Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis

Luckner, Manja; Wanner, Gerhard

doi:10.1007/s00418-018-1681-x

Cited by 27 publications

(20 citation statements)

References 85 publications

(92 reference statements)

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“…The massive accumulation of lysosomes reduced the space available for rough ER and implies steric hindrance of formation of rough ER. Similar data were recently published for large volume FIB/SEM reconstructions of HeLa cells: the dictyosomes, endosomes, lipid bodies and lysosomes form an interconnected system for Golgi degradation and reconstitution 36 . The massive increase of lysosomes, both in number and volume, may have different reasons.…”

Section: Discussionsupporting

confidence: 85%

Insights into replicative senescence of human testicular peritubular cells

Schmid

Flenkenthaler²,

Stöckl³

et al. 2019

Sci Rep

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There is evidence for an age-related decline in male reproductive functions, yet how the human testis may age is not understood. Human testicular peritubular cells (HTPCs) transport sperm, contribute to the spermatogonial stem cell (SSC) niche and immune surveillance, and can be isolated and studied in vitro. Consequences of replicative senescence of HTPCs were evaluated to gain partial insights into human testicular aging. To this end, early and advanced HTPC passages, in which replicative senescence was indicated by increased cell size, altered nuclear morphology, enhanced β-galactosidase activity, telomere attrition and reduced mitochondrial DNA (mtDNA), were compared. These alterations are typical for senescent cells, in general. To examine HTPC-specific changes, focused ion beam scanning electron microscopy (FIB/SEM) tomography was employed, which revealed a reduced mitochondrial network and an increased lysosome population. The results coincide with the data of a parallel proteomic analysis and indicate deranged proteostasis. The mRNA levels of typical contractility markers and growth factors, important for the SSC niche, were not significantly altered. A secretome analysis identified, however, elevated levels of macrophage migration inhibitory factor (MIF) and dipeptidyl peptidase 4 (DPP4), which may play a role in spermatogenesis. Testicular DPP4 may further represent a possible drug target.

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Section: Discussionsupporting

confidence: 85%

Insights into replicative senescence of human testicular peritubular cells

Schmid

Flenkenthaler²,

Stöckl³

et al. 2019

Sci Rep

Self Cite

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“…Monitoring the constantly increasing block face every 0.5–1 μm provides essential information about the position of the relevant landmarks, which can be reconstructed in 3D and compared with the LM stacks for possible corrections or fine adjustments ( Figures 4 C and 4D). Since the cross-section of the glass slide serves as absolute reference for precise alignment, an exact correlation is ensured ( Figure 4 C) ( Luckner and Wanner, 2018 ). These improvements reduce the CLEM workflow and make artificial fiducials and delicate trimming needless ( Kolotuev et al., 2012 ).…”

Section: Discussionmentioning

confidence: 99%

Label-free 3D-CLEM Using Endogenous Tissue Landmarks

et al. 2018

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SummaryEmerging 3D correlative light and electron microscopy approaches enable studying neuronal structure-function relations at unprecedented depth and precision. However, established protocols for the correlation of light and electron micrographs rely on the introduction of artificial fiducial markers, such as polymer beads or near-infrared brandings, which might obscure or even damage the structure under investigation. Here, we report a general applicable “flat embedding” preparation, enabling high-precision overlay of light and scanning electron micrographs, using exclusively endogenous landmarks in the brain: blood vessels, nuclei, and myelinated axons. Furthermore, we demonstrate feasibility of the workflow by combining in vivo 2-photon microscopy and focused ion beam scanning electron microscopy to dissect the role of astrocytic coverage in the persistence of dendritic spines.

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“…NEBD requires the activity of various kinases, including cyclin-dependent kinase 1 and Polo-like kinase 1, which drive the disassembly of the NPCs and nuclear lamina (Heald and McKeon, 1990; Laurell et al, 2011; Rahman et al, 2015; Martino et al, 2017; Linder et al, 2017). However, in many cell types, the membranes of the NE persist and become highly fenestrated such that they exclude organelles but no longer pose a diffusion barrier (Ellenberg et al, 1997; Yang et al, 1997; Hepler and Wolniak, 1984; Schweizer et al, 2015; Luckner and Wanner, 2018).…”

Section: Introductionmentioning

confidence: 99%

C. elegans pronuclei fuse after fertilization through a novel membrane structure

Rahman

Chang

Harned

et al. 2019

Journal of Cell Biology

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After fertilization, parental genomes are enclosed in two separate pronuclei. In Caenorhabditis elegans, and possibly other organisms, when the two pronuclei first meet, the parental genomes are separated by four pronuclear membranes. To understand how these membranes are breached to allow merging of parental genomes we used focused ion beam scanning electron microscopy (FIB-SEM) to study the architecture of the pronuclear membranes at nanometer-scale resolution. We find that at metaphase, the interface between the two pronuclei is composed of two membranes perforated by fenestrations ranging from tens of nanometers to several microns in diameter. The parental chromosomes come in contact through one of the large fenestrations. Surrounding this fenestrated, two-membrane region is a novel membrane structure, a three-way sheet junction, where the four membranes of the two pronuclei fuse and become two. In the plk-1 mutant, where parental genomes fail to merge, these junctions are absent, suggesting that three-way sheet junctions are needed for formation of a diploid genome.

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Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis

Cited by 27 publications

References 85 publications

Insights into replicative senescence of human testicular peritubular cells

Insights into replicative senescence of human testicular peritubular cells

Label-free 3D-CLEM Using Endogenous Tissue Landmarks

C. elegans pronuclei fuse after fertilization through a novel membrane structure

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