Name: Cryo-elektrontomografie workflow voor dik weefsel aangetoond bij de muishippocampus (Video) | JoVE Journal
Uploaded: 2026-03-27T14:30:00.000+00:00
Duration: 10 min 6 s
Description: Leer een gestandaardiseerde cryo-ET-workflow voor dik weefsel toegepast op de muishippocampus. Deze methode combineert hoogdrukbevriezing, cryo-FIB-frezen en geautomatiseerde beeldvorming voor in situ structurele analyse.

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</ol>

<table><tbody><tr><td>1-Hexadeceen</td><td>Sigma-Aldrich</td><td>H2131</td><td>Chemicaliën &amp; Reagentia</td></tr><tr><td>2-Methylpentaan</td><td>Sigma-Aldrich</td><td>M65807</td><td>Chemicaliën &amp; Reagentia</td></tr><tr><td>Clip-ring</td><td>Thermo Fisher Scientific</td><td>1131750;1188953</td><td>Verbruiksartikelen</td></tr><tr><td>Koperfolie</td><td>Fengcheng Metal Materials</td><td>1 &micro;m dikte</td><td>Verbruiksartikelen</td></tr><tr><td>Koperondersteuning TEM Grids (100 mesh)</td><td>Beijing Zhongjingkeyi Technology Co., Ltd</td><td>BZ10021b</td><td>Verbruiksartikelen</td></tr><tr><td>Cryo-EM</td><td>Thermo Fisher Scientific</td><td>Krios G4</td><td>Apparatuur&nbsp;</td></tr><tr><td>Cryo-FIB</td><td>Thermo Fisher Scientific</td><td>Aquilos 2</td><td>Apparatuur&nbsp;</td></tr><tr><td>Cryo-FIB Autogrid</td><td>Thermo Fisher Scientific</td><td>1205101</td><td>Verbruiksartikelen</td></tr><tr><td>Grids (UltrAufoil R 1.2/1.3 300 mesh)</td><td>Quantifoil</td><td>AltrAufoil</td><td>Verbruiksartikelen</td></tr><tr><td>Hogedrukvriesmachine</td><td>Leica Microsystems</td><td>EM ICE</td><td>Apparatuur&nbsp;</td></tr><tr><td>HPF drager, koper</td><td>Leica Microsystems</td><td>type B (6 mm)</td><td>Verbruiksartikelen</td></tr><tr><td>Ionenspuitercoater</td><td>Quorum</td><td>Q150RS plus</td><td>Apparatuur&nbsp;</td></tr><tr><td>Metallografische polijstpasta</td><td>Lab-testec</td><td>W0.5</td><td>Verbruiksartikelen</td></tr><tr><td>Polijstdoek</td><td>GORAL</td><td>B00809</td><td>Verbruiksartikelen</td></tr><tr><td>Schuurpapier</td><td>Eagle Brand Schuurpapier</td><td>8000-grit, 12000-grit</td><td>Verbruiksartikelen</td></tr><tr><td>Stereomicroscoop</td><td>Leica Microsystems</td><td>S9i</td><td><a name="RANGE!D17">Apparatuur&nbsp;</a></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>200-334-9</td><td>Chemicaliën &amp; Reagentia</td></tr><tr><td>Tilt-Series Verwervingssoftware</td><td>https://bio3d.colorado.edu/SerialEM/</td><td>SerialEM 4.0.9</td><td>Software</td></tr><tr><td>Tilt-Series Uitlijning &amp; Reconstructie Software</td><td>https://github.com/czimaginginstitute/AreTomo3&nbsp;</td><td>AreTomo 3</td><td>Software</td></tr><tr><td>Tilt-Series Verwervingssoftware</td><td>https://github.com/eisfabian/PACEtomo</td><td>PaceTomo</td><td>Software</td></tr><tr><td>Tilt-Series Uitlijning &amp; Reconstructie Software</td><td>https://github.com/IsoNet-cryoET/IsoNet</td><td>IsoNet</td><td>Software</td></tr><tr><td>Tilt-Series Uitlijning &amp; Reconstructie Software</td><td>https://github.com/cryoem/eman2</td><td>EMAN2</td><td>Software</td></tr><tr><td>Tilt-Series Uitlijning &amp; Reconstructie Software</td><td>https://relion.readthedocs.io/en/release-4.0/index.html</td><td>Relion 4</td><td>Software</td></tr></tbody></table>

cryo-elektrontomografie workflow voor dik weefsel aangetoond bij de muishippocampus

Cryo-Electron Tomography Workflow for Thick Tissues Demonstrated in Mouse Hippocampus

Cryo-elektrontomografie workflow voor dik weefsel aangetoond bij de muishippocampus

Cryo&#8209;electron tomography (cryo&#8209;ET) enables 3D visualization of native cellular ultrastructure, but its application to thick tissues is limited ...

cryo-electron-tomography-workflow-for-thick-tissues-demonstrated

Research

JoVE Journal

Neuroscience

Neurowetenschap

Aangetoond in deze video zijn de technieken voor flash invriezen en snijden embryonale hersenweefsel van de muis. Nuttige tips voor het gebruik van de cryostaat worden gegeven, waaronder het oplossen van problemen methoden die gebruikt kunnen worden tijdens het snijden om ervoor te zorgen dat de resulterende weefsel secties zijn vrij van scheuren en andere vervormingen.

Flash Invriezen en Cryosectioning E12.5 hersenen van muizen

Hier beschrijven we een snelle en eenvoudige methode om de afbeelding fluorescent gelabelde cellen in semi-dikke hersenen plakken. Door de vaststelling van, snijden, en optisch clearing hersenweefsel beschrijven we hoe standaard epifluorescerende of confocale beeldvorming kan worden gebruikt om individuele cellen en neuronale netwerken binnen intact zenuwweefsel te visualiseren.

Een snelle aanpak van High-Resolution Imaging fluorescentie in Semi-Dik Brain Slices

In this video, we demonstrate the sectioning of a frozen mouse brain tumor tissue using a cryostat. The frozen brain tissue sections so obtained are stored at low temperatures until further analysis.

Frozen Mouse Brain Tissue Sectioning: A Procedure to Obtain Thin Frozen Tissue Sections from Frozen Murine Brain Tissue

Neuronale processen bewaren voor ultrastructurele analyse beschrijven we een protocol voor beplating van primaire neuronen van elektronenmicroscopie netten gevolgd door flash-bevriezen, waardoor monsters gesuspendeerd in een laag van glasachtige ijs. Deze monsters kunnen worden onderzocht met een cryo-elektronenmicroscoop om structuren te visualiseren op de nanometer schaal.

Voorbereiding van de primaire neuronen voor het visualiseren neurites in een bevroren gehydrateerd Staat Met behulp van Cryo-electron tomography

Hier wordt een protocol gepresenteerd dat de monstervoorbereiding en gegevensverzamelingsstappen beschrijft die nodig zijn in cryo zachte röntgentomografie (SXT) om de ultrastructuur van hele cryo-geconserveerde cellen in beeld te brengen met een resolutie van 25 nm halve toonhoogte.

Een 3D cartografische beschrijving van de cel door Cryo Soft X-ray Tomography

Met behulp van gefocusseerde ionenbundelfrezen om glasvochtlamellen op het rooster te produceren uit bevroren biologische monsters voor cryo-elektronentomografie.

Lamellen bereiden van glasvochtbiologische monsters met behulp van een dual-beam scanning elektronenmicroscoop voor cryo-elektronentomografie

We presenteren een protocol voor lamellenpreparaat van diepgevroren biologische monsters door cryo-gerichte ionenbundelmicromachining voor structurele studies met hoge resolutie van macromoleculen in situ met cryo-elektronentomografie. Het gepresenteerde protocol biedt richtlijnen voor de bereiding van hoogwaardige lamellen met een hoge reproduceerbaarheid voor structurele karakterisering van macromoleculen in de Saccharomyces cerevisiae.

Bereiding en Cryo-FIB microbewerking van Saccharomyces cerevisiae voor Cryo-Elektronen Tomografie

De toenemende vraag naar grootschalige gegevensverzameling in cryogene elektronentomografie vereist routines voor beeldverwerving met hoge doorvoer. Hier wordt een protocol beschreven dat de recente ontwikkelingen van geavanceerde acquisitiestrategieën implementeert die gericht zijn op het maximaliseren van de tijdsefficiëntie en doorvoer van tomografische gegevensverzameling.

Strategieën voor optimalisatie van cryogene elektronentomografiegegevensverwerving

Cryo-STEM-tomografie biedt een middel om organellen van intacte cellen te visualiseren zonder inbedding, sectie of andere invasieve preparaten. De verkregen 3D-resolutie ligt momenteel in het bereik van enkele nanometers, met een gezichtsveld van enkele micrometers en een toegankelijke dikte in de orde van grootte van 1 μm.

Visualisatie van organellen in situ door cryo-STEM tomografie

Deze workflow maakt lamellenproductie mogelijk die gericht is op fluorescerend gelabelde biologische structuren die klein (&lt;1 μm in axiale omvang) en zeldzaam (1 kopie per cel) zijn met behulp van een cryogeen tri-samenvallend beeldvormingsplatform. Dit platform integreert fluorescentiemicroscopie, gefocusseerd ionenbundelfrezen en scanning-elektronenmicroscopie op een enkele brandpuntspositie en maakt gelijktijdige fluorescentiemicroscopie tijdens het malen mogelijk.

Workflow met behulp van een cryogene samenvallende fluorescentie-, elektronen- en ionenbundelmicroscoop voor het gericht frezen van cellen

Leer een gestandaardiseerde cryo-ET-workflow voor dik weefsel toegepast op de muishippocampus. Deze methode combineert hoogdrukbevriezing, cryo-FIB-frezen en geautomatiseerde beeldvorming voor in situ structurele analyse.

Cryo-elektrontomografie workflow voor dik weefsel aangetoond bij de muishippocampus

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