Neurons on tape: Automated Tape Collecting Ultramicrotomy-mediated volume EM for targeting neuropathology

Kislinger, Georg; Niemann, Cornelia; Rodriguez, Lawrence A.; Jiang, Hanyi; Fard, Maryam K.; Snaidero, Nicolas; Schumacher, Adrian-Minh; Kerschensteiner, Martin; Misgeld, Thomas; Schifferer, Martina

doi:10.1016/bs.mcb.2023.01.012

Cited by 9 publications

(9 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We applied a standard reduced osmium thiocarbohydrazide osmium (rOTO) en bloc staining protocol (Kislinger et al ., 2020a) (Kislinger et al ., 2023) including post-fixation in 2% osmium tetroxide (EMS), 1.5% potassium ferrocyanide (Sigma) in 0.1 M sodium cacodylate (Science Services) buffer (pH 7.4). Staining was enhanced by reaction with 1% thiocarbohydrazide (Sigma) for 45 min at 40°C.…”

Section: Methodsmentioning

confidence: 99%

“…For this end, we built a plasma treatment instrument for tape reels based on a standard instrument (easiGlow, PELCO) with a custom-made vacuum chamber according to the plans proposed by Jeff Lichtman (Schalek et al, 2012) and Mark Terasaki (Baena et al, 2019), with slight modifications regarding the motor placement and the type of motor (Figure 2 - figure supplement 6, see also (Kislinger et al ., 2023)). We used a dynamic O-ring shaft seal (Kurt J. Lesker Company) and a gear motor with a 150:1 reduction and a suitable controller (Pololu Corporation, Las Vegas, USA) and fixed it directly to the enclosure.…”

Section: Methodsmentioning

confidence: 99%

“…In ATUM-SEM, a reel-to-reel system transfers the sections from the diamond knife onto tape (Kubota et al ., 2018). The tape strips are assembled on silicon wafers (Baena et al ., 2019; Kislinger et al ., 2023), thus generating tissue libraries that can be repetitively imaged by SEM at different resolution regimes. This separates the correlation task into two phases: (1) low-resolution screening for the region previously imaged by fluorescence microscopy and (2) imaging of re-located structures by high-resolution volume EM.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

ATUM-Tomo: A multi-scale approach to cellular ultrastructure by combined volume scanning electron microscopy and electron tomography

Kislinger¹,

Fabig

Wehn³

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Like other volume electron microscopy approaches, Automated Tape Collecting Ultramicrotomy (ATUM) enables imaging of serial sections deposited on thick plastic tapes by scanning electron microscopy (SEM). However, ATUM is unique by enabling hierarchical imaging and thus efficient screening for target structures as needed e.g., for correlated light and electron microscopy. However, SEM of sections on tape can only access the section surface, thereby limiting the axial resolution to the typical size of cellular vesicles, an order of magnitude lower than the acquired xy resolution. In contrast, serial-section electron tomography (ET), a transmission electron microscopy-based approach, yields isotropic voxels at full EM resolution, but requires deposition of sections on electron-permeant thin and fragile monolayer films - thus making screening of large section libraries difficult and prone to section loss. To combine the strength of both approaches, we developed ATUM-Tomo, a hybrid method, where sections are first reversibly attached to plastic tape via a dissolvable coating, and after screening detached and transferred to the ET-compatible thin films. Thus, ATUM-SEM of serial semi-thick sections and consecutive ET of one selected section combines SEM's fast target recognition and coarse rendering capability with ET's high-resolution volume visualizations - thus enabling multi-scale interrogation of cellular ultrastructure. As a proof-of-principle, we applied correlative ATUM-Tomo to study ultrastructural features of blood brain barrier (BBB) leakiness around microthrombi in a mouse model of traumatic brain injury. Microthrombi and associated sites of BBB leakiness were identified by confocal imaging of injected fluorescent and electron-dense nanoparticles, then relocalized by ATUM-SEM, and finally interrogated by correlated ATUM-Tomo, a workflow which created a seamless zoom-in on structural BBB pathology from the micro- to the nanometer scale. Overall, our new ATUM-Tomo approach will substantially advance ultrastructural analysis of biological phenomena that require cell- and tissue-level contextualization of the finest subcellular textures.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

ATUM-Tomo: A multi-scale approach to cellular ultrastructure by combined volume scanning electron microscopy and electron tomography

Kislinger¹,

Fabig

Wehn³

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Subsequently, micrographs of serial sections were acquired at a lateral resolution of 5 nm. To align the serial section imaging data, a hybrid approach combining both automated and manual processing procedures was employed within Fiji TrakEM2 (44). Following the alignment, single sections containing the organellar structures of interest were chosen.…”

Section: Correlative Light and Scanning Electron Microscopymentioning

confidence: 99%

“…The tape stripes were affixed onto adhesive carbon tape (Science Services), and this assembly was then secured onto 4-inch silicon wafers (Siegert Wafer), and grounded through adhesive carbon tape stripes (Science Services). EM micrographs were acquired using an Apreo S2 SEM (Thermo Fisher Scientific) as previously described ( 44 ). Hierarchical imaging of serial sections was achieved through initial mapping of the plastic tape stripes at low lateral resolution and capture of the entire tissue sections at a medium resolution (100-200 nm).…”

Section: Correlative Light and Scanning Electron Microscopymentioning

confidence: 99%

ER-associated biogenesis of PINK1 preprotein for neuronal mitophagy

Hees,

Segura,

Schneider

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

A central role in mitochondrial quality control is played by the Parkinson-related mitochondrial kinase PINK1, whose mRNA is transported in neurons by mitochondrial hitch- hiking. Using a live-cell imaging assay for the translation of the PINK1 precursor, we show that local translation of PINK1 requires a concerted interplay between mitochondria and the ER in neurons. For efficient translation, thePink1mRNA needs to relocate to ribosomes located near endolysosomes and the ER. The ER membrane-tethered chaperone DNAJB6 then shields the PINK1 precursor on transit to mitochondria following the ER-SURF pathway. Loss of DNAJB6 hence leads to persistence of ER/endolysosome-associated PINK1 precursor stores and failure of mitophagy upon mitochondrial damage.

show abstract

Nonvesicular lipid transfer drives myelin growth in the central nervous system

Wu,

Kislinger,

Duschek

et al. 2024

Nat Commun

View full text Add to dashboard Cite

Oligodendrocytes extend numerous cellular processes that wrap multiple times around axons to generate lipid-rich myelin sheaths. Myelin biogenesis requires an enormously productive biosynthetic machinery for generating and delivering these large amounts of newly synthesized lipids. Yet, a complete understanding of this process remains elusive. Utilizing volume electron microscopy, we demonstrate that the oligodendroglial endoplasmic reticulum (ER) is enriched in developing myelin, extending into and making contact with the innermost myelin layer where growth occurs. We explore the possibility of transfer of lipids from the ER to myelin, and find that the glycolipid transfer protein (GLTP), implicated in nonvesicular lipid transport, is highly enriched in the growing myelin sheath. Mice with a specific knockout of Gltp in oligodendrocytes exhibit ER pathology, hypomyelination and a decrease in myelin glycolipid content. In summary, our results demonstrate a role for nonvesicular lipid transport in CNS myelin growth, revealing a cellular pathway in developmental myelination.

show abstract

Neurons on tape: Automated Tape Collecting Ultramicrotomy-mediated volume EM for targeting neuropathology

Cited by 9 publications

References 56 publications

ATUM-Tomo: A multi-scale approach to cellular ultrastructure by combined volume scanning electron microscopy and electron tomography

ATUM-Tomo: A multi-scale approach to cellular ultrastructure by combined volume scanning electron microscopy and electron tomography

ER-associated biogenesis of PINK1 preprotein for neuronal mitophagy

Nonvesicular lipid transfer drives myelin growth in the central nervous system

Contact Info

Product

Resources

About