber, K. L., Grant, D. G., White, T., Frisard, M. I., Sukhanov, S., Chandrasekar, B., Delafontaine, P., & Yoshida, T. (2019) · Journal of Applied Physiology · doi:10.1152/japplphysiol.00898.2018 · Cited by 32 · see citing works
“Autophagic vacuoles were identified and counted as previously reported (19, 63).”
Nanoparticles Induce Autophagy via mTOR Pathway Inhibition and Reactive Oxygen Species Generation
Lu, J. Q., Hao, S.-L., & Yang, W. (2020) · Nanomedicine · doi:10.2217/nnm-2019-0387 · Cited by 34 · see citing works
Standard Immunohistochemical Assays to Assess Autophagy in Mammalian Tissue
Martinet, W., Roth, L., & Meyer, G. R. Y. D. (2017) · Cells · doi:10.3390/cells6030017 · Cited by 31 · see citing works
“However, because the correct iden ification of autophagic vacuoles by TEM is challengi g [25,74], we definitely need more iomarkers for autophagosome formation.”
BackgroundHoward, M., Erickson, J. W., Cuba, Z., Kim, S., Zhou, W., Gade, P. V., Carter, R., Mitchell, K. C., Branscome, H., Siddhi, D., et al. (2022) · Journal of Extracellular Vesicles · doi:10.1002/jev2.12244 · Cited by 32 · see citing works
Rossiter, J. P., Hsu, L., & Jackson, A. C. (2009) · Acta Neuropathologica · doi:10.1007/s00401-009-0503-6 · Cited by 29 · see citing works
Kumar, A., Dailey, L. A., Swedrowska, M., Siow, R. C. M., Mann, G. E., Vizcay‐Barrena, G., Arno, M. J., Mudway, I., & Forbes, B. (2015) · FEBS Letters · doi:10.1002/1873-3468.12026 · Cited by 29 · see citing works
A role for Glucagon-Like Peptide-1 in the regulation of β-cell autophagy
Arden, C. (2018) · Peptides · doi:10.1016/j.peptides.2017.12.002 · Cited by 28 · see citing works
“It is often used to determine the effect of stresses/disease on the autophagic pathway but lacks detail on autophagic flux [24,25].”
BackgroundShintaku, M. (2011) · Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin · doi:10.1007/s00428-011-1104-z · Cited by 28 · see citing works
“However, as pointed out by some investigators [13], the electron microscopic identification of intracytoplasmic inclusions as autophagy-related structures Table 1 Clinicopathological findings and results of LC3 immunohistochemistry in granular cell tumors”
BackgroundCarzaniga, R., Domart, M., Duke, E., & Collinson, L. (2014) · Methods in cell biology · doi:10.1016/b978-0-12-801075-4.00008-2 · Cited by 25 · see citing works
“After 10 min, the holder was moved to dry filter paper and a grid was checked again in the TEM to evaluate the “etching” process.”
MethodInfluentialRole of oxidative stress in cytotoxicity of grape seed extract in human bladder cancer cells
Raina, K., Tyagi, A., Kumar, D., Agarwal, R., & Agarwal, C. (2013) · Food and Chemical Toxicology · doi:10.1016/j.fct.2013.06.039 · Cited by 27 · see citing works
“Transmission electron microscope (TEM, FEI Technai G2 BioTwin at 80KV) was used to visualize cellular vacuoles and other cellular changes, and images were captured with Gatan First Lite digital camera (Eskelinen, 2008; Klionsky et al., 2008; Mizushima and Yoshimori, 2007; Yla-Anttila et al., 2009).”
MethodSigmond, T., Fehér, J., Baksa, A., Pásti, G., Pálfia, Z., Takács-Vellai, K., Kovács, J., Vellai, T., & Kovács, A. L. (2008) · Methods in Enzymology · doi:10.1016/s0076-6879(08)03228-x · Cited by 26 · see citing works
Lumkwana, D., Peddie, C. J., Kriel, J., Michie, L. L., Heathcote, N., Collinson, L., Kinnear, C. J., & Loos, B. (2022) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2022.819571 · Cited by 25 · see citing works
“After conversion, an outline was drawn using a drawing pen circulating autophagosomal and autolysosomal structures collectively known as autophagic vacuoles (AVs) (Eskelinen, 2008; Klionsky et al., 2016).”
Method
- Authors
- Eskelinen, E.-L.
- Affiliations
- University of Helsinki (FI)
- Corresponding author
- Eskelinen, Eeva-Liisa
- Venue
- Autophagy
- Publisher
- Informa UK Limited
- Published
- 2008-02-16
- Volume
- 4
- Issue
- 2
- Pages
- 257-260
- ISSN
- 1554-8627 (ISSN-L)
- 1554-8635
- OpenAlex
- W1972517547
- Cited by
- 197
- References
- 0
- Citations by year
- 2026: 6 · 2025: 8 · 2024: 10 · 2023: 8 · 2022: 16 · 2021: 15 · 2020: 7 · 2019: 17 · 2018: 16 · 2017: 13 · 2016: 11 · 2015: 6 · 2014: 12 · 2013: 14 · 2012: 12
- FWCI
- 6.43
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Autophagy (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Health Sciences › Medicine › Epidemiology › Autophagy in Disease and Therapy
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
- Keywords
- transmission electron microscopy, mammalian cells, autophagosomes
- MeSH terms
- Animals, Autophagy, Cell Compartmentation, Endoplasmic Reticulum, Humans, Mammals, Phagosomes, Research Design, Microscopy, Electron, Transmission
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