Name: Flusso di lavoro di crio-tomografia elettronica per tessuti spessi dimostrato nellippocampo del topo (Video) | JoVE Journal
Uploaded: 2026-03-27T14:30:00.000+00:00
Duration: 10 min 6 s
Description: Scopri un flusso di lavoro standardizzato di crio-tomografia elettronica per tessuti spessi nell'ippocampo del topo. Questo metodo consente analisi strutturali in situ ad alta risoluzione di ambienti cellulari nativi.

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<ol><li>Young, L. N., Villa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bringing+structure+to+cell+biology+with+cryo-electron+tomography%5BTitle%5D)+AND+%22Annu+Rev+Biophys%22%5BJournal%5D)">Bringing structure to cell biology with cryo-electron tomography.</a> Annu Rev Biophys. 52 (1), 573-595 (2023).</li>
<li>Baumeister, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryo-electron+tomography%3A+A+long+journey+to+the+inner+space+of+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Cryo-electron tomography: A long journey to the inner space of cells.</a> Cell. 185 (15), 2649-2652 (2022).</li>
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<li>Schur, F. K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toward+high-resolution+in+situ+structural+biology+with+cryo-electron+tomography+and+subtomogram+averaging%5BTitle%5D)+AND+%22Curr+Opin+Struct+Biol%22%5BJournal%5D)">Toward high-resolution in situ structural biology with cryo-electron tomography and subtomogram averaging.</a> Curr Opin Struct Biol. 58, 1-9 (2019).</li>
<li>Briggs, J. A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+biology+in+situ%E2%80%94the+potential+of+subtomogram+averaging%5BTitle%5D)+AND+%22Curr+Opin+Struct+Biol%22%5BJournal%5D)">Structural biology in situ—the potential of subtomogram averaging.</a> Curr Opin Struct Biol. 23 (2), 261-267 (2013).</li>
<li>Berger, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryo-electron+tomography+on+focused+ion+beam+lamellae+transforms+structural+cell+biology%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Cryo-electron tomography on focused ion beam lamellae transforms structural cell biology.</a> Nat Methods. 20 (4), 499-511 (2023).</li>
<li>McCafferty, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrating+cellular+electron+microscopy+with+multimodal+data+to+explore+biology+across+space+and+time%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Integrating cellular electron microscopy with multimodal data to explore biology across space and time.</a> Cell. 187 (3), 563-584 (2024).</li>
<li>Pöge, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Making+plant+tissue+accessible+for+cryo-electron+tomography%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Making plant tissue accessible for cryo-electron tomography.</a> Elife. 106455, 1-41 (2025).</li>
<li>Bäuerlein, F. J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryo-electron+tomography+of+large+biological+specimens+vitrified+by+plunge+freezing%5BTitle%5D)+AND+%22Preprint.+bioRxiv%22%5BJournal%5D)">Cryo-electron tomography of large biological specimens vitrified by plunge freezing.</a> Preprint. bioRxiv. , (2023).</li>
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<li>Dubochet, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryo-electron+microscopy+of+vitrified+specimens%5BTitle%5D)+AND+%22Q+Rev+Biophys%22%5BJournal%5D)">Cryo-electron microscopy of vitrified specimens.</a> Q Rev Biophys. 21 (2), 129-228 (2009).</li>
<li>Matsui, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryo-electron+tomographic+investigation+of+native+hippocampal+glutamatergic+synapses%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Cryo-electron tomographic investigation of native hippocampal glutamatergic synapses.</a> Elife. 13, RP98458(2024).</li>
<li>Ning, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Uncovering+synaptic+and+cellular+nanoarchitecture+of+brain+tissue+via+seamless+in+situ+trimming+and+milling+for+cryo-electron+tomography%5BTitle%5D)+AND+%22Preprint.+bioRxiv%22%5BJournal%5D)">Uncovering synaptic and cellular nanoarchitecture of brain tissue via seamless in situ trimming and milling for cryo-electron tomography.</a> Preprint. bioRxiv. , (2025).</li>
<li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vhut-cryo-fib%2C+a+method+to+fabricate+frozen+hydrated+lamellae+from+tissue+specimens+for+in+situ+cryo-electron+tomography%5BTitle%5D)+AND+%22J+Struct+Biol%22%5BJournal%5D)">Vhut-cryo-fib, a method to fabricate frozen hydrated lamellae from tissue specimens for in situ cryo-electron tomography.</a> J Struct Biol. 213 (3), 107763(2021).</li>
<li>Glynn, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+generalizable+and+targeted+molecular+biopsy+approach+for+in+situ+cryogenic+electron+tomography+of+vitreous+brain+tissue%5BTitle%5D)+AND+%22Cell+Rep+Methods%22%5BJournal%5D)">A generalizable and targeted molecular biopsy approach for in situ cryogenic electron tomography of vitreous brain tissue.</a> Cell Rep Methods. 5 (7), 101080(2025).</li>
<li>Kelley, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Waffle+method%3A+A+general+and+flexible+approach+for+improving+throughput+in+fib-milling%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Waffle method: A general and flexible approach for improving throughput in fib-milling.</a> Nat Commun. 13 (1), 1857(2022).</li>
<li>Pierson, J., Ziese, U., Sani, M., Peters, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exploring+vitreous+cryo-section-induced+compression+at+the+macromolecular+level+using+electron+cryo-tomography%3B+80s+yeast+ribosomes+appear+unaffected%5BTitle%5D)+AND+%22J+Struct+Biol%22%5BJournal%5D)">Exploring vitreous cryo-section-induced compression at the macromolecular level using electron cryo-tomography; 80s yeast ribosomes appear unaffected.</a> J Struct Biol. 173 (2), 345-349 (2011).</li>
<li>Alamoudi, A., Studer, D., Dubochet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cutting+artefacts+and+cutting+process+in+vitreous+sections+for+cryo-electron+microscopy%5BTitle%5D)+AND+%22J+Struct+Biol%22%5BJournal%5D)">Cutting artefacts and cutting process in vitreous sections for cryo-electron microscopy.</a> J Struct Biol. 150 (1), 109-121 (2005).</li>
<li>Rigort, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Focused+ion+beam+micromachining+of+eukaryotic+cells+for+cryoelectron+tomography%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography.</a> Proc Natl Acad Sci U S A. 109 (12), 4449-4454 (2012).</li>
<li>Mahamid, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+focused+ion+beam+milling+and+lift-out+approach+for+site-specific+preparation+of+frozen-hydrated+lamellas+from+multicellular+organisms%5BTitle%5D)+AND+%22J+Struct+Biol%22%5BJournal%5D)">A focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms.</a> J Struct Biol. 192 (2), 262-269 (2015).</li>
<li>Marko, M., Hsieh, C., Schalek, R., Frank, J., Mannella, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Focused-ion-beam+thinning+of+frozen-hydrated+biological+specimens+for+cryo-electron+microscopy%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy.</a> Nat Methods. 4 (3), 215-217 (2007).</li>
<li>Engel, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Native+architecture+of+the+Chlamydomonas+chloroplast+revealed+by+in+situ+cryo-electron+tomography%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography.</a> Elife. 4, e04889(2015).</li>
<li>Hayles, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+making+of+frozen-hydrated%2C+vitreous+lamellas+from+cells+for+cryo-electron+microscopy%5BTitle%5D)+AND+%22J+Struct+Biol%22%5BJournal%5D)">The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy.</a> J Struct Biol. 172 (2), 180-190 (2010).</li>
<li>Rubino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+site-specific+focused-ion-beam+lift-out+method+for+cryo+transmission+electron+microscopy%5BTitle%5D)+AND+%22J+Struct+Biol%22%5BJournal%5D)">A site-specific focused-ion-beam lift-out method for cryo transmission electron microscopy.</a> J Struct Biol. 180 (3), 572-576 (2012).</li>
<li>Schiøtz, O. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serial+lift-out%3A+Sampling+the+molecular+anatomy+of+whole+organisms%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Serial lift-out: Sampling the molecular anatomy of whole organisms.</a> Nat Methods. 21 (9), 1684-1692 (2023).</li>
<li>Nguyen, H. T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serialized+on-grid+lift-in+sectioning+for+tomography+%28solist%29+enables+a+biopsy+at+the+nanoscale%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Serialized on-grid lift-in sectioning for tomography (solist) enables a biopsy at the nanoscale.</a> Nat Methods. 21 (9), 1693-1701 (2024).</li>
<li>Tang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Step-by-step+procedure+for+an+optimized+serial+lift-out+cryo-focused+ion+beam+milling+technique+in+tissue+analysis%5BTitle%5D)+AND+%22Biophys+Rep%22%5BJournal%5D)">Step-by-step procedure for an optimized serial lift-out cryo-focused ion beam milling technique in tissue analysis.</a> Biophys Rep. 11, 1-13 (2025).</li>
<li>Wolff, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mind+the+gap%3A+Micro-expansion+joints+drastically+decrease+the+bending+of+fib-milled+cryo-lamellae%5BTitle%5D)+AND+%22J+Struct+Biol%22%5BJournal%5D)">Mind the gap: Micro-expansion joints drastically decrease the bending of fib-milled cryo-lamellae.</a> J Struct Biol. 208 (3), 107389(2019).</li>
<li>Berger, C., Watson, H., Naismith, J. H., Dumoux, M., Grange, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xenon+plasma+focused+ion+beam+lamella+fabrication+on+high-pressure+frozen+specimens+for+structural+cell+biology%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Xenon plasma focused ion beam lamella fabrication on high-pressure frozen specimens for structural cell biology.</a> Nat Commun. 16 (1), 2286(2025).</li>
<li>Eisenstein, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parallel+cryo+electron+tomography+on+in+situ+lamellae%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Parallel cryo electron tomography on in situ lamellae.</a> Nat Methods. 20 (1), 131-138 (2022).</li>
<li>Peck, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aretomolive%3A+Automated+reconstruction+of+comprehensively-corrected+and+denoised+cryo-electron+tomograms+in+real-time+and+at+high+throughput%5BTitle%5D)+AND+%22Preprint.+bioRxiv%22%5BJournal%5D)">Aretomolive: Automated reconstruction of comprehensively-corrected and denoised cryo-electron tomograms in real-time and at high throughput.</a> Preprint. bioRxiv. , (2025).</li>
<li>Liu, Y. -T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isotropic+reconstruction+for+electron+tomography+with+deep+learning%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Isotropic reconstruction for electron tomography with deep learning.</a> Nat Commun. 13 (1), 6482(2022).</li>
<li>Chen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+complete+data+processing+workflow+for+cryo-ET+and+subtomogram+averaging%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">A complete data processing workflow for cryo-ET and subtomogram averaging.</a> Nat Methods. 16 (11), 1161-1168 (2019).</li>
<li>Yang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+reduction+of+fib+damage+on+cryo-lamella+by+lowering+energy+of+ion+beam+revealed+by+a+quantitative+analysis%5BTitle%5D)+AND+%22Structure%22%5BJournal%5D)">The reduction of fib damage on cryo-lamella by lowering energy of ion beam revealed by a quantitative analysis.</a> Structure. 31 (10), 1275-1281.e4 (2023).</li>
<li>Tacke, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+streamlined+workflow+for+automated+cryo+focused+ion+beam+milling%5BTitle%5D)+AND+%22J+Struct+Biol%22%5BJournal%5D)">A streamlined workflow for automated cryo focused ion beam milling.</a> J Struct Biol. 213 (3), 107743(2021).</li>
<li>Boltje, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+cryogenic%2C+coincident+fluorescence%2C+electron%2C+and+ion+beam+microscope%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">A cryogenic, coincident fluorescence, electron, and ion beam microscope.</a> Elife. 11, e82891(2022).</li>
<li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrated+fluorescence+microscopy+%28IFLM%29+for+cryo-fib-milling+and+in-situ+cryo-ET%5BTitle%5D)+AND+%22Preprint.+bioRxiv%22%5BJournal%5D)">Integrated fluorescence microscopy (IFLM) for cryo-fib-milling and in-situ cryo-ET.</a> Preprint. bioRxiv. , (2023).</li>
<li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eli+trifocal+microscope%3A+A+precise+system+to+prepare+target+cryo-lamellae+for+in+situ+cryo-ET+study%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Eli trifocal microscope: A precise system to prepare target cryo-lamellae for in situ cryo-ET study.</a> Nat Methods. 20 (2), 276-283 (2023).</li>
<li>Medeiros, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Robust+workflow+and+instrumentation+for+cryo-focused+ion+beam+milling+of+samples+for+electron+cryotomography%5BTitle%5D)+AND+%22Ultramicroscopy%22%5BJournal%5D)">Robust workflow and instrumentation for cryo-focused ion beam milling of samples for electron cryotomography.</a> Ultramicroscopy. 190, 1-11 (2018).</li>
<li>Goetz, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+modular+platform+for+automated+cryo-fib+workflows%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">A modular platform for automated cryo-fib workflows.</a> Elife. 10, e70506(2021).</li>
<li>Eisenstein, F., Fukuda, Y., Danev, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Smart+parallel+automated+cryo-electron+tomography%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Smart parallel automated cryo-electron tomography.</a> Nat Methods. 21 (9), 1612-1615 (2024).</li>
<li>Swulius, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deep+learning-based+segmentation+of+cryo-electron+tomograms%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Deep learning-based segmentation of cryo-electron tomograms.</a> J Vis Exp. (189), e64435(2022).</li>
</ol>

<table><tbody><tr><td>1-Esadeceno</td><td>Sigma-Aldrich</td><td>H2131</td><td>Sostanze chimiche e Reagenti</td></tr><tr><td>2-metilpentano</td><td>Sigma-Aldrich</td><td>M65807</td><td>Sostanze chimiche e Reagenti</td></tr><tr><td>Anello a clip</td><td>Thermo Fisher Scientific</td><td>1131750; 1188953</td><td>Consumabili</td></tr><tr><td>Lamina di rame</td><td>Materiali metallici Fengcheng</td><td>1 & micro; Spessore m</td><td>Consumabili</td></tr><tr><td>Griglia TEM di supporto in rame (100 mesh)</td><td>Beijing Zhongjingkeyi Technology Co., Ltd</td><td>BZ10021b</td><td>Consumabili</td></tr><tr><td>Cryo-EM</td><td>Thermo Fisher Scientific</td><td>Krios G4</td><td>Attrezzature &nbsp;</td></tr><tr><td>Crio-FIB</td><td>Thermo Fisher Scientific</td><td>Aquilos 2</td><td>Attrezzature &nbsp;</td></tr><tr><td>Autogrid crio-FIB</td><td>Thermo Fisher Scientific</td><td>1205101</td><td>Consumabili</td></tr><tr><td>Griglie (UltrAufoil R 1.2/1.3 300 mesh)</td><td>Quantifoil</td><td>AltrAufoil</td><td>Consumabili</td></tr><tr><td>Congelatore ad alta pressione</td><td>Leica Microsystems</td><td>EM ICE</td><td>Attrezzature &nbsp;</td></tr><tr><td>Portatore HPF, rame</td><td>Leica Microsystems</td><td>Tipo B (6 mm)</td><td>Consumabili</td></tr><tr><td>Rivestimento a Sput Ionico</td><td>Quorum</td><td>Q150RS plus</td><td>Attrezzature &nbsp;</td></tr><tr><td>Pasta di lucidatura metallografica</td><td>Lab-testec</td><td>W0.5</td><td>Consumabili</td></tr><tr><td>Lucidatura del tessuto</td><td>GORAL</td><td>B00809</td><td>Consumabili</td></tr><tr><td>Carta vetrata</td><td>Carta vetrata Eagle Brand</td><td>8000 grana, 12000 grana</td><td>Consumabili</td></tr><tr><td>Stereomicroscopio</td><td>Leica Microsystems</td><td>S9i</td><td><un nome="RANGE! D17">Equipaggiamento&nbsp; </a></td></tr><tr><td>Sucrosio</td><td>Sigma-Aldrich</td><td>200-334-9</td><td>Sostanze chimiche e Reagenti</td></tr><tr><td>Software per l'acquisizione della serie Tilt</td><td>https://bio3d.colorado.edu/SerialEM/</td><td>SerialEM 4.0.9</td><td>Software</td></tr><tr><td>Allineamento e allineamento della serie Tilt Software di ricostruzione</td><td>https://github.com/czimaginginstitute/AreTomo3&nbsp;</td><td>AreTomo 3</td><td>Software</td></tr><tr><td>Software per l'acquisizione della serie Tilt</td><td>https://github.com/eisfabian/PACEtomo</td><td>PaceTomo</td><td>Software</td></tr><tr><td>Allineamento e allineamento della serie Tilt Software di ricostruzione</td><td>https://github.com/IsoNet-cryoET/IsoNet</td><td>IsoNet</td><td>Software</td></tr><tr><td>Allineamento e allineamento della serie Tilt Software di ricostruzione</td><td>https://github.com/cryoem/eman2</td><td>EMAN2</td><td>Software</td></tr><tr><td>Allineamento e allineamento della serie Tilt Software di ricostruzione</td><td>https://relion.readthedocs.io/en/release-4.0/index.html</td><td>Relion 4</td><td>Software</td></tr></tbody></table>

flusso di lavoro di crio-tomografia elettronica per tessuti spessi dimostrato nell'ippocampo del topo

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

Flusso di lavoro di crio-tomografia elettronica per tessuti spessi dimostrato nell'ippocampo del topo

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

Ricerca

JoVE Journal

Neuroscience

Neuroscienze

Dimostrato in questo video sono le tecniche per flash congelamento e sezionamento del tessuto embrionale del cervello di topo. Consigli utili per l'utilizzo del criostato sono dati, compresi i metodi di risoluzione dei problemi che possono essere utilizzati durante il taglio per assicurare che le sezioni risultanti tessuti sono privi di crepe e altre distorsioni.

Flash Congelamento e criosezionamento E12.5 cervello di topo

Qui si descrive un metodo rapido e semplice per celle di immagine fluorescente in semi-densi fettine di cervello. Fissando, affettare, e otticamente compensazione tessuto cerebrale si descrivono le modalità standard di imaging epifluorescente o confocale può essere utilizzato per visualizzare le singole cellule e reti neuronali all'interno intatto tessuto nervoso.

Un metodo rapido per High-Resolution Imaging di fluorescenza in semi-spessa fettine di cervello

In questo video, dimostriamo il sezionamento di un tessuto tumorale cerebrale di topo congelato utilizzando un criostato. Le sezioni di tessuto cerebrale congelate così ottenute vengono conservate a basse temperature fino a ulteriori analisi.

Sezionamento del tessuto cerebrale del topo congelato: una procedura per ottenere sottili sezioni di tessuto congelato dal tessuto cerebrale murino congelato

Per conservare processi neuronali per l'analisi ultrastrutturale, si descrive un protocollo per la placcatura di neuroni primari su Electron griglie microscopia seguite da congelamento flash, ottenendo campioni sospesi in uno strato di ghiaccio vitreo. Questi campioni possono essere esaminate con un microscopio crio-elettroni per visualizzare le strutture in scala nanometrica.

Preparazione di primaria neuroni per visualizzare neuriti in uno Stato Frozen-idratata Utilizzo Cryo-Electron Positron

Qui viene presentato un protocollo che descrive le fasi di preparazione del campione e di raccolta dei dati richieste nella tomografia a raggi X criomoscido morbido (SXT) per visualizzare l'ultrastruttura di intere cellule crioconservate con una risoluzione di 25 nm a mezzo passo.

Una descrizione cartografica 3D della cellula mediante tomografia a raggi X Cryo Soft

Utilizzo della fresatura a fascio ionico focalizzato per produrre lamelle vetrose su griglia da campioni biologici congelati a immersione per la tomografia crioelettronica.

Preparazione di lamelle da campioni biologici vitreali utilizzando un microscopio elettronico a scansione a doppio raggio per la tomografia crioelettronica

Presentiamo un protocollo per la preparazione lamellare di campioni biologici surgelati a tuffo mediante microlavorazione a fascio ionico crio-focalizzato per studi strutturali ad alta risoluzione di macromolecole in situ con tomografia crioeletnico. Il protocollo presentato fornisce linee guida per la preparazione di lamelle di alta qualità ad alta riproducibilità per la caratterizzazione strutturale di macromolecole all'interno del Saccharomyces cerevisiae.

Preparazione e microlavorazione Cryo-FIB di Saccharomyces cerevisiae per Tomografia Crioeletnico

La crescente domanda di raccolta dati su larga scala nella tomografia elettronica criogenica richiede routine di acquisizione delle immagini ad alto rendimento. Qui è descritto un protocollo che implementa i recenti sviluppi di strategie di acquisizione avanzate volte a massimizzare l'efficienza temporale e il throughput della raccolta dei dati tomografici.

Strategie per l'ottimizzazione dell'acquisizione dei dati della tomografia elettronica criogenica

La tomografia crio-STEM fornisce un mezzo per visualizzare organelli di cellule intatte senza incorporamento, sezionamento o altre preparazioni invasive. La risoluzione 3D ottenuta è attualmente nell'intervallo di pochi nanometri, con un campo visivo di diversi micrometri e uno spessore accessibile nell'ordine di 1 μm.

Visualizzazione di organelli in situ mediante tomografia crio-STEM

Questo flusso di lavoro consente la produzione di lamelle mirate a strutture biologiche marcate in fluorescenza che sono piccole (&lt;1 μm di estensione assiale) e rare (1 copia per cellula) utilizzando una piattaforma di imaging criogenica tri-coincidente. Questa piattaforma integra la microscopia a fluorescenza, la fresatura a fascio ionico focalizzato e la microscopia elettronica a scansione in un'unica posizione focale e consente la microscopia a fluorescenza simultanea durante la fresatura.

Flusso di lavoro con un microscopio criogenico a fluorescenza coincidente, a fascio di elettroni e ioni per la fresatura mirata delle cellule

Scopri un flusso di lavoro standardizzato di crio-tomografia elettronica per tessuti spessi nell'ippocampo del topo. Questo metodo consente analisi strutturali in situ ad alta risoluzione di ambienti cellulari nativi.

Flusso di lavoro di crio-tomografia elettronica per tessuti spessi dimostrato nell'ippocampo del topo

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