The versatile electron microscope: an ultrastructural overview of autophagy

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Sammanfattning

Both light microscopy (LM) and electron microscopy (EM) are able to reveal important information about the formation and function of various autophagic compartments. In this article we will outline the various techniques that are emerging in EM, focusing on analyzing three-dimensional morphology, collectively
known as volume electron microscopy (volume EM), as well as on methods that can be used to localize proteins and antigenic epitopes. Large cell volumes can now be visualized at the EM level by using one of the two complementary imaging techniques, namely Serial Block-face Scanning Electron Microscopy (SB-SEM) or Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). These two blockface imaging methods reveal ultrastructural information from all membrane-bound organelles such as autophagic compartments to be visualized in a three-dimensional space, in association with their surrounding organelles. Another method which falls into the volume EM category is dual-axis electron
tomography (ET). This method is more suited to reconstructing smaller volumes from areas of interest that require nano-structural detail to be confirmed such as membrane contact sites (MCSs) between autophagic compartments and various organelles. Further to this, to complement the morphological identification of autophagic compartments, immunolabeling can be carried out at the EM level to confirm the nature of various autophagic compartments depending on the localization of various antigens at a sub-cellular level. To determine this, various immunolabeling techniques can be carried out, namely the pre-embedding or the post-embedding immunolabeling methods. Examples of both of these methods will be described in this chapter. Correlative light-electron microscopy (CLEM) can be used to visualize the same autophagic organelles under the LM, followed by high-resolution imaging under the EM. Finally, cryofixation has revolutionized the EM field by allowing rapid immobilization of cells and tissue in the near native state, so samples are no longer prone to artefacts induced by chemical fixation. Collectively, this chapter will discuss the aforementioned capabilities of the EM in more detail, with a particular focus on autophagy, namely the impact of EM in the study of the morphology and biogenesis of the phagophore/isolation membrane (referred to as the phagophore hereafter).
Originalspråkengelska
TidskriftMethods
Volym75
Sidor (från-till)44-53
Antal sidor10
ISSN1046-2023
DOI
StatusPublicerad - 2015
MoE-publikationstypA2 Granska artikel i en vetenskaplig tidskrift

Vetenskapsgrenar

  • 1182 Biokemi, cell- och molekylärbiologi

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title = "The versatile electron microscope: an ultrastructural overview of autophagy",
abstract = "Both light microscopy (LM) and electron microscopy (EM) are able to reveal important information about the formation and function of various autophagic compartments. In this article we will outline the various techniques that are emerging in EM, focusing on analyzing three-dimensional morphology, collectivelyknown as volume electron microscopy (volume EM), as well as on methods that can be used to localize proteins and antigenic epitopes. Large cell volumes can now be visualized at the EM level by using one of the two complementary imaging techniques, namely Serial Block-face Scanning Electron Microscopy (SB-SEM) or Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). These two blockface imaging methods reveal ultrastructural information from all membrane-bound organelles such as autophagic compartments to be visualized in a three-dimensional space, in association with their surrounding organelles. Another method which falls into the volume EM category is dual-axis electrontomography (ET). This method is more suited to reconstructing smaller volumes from areas of interest that require nano-structural detail to be confirmed such as membrane contact sites (MCSs) between autophagic compartments and various organelles. Further to this, to complement the morphological identification of autophagic compartments, immunolabeling can be carried out at the EM level to confirm the nature of various autophagic compartments depending on the localization of various antigens at a sub-cellular level. To determine this, various immunolabeling techniques can be carried out, namely the pre-embedding or the post-embedding immunolabeling methods. Examples of both of these methods will be described in this chapter. Correlative light-electron microscopy (CLEM) can be used to visualize the same autophagic organelles under the LM, followed by high-resolution imaging under the EM. Finally, cryofixation has revolutionized the EM field by allowing rapid immobilization of cells and tissue in the near native state, so samples are no longer prone to artefacts induced by chemical fixation. Collectively, this chapter will discuss the aforementioned capabilities of the EM in more detail, with a particular focus on autophagy, namely the impact of EM in the study of the morphology and biogenesis of the phagophore/isolation membrane (referred to as the phagophore hereafter).",
keywords = "1182 Biochemistry, cell and molecular biology, autophagy, ELECTRON MICROSCOPY, electron tomography, serial block face SEM, immuno electron microscopy",
author = "Joanna Biazik and Helena Vihinen and Tahira Anwar and Eija Jokitalo and Eeva-Liisa Eskelinen",
year = "2015",
doi = "10.1016/j.ymeth.2014.11.013",
language = "English",
volume = "75",
pages = "44--53",
journal = "Methods",
issn = "1046-2023",
publisher = "ACADEMIC PRESS INC ELSEVIER SCIENCE",

}

The versatile electron microscope : an ultrastructural overview of autophagy. / Biazik, Joanna; Vihinen, Helena; Anwar, Tahira; Jokitalo, Eija; Eskelinen, Eeva-Liisa.

I: Methods, Vol. 75, 2015, s. 44-53.

Forskningsoutput: TidskriftsbidragÖversiktsartikelVetenskapligPeer review

TY - JOUR

T1 - The versatile electron microscope

T2 - an ultrastructural overview of autophagy

AU - Biazik, Joanna

AU - Vihinen, Helena

AU - Anwar, Tahira

AU - Jokitalo, Eija

AU - Eskelinen, Eeva-Liisa

PY - 2015

Y1 - 2015

N2 - Both light microscopy (LM) and electron microscopy (EM) are able to reveal important information about the formation and function of various autophagic compartments. In this article we will outline the various techniques that are emerging in EM, focusing on analyzing three-dimensional morphology, collectivelyknown as volume electron microscopy (volume EM), as well as on methods that can be used to localize proteins and antigenic epitopes. Large cell volumes can now be visualized at the EM level by using one of the two complementary imaging techniques, namely Serial Block-face Scanning Electron Microscopy (SB-SEM) or Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). These two blockface imaging methods reveal ultrastructural information from all membrane-bound organelles such as autophagic compartments to be visualized in a three-dimensional space, in association with their surrounding organelles. Another method which falls into the volume EM category is dual-axis electrontomography (ET). This method is more suited to reconstructing smaller volumes from areas of interest that require nano-structural detail to be confirmed such as membrane contact sites (MCSs) between autophagic compartments and various organelles. Further to this, to complement the morphological identification of autophagic compartments, immunolabeling can be carried out at the EM level to confirm the nature of various autophagic compartments depending on the localization of various antigens at a sub-cellular level. To determine this, various immunolabeling techniques can be carried out, namely the pre-embedding or the post-embedding immunolabeling methods. Examples of both of these methods will be described in this chapter. Correlative light-electron microscopy (CLEM) can be used to visualize the same autophagic organelles under the LM, followed by high-resolution imaging under the EM. Finally, cryofixation has revolutionized the EM field by allowing rapid immobilization of cells and tissue in the near native state, so samples are no longer prone to artefacts induced by chemical fixation. Collectively, this chapter will discuss the aforementioned capabilities of the EM in more detail, with a particular focus on autophagy, namely the impact of EM in the study of the morphology and biogenesis of the phagophore/isolation membrane (referred to as the phagophore hereafter).

AB - Both light microscopy (LM) and electron microscopy (EM) are able to reveal important information about the formation and function of various autophagic compartments. In this article we will outline the various techniques that are emerging in EM, focusing on analyzing three-dimensional morphology, collectivelyknown as volume electron microscopy (volume EM), as well as on methods that can be used to localize proteins and antigenic epitopes. Large cell volumes can now be visualized at the EM level by using one of the two complementary imaging techniques, namely Serial Block-face Scanning Electron Microscopy (SB-SEM) or Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). These two blockface imaging methods reveal ultrastructural information from all membrane-bound organelles such as autophagic compartments to be visualized in a three-dimensional space, in association with their surrounding organelles. Another method which falls into the volume EM category is dual-axis electrontomography (ET). This method is more suited to reconstructing smaller volumes from areas of interest that require nano-structural detail to be confirmed such as membrane contact sites (MCSs) between autophagic compartments and various organelles. Further to this, to complement the morphological identification of autophagic compartments, immunolabeling can be carried out at the EM level to confirm the nature of various autophagic compartments depending on the localization of various antigens at a sub-cellular level. To determine this, various immunolabeling techniques can be carried out, namely the pre-embedding or the post-embedding immunolabeling methods. Examples of both of these methods will be described in this chapter. Correlative light-electron microscopy (CLEM) can be used to visualize the same autophagic organelles under the LM, followed by high-resolution imaging under the EM. Finally, cryofixation has revolutionized the EM field by allowing rapid immobilization of cells and tissue in the near native state, so samples are no longer prone to artefacts induced by chemical fixation. Collectively, this chapter will discuss the aforementioned capabilities of the EM in more detail, with a particular focus on autophagy, namely the impact of EM in the study of the morphology and biogenesis of the phagophore/isolation membrane (referred to as the phagophore hereafter).

KW - 1182 Biochemistry, cell and molecular biology

KW - autophagy

KW - ELECTRON MICROSCOPY

KW - electron tomography

KW - serial block face SEM

KW - immuno electron microscopy

U2 - 10.1016/j.ymeth.2014.11.013

DO - 10.1016/j.ymeth.2014.11.013

M3 - Review Article

VL - 75

SP - 44

EP - 53

JO - Methods

JF - Methods

SN - 1046-2023

ER -