Name: Flusso di lavoro con un microscopio criogenico a fluorescenza coincidente, a fascio di elettroni e ioni per la fresatura mirata delle cellule (Video) | JoVE Journal
Uploaded: 2025-10-17T13:50:00.000+00:00
Duration: 8 min 29 s
Description: Impara la microscopia criogenica correlata per preservare strutture cellulari fluorescenti durante la fresatura a fascio di ioni. Questo metodo consente l'analisi dettagliata di organelli come i centrioli mediante tomografia crioelettronica.

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<ol><li>Komeili, A., Li, Z., Newman, D. K., Jensen, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magnetosomes+are+cell+membrane+invaginations+organized+by+the+actin-like+protein+MamK%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Magnetosomes are cell membrane invaginations organized by the actin-like protein MamK.</a> Science. 311 (5758), 242-245 (2006).</li>
<li>Bykov, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+structure+of+the+COPI+coat+determined+within+the+cell%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">The structure of the COPI coat determined within the cell.</a> eLife. 6, e32493(2017).</li>
<li>Mahamid, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+the+molecular+sociology+at+the+HeLa+cell+nuclear+periphery%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Visualizing the molecular sociology at the HeLa cell nuclear periphery.</a> Science. 351 (6276), 969-972 (2016).</li>
<li>Xue, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+translation+dynamics+at+atomic+detail+inside+a+bacterial+cell%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Visualizing translation dynamics at atomic detail inside a bacterial cell.</a> Nature. 610 (7930), 205-211 (2022).</li>
<li>Hampton, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correlated+fluorescence+microscopy+and+cryo-electron+tomography+of+virus-infected+or+transfected+mammalian+cells%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells.</a> Nat Protoc. 12 (1), 150-167 (2017).</li>
<li>Oikonomou, C. M., Jensen, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+electron+cryotomography%3A+Toward+structural+biology+in+situ%5BTitle%5D)+AND+%22Annu+Rev+Biochem%22%5BJournal%5D)">Cellular electron cryotomography: Toward structural biology in situ.</a> Annu Rev Biochem. 86 (1), 873-896 (2017).</li>
<li>Grimm, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electron+tomography+of+ice-embedded+prokaryotic+cells%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Electron tomography of ice-embedded prokaryotic cells.</a> Biophys J. 74 (2), 1031-1042 (1998).</li>
<li>Medalia, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macromolecular+architecture+in+eukaryotic+cells+visualized+by+cryoelectron+tomography%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography.</a> Science. 298 (5596), 1209-1213 (2002).</li>
<li>Tamborrini, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+of+the+native+myosin+filament+in+the+relaxed+cardiac+sarcomere%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Structure of the native myosin filament in the relaxed cardiac sarcomere.</a> Nature. 623 (7988), 863-871 (2023).</li>
<li>Kaplan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bdellovibrio+predation+cycle+characterized+at+nanometre-scale+resolution+with+cryo-electron+tomography%5BTitle%5D)+AND+%22Nat+Microbiol%22%5BJournal%5D)">Bdellovibrio predation cycle characterized at nanometre-scale resolution with cryo-electron tomography.</a> Nat Microbiol. 8 (7), 1267-1279 (2023).</li>
<li>Schur, F. K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+atomic+model+of+HIV-1+capsid-SP1+reveals+structures+regulating+assembly+and+maturation%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation.</a> Science. 353 (6298), 506-508 (2016).</li>
<li>Arnold, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Site-specific+cryo-focused+ion+beam+sample+preparation+guided+by+3D+correlative+microscopy%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Site-specific cryo-focused ion beam sample preparation guided by 3D correlative microscopy.</a> Biophys J. 110 (4), 860-869 (2016).</li>
<li>Lam, V., Villa, E. Practical approaches for cryo-FIB milling and applications for cellular cryo-electron tomography. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLam%2C+V+%22cryoEM%3A+Methods+and+Protocols%22">cryoEM: Methods and Protocols</a>. , Springer. 49-82 (2020).</li>
<li>Rigort, A., Plitzko, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryo-focused-ion-beam+applications+in+structural+biology%5BTitle%5D)+AND+%22Arch+Biochem+Biophys%22%5BJournal%5D)">Cryo-focused-ion-beam applications in structural biology.</a> Arch Biochem Biophys. 581, 122-130 (2015).</li>
<li>Noble, A. J., de Marco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryo-focused+ion+beam+for+in+situ+structural+biology%3A+State+of+the+art%2C+challenges%2C+and+perspectives%5BTitle%5D)+AND+%22Curr+Opin+Struct+Biol%22%5BJournal%5D)">Cryo-focused ion beam for in situ structural biology: State of the art, challenges, and perspectives.</a> Curr Opin Struct Biol. 87, 102864(2024).</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>Iancu, C. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electron+cryotomography+sample+preparation+using+the+Vitrobot%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Electron cryotomography sample preparation using the Vitrobot.</a> Nat Protoc. 1 (6), 2813-2819 (2006).</li>
<li>Dobro, M. J., Melanson, L. A., Jensen, G. J., McDowall, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plunge+freezing+for+electron+cryomicroscopy%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Plunge freezing for electron cryomicroscopy.</a> Methods Enzymol. 481, 63-82 (2010).</li>
<li>Chang, Y. -W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correlated+cryogenic+photoactivated+localization+microscopy+and+cryo-electron+tomography%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography.</a> Nat Methods. 11 (7), 737-739 (2014).</li>
<li>Wu, G. -H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-scale+3D+cryo-correlative+microscopy+for+vitrified+cells%5BTitle%5D)+AND+%22Structure%22%5BJournal%5D)">Multi-scale 3D cryo-correlative microscopy for vitrified cells.</a> Structure. 28 (11), 1231-1237.e3 (2020).</li>
<li>Lin, C., Zhang, L., Zhang, Z., Jiang, Y., Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Locating+cellular+contents+during+cryo-FIB+milling+using+cellular+secondary-electron+imaging%5BTitle%5D)+AND+%22J+Struct+Biol%22%5BJournal%5D)">Locating cellular contents during cryo-FIB milling using cellular secondary-electron imaging.</a> J Struct Biol. 215 (3), 108005(2023).</li>
<li>Wagner, F. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparing+samples+from+whole+cells+using+focused-ion-beam+milling+for+cryo-electron+tomography%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography.</a> Nat Protoc. 15 (6), 2041-2070 (2020).</li>
<li>Carter, S. D., Mamede, J. I., Hope, T. J., Jensen, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correlated+cryogenic+fluorescence+microscopy+and+electron+cryo-tomography+shows+that+exogenous+TRIM5%CE%B1+can+form+hexagonal+lattices+or+autophagy+aggregates+in+vivo%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Correlated cryogenic fluorescence microscopy and electron cryo-tomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo.</a> Proc Natl Acad Sci U S A. 117 (47), 29702-29711 (2020).</li>
<li>Guo, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+situ+structure+of+neuronal+C9orf72+poly-GA+aggregates+reveals+proteasome+recruitment%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">In situ structure of neuronal C9orf72 poly-GA aggregates reveals proteasome recruitment.</a> Cell. 172 (4), 696-705.e12 (2018).</li>
<li>Perez, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+demonstration+of+roGFP2+as+an+environmental+sensor+for+cryogenic+correlative+light+and+electron+microscopy%5BTitle%5D)+AND+%22J+Struct+Biol%22%5BJournal%5D)">Identification and demonstration of roGFP2 as an environmental sensor for cryogenic correlative light and electron microscopy.</a> J Struct Biol. 214 (3), 107881(2022).</li>
<li>Sartor, A. M., Dahlberg, P. D., Perez, D., Moerner, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+mApple+as+a+red+fluorescent+protein+for+cryogenic+single-molecule+imaging+with+turn-off+and+turn-on+active+control+mechanisms%5BTitle%5D)+AND+%22J+Phys+Chem+B%22%5BJournal%5D)">Characterization of mApple as a red fluorescent protein for cryogenic single-molecule imaging with turn-off and turn-on active control mechanisms.</a> J Phys Chem B. 127 (12), 2690-2700 (2023).</li>
<li>Sexton, D. L., Burgold, S., Schertel, A., Tocheva, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Super-resolution+confocal+cryo-CLEM+with+cryo-FIB+milling+for+in+situ+imaging+of+Deinococcus+radiodurans%5BTitle%5D)+AND+%22Curr+Res+Struct+Biol%22%5BJournal%5D)">Super-resolution confocal cryo-CLEM with cryo-FIB milling for in situ imaging of Deinococcus radiodurans.</a> Curr Res Struct Biol. 4, 1-9 (2022).</li>
<li>Dahlberg, P. D., Moerner, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryogenic+super-resolution+fluorescence+and+electron+microscopy+correlated+at+the+nanoscale%5BTitle%5D)+AND+%22Annu+Rev+Phys+Chem%22%5BJournal%5D)">Cryogenic super-resolution fluorescence and electron microscopy correlated at the nanoscale.</a> Annu Rev Phys Chem. 72 (1), 253-278 (2021).</li>
<li>Perez, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exploring+transient+states+of+PAmKate+to+enable+improved+cryogenic+single-molecule+imaging%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">Exploring transient states of PAmKate to enable improved cryogenic single-molecule imaging.</a> J Am Chem Soc. 146 (42), 28707-28716 (2024).</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>Loginov, S. V., Boltje, D. B., Hensgens, M. N. F., Hoogenboom, J. P., vander Wee, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Depth-dependent+scaling+of+axial+distances+in+light+microscopy%5BTitle%5D)+AND+%22Optica%22%5BJournal%5D)">Depth-dependent scaling of axial distances in light microscopy.</a> Optica. 11 (4), 553-568 (2024).</li>
<li>Petrov, P. N., Moerner, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Addressing+systematic+errors+in+axial+distance+measurements+in+single-emitter+localization+microscopy%5BTitle%5D)+AND+%22Opt+Express%22%5BJournal%5D)">Addressing systematic errors in axial distance measurements in single-emitter localization microscopy.</a> Opt Express. 28 (13), 18616-18632 (2020).</li>
<li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Precise+3D+localization+by+integrated+fluorescence+microscopy+%28iFLM%29+for+cryo-FIB-milling+and+in+situ+cryo-ET%5BTitle%5D)+AND+%22Microsc+Microanal%22%5BJournal%5D)">Precise 3D localization by integrated fluorescence microscopy (iFLM) for cryo-FIB-milling and in situ cryo-ET.</a> Microsc Microanal. 29 (Suppl. 1), 1055-1057 (2023).</li>
<li>Perton, E., Boltje, D., Jakobi, A., Hoogenboom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Workflow+for+fluorescence-targeted+lamella+milling+from+vitrified+cells+with+a+coincident+fluorescence%2C+electron%2C+and+ion+beam+microscope%5BTitle%5D)+AND+%22Bio+Protoc%22%5BJournal%5D)">Workflow for fluorescence-targeted lamella milling from vitrified cells with a coincident fluorescence, electron, and ion beam microscope.</a> Bio Protoc. 15 (14), e5390(2025).</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>Sica, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+interference+for+the+guidance+of+cryogenic+focused+ion+beam+milling+beyond+the+axial+diffraction+limit%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">Optical interference for the guidance of cryogenic focused ion beam milling beyond the axial diffraction limit.</a> bioRxiv. , (2024).</li>
<li>Dahlberg, P. D., Perez, D., Hecksel, C. W., Chiu, W., Moerner, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metallic+support+films+reduce+optical+heating+in+cryogenic+correlative+light+and+electron+tomography%5BTitle%5D)+AND+%22J+Struct+Biol%22%5BJournal%5D)">Metallic support films reduce optical heating in cryogenic correlative light and electron tomography.</a> J Struct Biol. 214 (4), 107901(2022).</li>
<li>Lukinavicius, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorogenic+probes+for+live-cell+imaging+of+the+cytoskeleton%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Fluorogenic probes for live-cell imaging of the cytoskeleton.</a> Nat Methods. 11 (7), 731-733 (2014).</li>
<li>Wang, J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWang%2C+J+%22Direct+visualization+of+cellular+protein+complexes+in+situ+by+fluorescence-guided+cryo-FIB-SEM+and+cryo-ET%22">Direct visualization of cellular protein complexes in situ by fluorescence-guided cryo-FIB-SEM and cryo-ET</a>. , California Institute of Technology. (2025).</li>
<li>Last, M. G., Tuijtel, M. W., Voortman, L. M., Sharp, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selecting+optimal+support+grids+for+super-resolution+cryogenic+correlated+light+and+electron+microscopy%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Selecting optimal support grids for super-resolution cryogenic correlated light and electron microscopy.</a> Sci Rep. 13 (1), 8270(2023).</li>
<li>Mastronarde, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+electron+microscope+tomography+using+robust+prediction+of+specimen+movements%5BTitle%5D)+AND+%22J+Struct+Biol%22%5BJournal%5D)">Automated electron microscope tomography using robust prediction of specimen movements.</a> J Struct Biol. 152 (1), 36-51 (2005).</li>
<li>Mastronarde, D. N., Held, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+tilt+series+alignment+and+tomographic+reconstruction+in+IMOD%5BTitle%5D)+AND+%22J+Struct+Biol%22%5BJournal%5D)">Automated tilt series alignment and tomographic reconstruction in IMOD.</a> J Struct Biol. 197 (2), 102-113 (2017).</li>
<li>Kremer, J. R., Mastronarde, D. N., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computer+visualization+of+three-dimensional+image+data+using+IMOD%5BTitle%5D)+AND+%22J+Struct+Biol%22%5BJournal%5D)">Computer visualization of three-dimensional image data using IMOD.</a> J Struct Biol. 116 (1), 71-76 (1996).</li>
<li>Meng, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((UCSF+ChimeraX%3A+Tools+for+structure+building+and+analysis%5BTitle%5D)+AND+%22Protein+Sci%22%5BJournal%5D)">UCSF ChimeraX: Tools for structure building and analysis.</a> Protein Sci. 32 (11), e4792(2023).</li>
<li>Heebner, J. E., 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>
<li>Klumpe, S., 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>Li, Y., Kang, D. -D., Dai, J. -Y., Wang, L. -W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cage+effect+of+electron+beam+irradiation+damage+in+cryo-electron+microscopy%5BTitle%5D)+AND+%22npj+Comput+Mater%22%5BJournal%5D)">The cage effect of electron beam irradiation damage in cryo-electron microscopy.</a> npj Comput Mater. 10 (1), 115(2024).</li>
</ol>

<table><tbody><tr><td>Crio-FIB Aquilos 2</td><td>ThermoFisher Scientific</td><td></td><td>https://www.thermofisher.com/us/en/home/electron-microscopy/products/dualbeam-fib-sem-microscopes/aquilos-cryo-fib.html?cid=msd_ls_xseg_xmkt_sdb-aquilos_285811_na_pso_gaw_pei90m & gad_source=1&; gad_campaignid=12 764255901& gbraid=0AAAAADxi_GS s7OvZmg4cgCUm0PjSiq9X9& gclid= Cj0KCQjwh5vFBhCyARIsAHBx2w xlIfYHMvqY3sSWu6oEAdUynCSf9 KRg9Jhjp7l0Dot9qTl6GyQaEo8aAiy 2EALw_wcB</td></tr><tr><td>Scatola cryo grid compatibile con Autogrid</td><td>Scienze della microscopia elettronica</td><td></td><td>https://www.emsdiasum.com/autogrid -compatible-cryo-grid-box?srsltid= AfmBOorQAd3esD8fLmnDDO2TvrNk cj4UINXFnPSMgrpEmkWd0H7ImUq7</td></tr><tr><td>Anelli Autogrid e clip a C</td><td>Nanosoft</td><td></td><td>https://www.nanosoftmaterials.com/product-page/100-pack-autogrid-rings-c-clips</td></tr><tr><td>CLEM Software Suite</td><td>Dahlberg Research Group presso Stanford/SLAC</td><td></td><td>https://github.com/pdahlb/CLEM_software_suite.git</td></tr><tr><td>Dragonfly 2022.1</td><td>Sistemi di Ricerca sugli Oggetti</td><td></td><td>https://dragonfly.comet.tech/</td></tr><tr><td>ENZEL</td><td>Delmic B. V.</td><td></td><td>Il sistema ENZEL è uno strumento personalizzato e attualmente non disponibile commercialmente</td></tr><tr><td>IMOD 4.12</td><td>Università del Colorado</td><td></td><td>https://bio3d.colorado.edu/imod/</td></tr><tr><td>LD4 dewar di azoto liquido</td><td>Nanosoft</td><td></td><td>https://www.nanosoftmaterials.com/product-page/ld4-liquid-dewar-4-liters</td></tr><tr><td>MATLAB 2023</td><td>MathWorks</td><td></td><td>https://www.mathworks.com/products/matlab.html</td></tr><tr><td>Strumento manipolatore del coperchio per perni</td><td>Nanosoft</td><td></td><td>https://www.nanosoftmaterials.com/product-page/pin-lid-manipulator-tool</td></tr><tr><td>Kit di strumenti Python</td><td>Dahlberg Research Group presso Stanford/SLAC</td><td></td><td>https://github.com/pdahlb/trico_softwaresuite</td></tr><tr><td>Quantifoil R2/2, LF, mesh 200, griglia EM Au</td><td>Scienze della microscopia elettronica</td><td></td><td>https://www.emsdiasum.com/quantifoil-r-22-lf-200-mesh-gold</td></tr><tr><td>EM seriale</td><td>Università del Colorado Boulder</td><td></td><td>https://bio3d.colorado.edu/SerialEM/</td></tr><tr><td>Nave standard Spearlab (transfer dewar)</td><td>Nanosoft</td><td></td><td>https://www.nanosoftmaterials.com/product-page/spearlab-standard-vessel</td></tr><tr><td>Pinzette</td><td>Scienze della microscopia elettronica</td><td></td><td>https://www.emsdiasum.com/style-3sa</td></tr></tbody></table>

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

Workflow Using a Cryogenic Coincident Fluorescence, Electron, and Ion Beam Microscope for Targeted Milling of Cells

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

Cryogenic focused ion beam milling with scanning electron microscopy (CryoFIB-SEM) has become the&#160;dominant approach for preparing sub-200-nm thick ...

workflow-using-cryogenic-coincident-fluorescence-electron-ion-beam

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Biologia

Illustriamo qui come utilizzare elettroni cryotomography (ECT) per studiare l'ultrastruttura delle cellule batteriche in quasi native stati, a "macromolecolari" (~ 4 nm) risoluzione.

Cryotomography elettrone di cellule batteriche

Cryo Electron Microscopi, sia a scansione (SEM) o trasmissione (TEM), sono ampiamente utilizzati per la caratterizzazione di campioni biologici o di altri materiali con un elevato contenuto di acqua 1. A SEM / Focused Ion Beam (FIB) viene utilizzato per identificare le caratteristiche di interesse in campioni ed estrarre una lamella sottile elettrone-trasparente per il trasferimento ad un crio-TEM.

Cryo-microscopia elettronica Preparazione del campione per mezzo di un Focused Ion Beam

Qui si descrivono i protocolli di perturbare le cellule di mammifero mediante fresatura a stato solido ad una temperatura criogenica, la produzione di un estratto cellulare dalla polvere di cellule risultante, e isolare complessi proteici di interesse per cattura affinità su perline paramagnetici micron scala anticorpi accoppiati.

Proteine ​​Complesso affinità Acquisizione da cellule di mammifero Cryomilled

Presentato qui è un protocollo per la coltura cellulare sulle membrane di nitrato di silicio e il congelamento del tuffo prima dell'imaging a fluorescenza a raggi X con una nanosonda a raggi X criogenica sincrotrone. Quando viene fornita solo una nano-analisi a temperatura ambiente, i campioni congelati possono essere ulteriormente liofilizzati. Questi sono i passaggi critici per ottenere informazioni sulla composizione elementale intracellulare.

Coltura cellulare sulle membrane di nitride di silicio e criopreparazione per la fluorescenza a raggi X del sincrotrone Nano-analisi

Le tecniche FIB (Cryogenic Focused Ion Beam) e SEM (Scanning Electron Microscopy) possono fornire informazioni chiave sulla chimica e la morfologia delle interfacce solido-liquido intatte. I metodi per la preparazione di mappe spettroscopiche a raggi X a dispersione di energia (EDX) di alta qualità di tali interfacce sono dettagliati, con particolare attenzione ai dispositivi di accumulo di energia.

Caratterizzazione su scala nanometrica di interfacce liquido-solido mediante accoppiamento di fresatura a fascio ionico crio-focalizzato con microscopia elettronica a scansione e spettroscopia

Il protocollo qui presentato descrive l'uso di Spotiton, un nuovo sistema robotico, per fornire due campioni di interesse su una griglia di nanofili auto-traspiranti che si mescolano per un minimo di 90 ms prima della vetrificazione nel criogeno liquido.

Preparazione della griglia crio-elettrone microscopica per studi risolti nel tempo utilizzando un nuovo sistema robotico, Spotiton

Questo protocollo dimostra come immaginare campioni biologici crio-conservati utilizzando la microscopia a illuminazione criostrutto. Dimostriamo la metodologia mediante l'imaging del citoscheletro delle cellule U2OS.

Raccolta di dati microscopici di illuminazione criostruzionata da cellule conservate criogenicamente

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

Qui, presentiamo una pipeline per la fresatura di fasci ionici focalizzati correlativi 3D sulla guida della preparazione di campioni cellulari per la tomografia crioelettronica. La posizione 3D delle proteine di interesse marcate con fluorescenza viene prima determinata mediante microscopia a criofluorescenza e quindi mirata per la fresatura. Il protocollo è adatto per cellule di mammiferi, lieviti e batteri.

Preparazione del campione mediante fresatura a fascio ionico focalizzato correlativo 3D per la tomografia crioelettronica ad alta risoluzione

Questo protocollo standardizza la criotomografia elettronica (crio-ET) per tessuti spessi integrando il congelamento ad alta pressione (HPF) con il metodo waffle, il sollevamento criogenico del fascio di ioni a fuoco (crio-FIB) e l'acquisizione parallela a simmetria dose, producendo lamelle uniformemente sottili e campioni vitrificati per analisi riproducibili in situ nell'ippocampo del topo.

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

Impara la microscopia criogenica correlata per preservare strutture cellulari fluorescenti durante la fresatura a fascio di ioni. Questo metodo consente l'analisi dettagliata di organelli come i centrioli mediante tomografia crioelettronica.

Flusso di lavoro con un microscopio criogenico a fluorescenza coincidente, a fascio di elettroni e ioni per la fresatura mirata delle cellule (Video) | JoVE Journal

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

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