title: Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.
abstract: In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells.
authors: Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.
authors: K
authors: K
authors: Höhn
authors: Carl Zeiss AG, Oberkochen, Germany.
authors: J
authors: J
authors: Fuchs
authors: Carl Zeiss AG, Oberkochen, Germany.
authors: A
authors: A
authors: Fröber
authors: Carl Zeiss Microscopy GmbH, Jena, Germany.
authors: R
authors: R
authors: Kirmse
authors: Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.
authors: B
authors: B
authors: Glass
authors: Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.
authors: M
authors: M
authors: Anders-Össwein
authors: Electron Microscopy Facility, Ulm University, Ulm, Germany.
authors: P
authors: P
authors: Walther
authors: Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.
authors: H-G
authors: HG
authors: Kräusslich
authors: Carl Zeiss AG, Oberkochen, Germany.
authors: C
authors: C
authors: Dietrich
mesh_terms: D002478:Cells, Cultured; D003713:Dendritic Cells / Q000648:ultrastructure* / Q000821:virology; D005453:Fluorescence; D017110:Freeze Substitution; D005615:Freezing; D006678:HIV; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008164:Luminescent Proteins / Q000032:analysis*; D018613:Microscopy, Confocal / Q000379:methods*; D008854:Microscopy, Electron / Q000379:methods*; D008855:Microscopy, Electron, Scanning / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D008867:Microtomy; D011994:Recombinant Proteins / Q000032:analysis; D016610:Tissue Embedding; D014771:Virion / Q000648:ultrastructure; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000032:analysis*; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D000097573:Red Fluorescent Protein
keywords: Confocal laser scanning microscopy; correlative light and electron microscopy; fluorescence preservation of embedded samples; focused ion beam; targeted FIB