Your browser doesn't support javascript.
loading
On-section correlative light and electron microscopy of large cellular volumes using STEM tomography.
Buerger, Korbinian; Schmidt, Kerstin N; Fokkema, Jantina; Gerritsen, Hans C; Maier, Olga; de Vries, Uwe; Zaytseva, Yulia; Rachel, Reinhard; Witzgall, Ralph.
Afiliação
  • Buerger K; Institute for Molecular and Cellular Anatomy, University of Regensburg, Regensburg, Germany. Electronic address: korbinian.buerger@vkl.uni-regensburg.de.
  • Schmidt KN; Institute for Molecular and Cellular Anatomy, University of Regensburg, Regensburg, Germany.
  • Fokkema J; Molecular Biophysics, Debye Institute for Nanomaterials Science, Utrecht University, Utrecht, The Netherlands.
  • Gerritsen HC; Molecular Biophysics, Debye Institute for Nanomaterials Science, Utrecht University, Utrecht, The Netherlands.
  • Maier O; Institute for Molecular and Cellular Anatomy, University of Regensburg, Regensburg, Germany.
  • de Vries U; Institute for Molecular and Cellular Anatomy, University of Regensburg, Regensburg, Germany.
  • Zaytseva Y; Institute for Molecular and Cellular Anatomy, University of Regensburg, Regensburg, Germany.
  • Rachel R; Center for Electron Microscopy, University of Regensburg, Regensburg, Germany.
  • Witzgall R; Institute for Molecular and Cellular Anatomy, University of Regensburg, Regensburg, Germany. Electronic address: ralph.witzgall@vkl.uni-regensburg.de.
Methods Cell Biol ; 162: 171-203, 2021.
Article em En | MEDLINE | ID: mdl-33707012
The application of both fluorescence and electron microscopy results in a powerful combination of imaging modalities called "correlative light and electron microscopy" (CLEM). Whereas conventional transmission electron microscopy (TEM) tomography is only able to image sections up to a thickness of ~300nm, scanning transmission electron microscopy (STEM) tomography at 200kV allows the analysis of sections up to a thickness of 900nm in three dimensions. In the current study we have successfully integrated STEM tomography into CLEM as demonstrated for human retinal pigment epithelial 1 (RPE1) cells expressing various fluorescent fusion proteins which were high-pressure frozen and then embedded in Lowicryl HM20. Fluorescently labeled gold nanoparticles were applied onto resin sections and imaged by fluorescence and electron microscopy. STEM tomograms were recorded at regions of interest, and overlays were generated using the eC-CLEM software package. Through the nuclear staining of living cells, the use of fluorescently labeled gold fiducials for the generation of overlays, and the integration of STEM tomography we have markedly extended the application of the Kukulski protocol (Kukulski et al., 2011, 2012). Various fluorescently tagged proteins localizing to different cellular organelles could be assigned to their ultrastructural compartments. By combining STEM tomography with on-section CLEM, fluorescently tagged proteins can be localized in three-dimensional ultrastructural environments with a volume of at least 2.7×2.7×0.5µm.
Assuntos
Palavras-chave

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Nanopartículas Metálicas / Tomografia com Microscopia Eletrônica Limite: Humans Idioma: En Ano de publicação: 2021 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Nanopartículas Metálicas / Tomografia com Microscopia Eletrônica Limite: Humans Idioma: En Ano de publicação: 2021 Tipo de documento: Article