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3D reconstruction of VZV infected cell nuclei and PML nuclear cages by serial section array scanning electron microscopy and electron tomography.

Reichelt M, Joubert L, Perrino J, Koh AL, Phanwar I, Arvin AM - PLoS Pathog. (2012)

Bottom Line: Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages.This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages.This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, California, United States of America. reichelt@stanford.edu

ABSTRACT
Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.

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Outline of the serial section array scanning electron microscopy (SSA-SEM) method.SSA-SEM enables the three-dimensional reconstruction of cell nuclei and PML domains combined with the visualization and quantification of VZV capsids with ultrastructural precision. (A) Ribbons of ultrathin serial sections are placed on gelatin-coated glass slides and then carbon-coated to prevent charging effects during SEM imaging. The indicated area (red square) contains about 60 consecutive sections. A standard TEM slot-grid (arrow) commonly used in serial section TEM and a ten-cent coin are shown for size comparison. (B) Low magnification view of a ribbon of serial sections imaged by SEM using a back-scattered electron detector (BSE). (C) Using BSE-SEM, regions of interest (ROI), such as whole cells, nuclei or PML-domains can be identified and then repeatedly imaged in consecutive sections, yielding a stack of unaligned digital images of the ROI. (D) The stack of digital images must be aligned, either manually or automatically, for later 3D reconstruction. (E) Structures of interest, such as electron dense heterochromatin (blue), PML domains (green) and VZV capsids (yellow) are manually or automatically (threshold) traced in each serial section for quantification of numbers, areas or volumes and for the visualization of size, shape and distribution of segmented structures in the final 3D model (F).
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ppat-1002740-g002: Outline of the serial section array scanning electron microscopy (SSA-SEM) method.SSA-SEM enables the three-dimensional reconstruction of cell nuclei and PML domains combined with the visualization and quantification of VZV capsids with ultrastructural precision. (A) Ribbons of ultrathin serial sections are placed on gelatin-coated glass slides and then carbon-coated to prevent charging effects during SEM imaging. The indicated area (red square) contains about 60 consecutive sections. A standard TEM slot-grid (arrow) commonly used in serial section TEM and a ten-cent coin are shown for size comparison. (B) Low magnification view of a ribbon of serial sections imaged by SEM using a back-scattered electron detector (BSE). (C) Using BSE-SEM, regions of interest (ROI), such as whole cells, nuclei or PML-domains can be identified and then repeatedly imaged in consecutive sections, yielding a stack of unaligned digital images of the ROI. (D) The stack of digital images must be aligned, either manually or automatically, for later 3D reconstruction. (E) Structures of interest, such as electron dense heterochromatin (blue), PML domains (green) and VZV capsids (yellow) are manually or automatically (threshold) traced in each serial section for quantification of numbers, areas or volumes and for the visualization of size, shape and distribution of segmented structures in the final 3D model (F).

Mentions: We developed SSA-SEM as a method that provided both a high enough resolution to identify and precisely locate virion capsids and at the same time allowed the efficient 3D reconstruction of large volumes of host cell nuclei and complete PML cages (Figure 2). SSA-SEM combines principles and strategies of related methods such as immunofluorescence (IF) array tomography [27], [28], serial block face-SEM [29] and focus ion beam (FIB) or iron abrasion SEM [30], [31]. Ribbons of ultrathin serial sections were acquired by ultramicrotomy. We used 100 nm sections to avoid double counting of VZV nucleocapsids, which have a diameter of approximately 100 nm, in consecutive sections. Ribbons of serial sections were transferred onto gelatin-coated glass-slides (Figure 2A), followed by heavy metal counterstaining and a final carbon coating step to avoid charging during SEM imaging. The serial section array was then imaged with a high-resolution SEM using a BSE detector, which generates TEM-like images of cell structures with a contrast dependent mainly on the high atomic weight and differential adsorption of heavy metal stains to cellular proteins, membranes and nucleic acids (Figure 2B). Consecutive SEM imaging of serial sections created ordered stacks of unaligned digital images (Figure 2C). These stacks were then computationally aligned (Figure 2D). The aligned images were then segmented by manual or automatic (threshold) tracing of the morphology of structures of interest, e.g. nucleocapsids and PML cages (Figure 2E). From this data, a 3D model was generated that shows the shape of PML cages and the distribution of virion capsids within the reconstructed nuclear volume (Figure 2F).


3D reconstruction of VZV infected cell nuclei and PML nuclear cages by serial section array scanning electron microscopy and electron tomography.

Reichelt M, Joubert L, Perrino J, Koh AL, Phanwar I, Arvin AM - PLoS Pathog. (2012)

Outline of the serial section array scanning electron microscopy (SSA-SEM) method.SSA-SEM enables the three-dimensional reconstruction of cell nuclei and PML domains combined with the visualization and quantification of VZV capsids with ultrastructural precision. (A) Ribbons of ultrathin serial sections are placed on gelatin-coated glass slides and then carbon-coated to prevent charging effects during SEM imaging. The indicated area (red square) contains about 60 consecutive sections. A standard TEM slot-grid (arrow) commonly used in serial section TEM and a ten-cent coin are shown for size comparison. (B) Low magnification view of a ribbon of serial sections imaged by SEM using a back-scattered electron detector (BSE). (C) Using BSE-SEM, regions of interest (ROI), such as whole cells, nuclei or PML-domains can be identified and then repeatedly imaged in consecutive sections, yielding a stack of unaligned digital images of the ROI. (D) The stack of digital images must be aligned, either manually or automatically, for later 3D reconstruction. (E) Structures of interest, such as electron dense heterochromatin (blue), PML domains (green) and VZV capsids (yellow) are manually or automatically (threshold) traced in each serial section for quantification of numbers, areas or volumes and for the visualization of size, shape and distribution of segmented structures in the final 3D model (F).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369938&req=5

ppat-1002740-g002: Outline of the serial section array scanning electron microscopy (SSA-SEM) method.SSA-SEM enables the three-dimensional reconstruction of cell nuclei and PML domains combined with the visualization and quantification of VZV capsids with ultrastructural precision. (A) Ribbons of ultrathin serial sections are placed on gelatin-coated glass slides and then carbon-coated to prevent charging effects during SEM imaging. The indicated area (red square) contains about 60 consecutive sections. A standard TEM slot-grid (arrow) commonly used in serial section TEM and a ten-cent coin are shown for size comparison. (B) Low magnification view of a ribbon of serial sections imaged by SEM using a back-scattered electron detector (BSE). (C) Using BSE-SEM, regions of interest (ROI), such as whole cells, nuclei or PML-domains can be identified and then repeatedly imaged in consecutive sections, yielding a stack of unaligned digital images of the ROI. (D) The stack of digital images must be aligned, either manually or automatically, for later 3D reconstruction. (E) Structures of interest, such as electron dense heterochromatin (blue), PML domains (green) and VZV capsids (yellow) are manually or automatically (threshold) traced in each serial section for quantification of numbers, areas or volumes and for the visualization of size, shape and distribution of segmented structures in the final 3D model (F).
Mentions: We developed SSA-SEM as a method that provided both a high enough resolution to identify and precisely locate virion capsids and at the same time allowed the efficient 3D reconstruction of large volumes of host cell nuclei and complete PML cages (Figure 2). SSA-SEM combines principles and strategies of related methods such as immunofluorescence (IF) array tomography [27], [28], serial block face-SEM [29] and focus ion beam (FIB) or iron abrasion SEM [30], [31]. Ribbons of ultrathin serial sections were acquired by ultramicrotomy. We used 100 nm sections to avoid double counting of VZV nucleocapsids, which have a diameter of approximately 100 nm, in consecutive sections. Ribbons of serial sections were transferred onto gelatin-coated glass-slides (Figure 2A), followed by heavy metal counterstaining and a final carbon coating step to avoid charging during SEM imaging. The serial section array was then imaged with a high-resolution SEM using a BSE detector, which generates TEM-like images of cell structures with a contrast dependent mainly on the high atomic weight and differential adsorption of heavy metal stains to cellular proteins, membranes and nucleic acids (Figure 2B). Consecutive SEM imaging of serial sections created ordered stacks of unaligned digital images (Figure 2C). These stacks were then computationally aligned (Figure 2D). The aligned images were then segmented by manual or automatic (threshold) tracing of the morphology of structures of interest, e.g. nucleocapsids and PML cages (Figure 2E). From this data, a 3D model was generated that shows the shape of PML cages and the distribution of virion capsids within the reconstructed nuclear volume (Figure 2F).

Bottom Line: Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages.This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages.This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, California, United States of America. reichelt@stanford.edu

ABSTRACT
Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.

Show MeSH
Related in: MedlinePlus