Name: Fluxo de trabalho usando um microscópio de fluorescência coincidente criogênica, elétron e feixe de íons para moagem direcionada de células (Vídeo) | JoVE Journal
Uploaded: 2025-10-17T13:50:00.000+00:00
Duration: 8 min 29 s
Description: Aprenda a produzir lamelas criogênicas de estruturas biológicas fluorescentes raras usando microscopia de fluorescência, FIB e SEM simultâneas. Este método preserva alvos marcados para análise detalhada por tomografia criogênica.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

<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>Criofibrilação 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>Caixa de grade criogênica compatível com Autogrid</td><td>Ciências da Microscopia Eletrônica</td><td></td><td>https://www.emsdiasum.com/autogrid -compatible-cryo-grid-box?srsltid= AfmBOorQAd3esD8fLmnDDO2TvrNk cj4UINXFnPSMgrpEmkWd0H7ImUq7</td></tr><tr><td>Anéis de autograde e C-clips</td><td>Nanosoft</td><td></td><td>https://www.nanosoftmaterials.com/product-page/100-pack-autogrid-rings-c-clips</td></tr><tr><td>Suíte de Software CLEM</td><td>Grupo de Pesquisa Dahlberg em Stanford/SLAC</td><td></td><td>https://github.com/pdahlb/CLEM_software_suite.git</td></tr><tr><td>Dragonfly 2022.1</td><td>Sistemas de Pesquisa de Objetos</td><td></td><td>https://dragonfly.comet.tech/</td></tr><tr><td>ENZEL</td><td>Delmic B. V.</td><td></td><td>O sistema ENZEL é um instrumento personalizado e atualmente não está disponível comercialmente</td></tr><tr><td>IMOD 4.12</td><td>Universidade do Colorado</td><td></td><td>https://bio3d.colorado.edu/imod/</td></tr><tr><td>LD4 dewar de nitrogênio líquido</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>Ferramenta manipuladora de tampa de pinos</td><td>Nanosoft</td><td></td><td>https://www.nanosoftmaterials.com/product-page/pin-lid-manipulator-tool</td></tr><tr><td>Kit de ferramentas Python</td><td>Grupo de Pesquisa Dahlberg em Stanford/SLAC</td><td></td><td>https://github.com/pdahlb/trico_softwaresuite</td></tr><tr><td>Quantifoil R2/2, LF, malha 200, grade EM Au</td><td>Ciências da Microscopia Eletrônica</td><td></td><td>https://www.emsdiasum.com/quantifoil-r-22-lf-200-mesh-gold</td></tr><tr><td>Serial EM</td><td>Universidade do Colorado Boulder</td><td></td><td>https://bio3d.colorado.edu/SerialEM/</td></tr><tr><td>Embarcação padrão Spearlab (transferência dewar)</td><td>Nanosoft</td><td></td><td>https://www.nanosoftmaterials.com/product-page/spearlab-standard-vessel</td></tr><tr><td>Pinça</td><td>Ciências da Microscopia Eletrônica</td><td></td><td>https://www.emsdiasum.com/style-3sa</td></tr></tbody></table>

fluxo de trabalho usando um microscópio de fluorescência coincidente criogênica, elétron e feixe de íons para moagem direcionada de células

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

Fluxo de trabalho usando um microscópio de fluorescência coincidente criogênica, elétron e feixe de íons para moagem direcionada de células

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

Research

JoVE Journal

Biology

Biologia

Que vamos mostrar aqui como usar elétron cryotomography (ECT) para estudar a ultra-estrutura das células bacterianas em quase nativo estados, a "macromolecular" (~ 4 nm) resolução.

Electron Cryotomography de células bacterianas

Cryo Electron Microscópios, ou de varredura (MEV) ou de transmissão (TEM), são amplamente utilizados para a caracterização de amostras biológicas ou outros materiais com alto teor de água 1. A SEM / Focused Ion Beam (FIB) é usado para identificar características de interesse em amostras e extrair, uma lamela elétron-fina e transparente de transferência para um crio-TEM.

Cryo-Microscopia Eletrônica de Preparação de Amostras por meio de um Focused Ion Beam

Aqui descrevemos protocolos para romper as células de mamíferos por moagem em estado sólido a uma temperatura criogénica, produzem um extracto celular a partir do pó de células resultante, e isolar complexos proteína de interesse por captura de afinidade sobre esferas paramagnéticas escala mícron acoplado com anticorpo.

Proteína de captura por afinidade complexos a partir de células de mamíferos Cryomilled

Apresentado aqui é um protocolo para a cultura celular em membranas de nitreto de silício e congelamento de mergulho antes da imagem de fluorescência de raios-X com uma nanossonda de raios-X criogênico síncrotron. Quando apenas a temperatura ambiente nano-análise é fornecida, as amostras congeladas podem ser mais liofilizadas. Estes são passos críticos para obter informações sobre a composição elementar intracelular.

Cultura celular em membranas de nitreto de silício e criopreparação para a fluorescência de raios-X synchrotron Nano-análise

As técnicas de Feixe de Íons Focados Criogênicos (FIB) e Microscopia eletrônica de varredura (SEM) podem fornecer insights-chave sobre a química e a morfologia de interfaces de líquido sólido intacto. Os métodos para preparar mapas espectroscópicos de raios-X dispersivos de alta qualidade (EDX) de tais interfaces são detalhados, com foco em dispositivos de armazenamento de energia.

Caracterização em nanoescala de interfaces líquidas sólidas acoplando fresagem de feixe de íons focada em crio-focal com microscopia eletrônica de varredura e espectroscopia

O protocolo aqui apresentado descreve o uso do Spotiton, um novo sistema robótico, para entregar duas amostras de interesse em uma grade de nanofios que se misturam por um mínimo de 90 ms antes da vitrificação em criogen líquido.

Preparação da grade microscópica crio-elétron para estudos resolvidos pelo tempo usando um novo sistema robótico, Spotiton

Este protocolo demonstra como imaginar amostras biológicas conservadas por crio-preservados usando microscopia de iluminação estruturada crioes. Demonstramos a metodologia ao fotografar o citoesqueleto das células U2OS.

Coleta de dados microscópicos de iluminação crioesticamente estruturadas de células criogenicamente preservadas

Usando fresagem por feixe de íons focalizado para produzir lamelas vítreas on-grid a partir de amostras biológicas congeladas por imersão para tomografia crio-eletrônica.

Preparação de Lamelas a partir de Amostras Biológicas Vítreas Usando um Microscópio Eletrônico de Varredura de Feixe Duplo para Tomografia Crio-Eletrônica

Apresentamos um protocolo para a preparação de lamella de espécimes biológicos congelados por micromaquingia de feixe de íons focado em crio-pia para estudos estruturais de alta resolução de macromoléculas in situ com tomografia crio-elétron. O protocolo apresentado fornece diretrizes para a preparação de lamellae de alta qualidade com alta reprodutibilidade para caracterização estrutural de macromoléculas dentro do Saccharomyces cerevisiae.

Preparação e micromaquinção Crio-FIB de Saccharomyces cerevisiae para Tomografia Crio-Elétron

Aqui, apresentamos um pipeline para fresagem de feixe de íons com foco correlativo 3D na orientação da preparação de amostras celulares para tomografia crio-eletrônica. A posição 3D das proteínas fluorescentemente marcadas de interesse é primeiramente determinada por microscopia de criofluorescência e, em seguida, direcionada para moagem. O protocolo é adequado para células de mamíferos, leveduras e bactérias.

Preparação de amostras por fresagem de feixe de íons com foco correlativo 3D para tomografia crioeletrônica de alta resolução

Esse protocolo padroniza a criotomografia eletrônica (crio-ET) para tecidos espessos, integrando congelamento de alta pressão (HPF) com o método waffle, levantamento por feixe de íons de foco criogênico (crio-FIB) e aquisição paralela e dose-simétrica, resultando em lamelas uniformemente finas e amostras vitrificadas para análise in situ reprodutível no hipocampo do camundongo.

Fluxo de trabalho de criotomografia eletrônica para tecidos espessos demonstrado em hipocampo de camundongos

Aprenda a produzir lamelas criogênicas de estruturas biológicas fluorescentes raras usando microscopia de fluorescência, FIB e SEM simultâneas. Este método preserva alvos marcados para análise detalhada por tomografia criogênica.

Fluxo de trabalho usando um microscópio de fluorescência coincidente criogênica, elétron e feixe de íons para moagem direcionada de células (Vídeo) | JoVE Journal

Fluxo de trabalho usando um microscópio de fluorescência coincidente criogênica, elétron e feixe de íons para moagem direcionada de células

Explorar mais vídeos