Name: Hücrelerin Hedeflenen Frezelenmesi için Kriyojenik Tesadüfi Floresan, Elektron ve İyon Işını Mikroskobu Kullanan İş Akışı (Video) | JoVE Journal
Uploaded: 2025-10-17T13:50:00.000+00:00
Duration: 8 min 29 s
Description: Keşfedin kriyojenik lamella üretimi için floresan mikroskopi, odaklanmış iyon demesi frezeleme ve taramalı elektron mikroskobu entegrasyonunu. Bu yöntem, floresanla etiketlenmiş hücresel yapıların hassas hedeflenmesini ve kriyojenik elektron tomografisi ile ayrıntılı analizini mümkün kılar.

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<table><tbody><tr><td>Aquilos 2 Cryo-FIB</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>Autogrid uyumlu kriyo ızgara kutusu</td><td>Elektron Mikroskopi Bilimleri</td><td></td><td>https://www.emsdiasum.com/autogrid -compatible-cryo-grid-box?srsltid= AfmBOorQAd3esD8fLmnDDO2TvrNk cj4UINXFnPSMgrpEmkWd0H7ImUq7</td></tr><tr><td>Autogrid halkaları ve C-klipsleri</td><td>Nanosoft</td><td></td><td>https://www.nanosoftmaterials.com/product-page/100-pack-autogrid-rings-c-clips</td></tr><tr><td>CLEM Yazılım Paketi</td><td>Stanford/SLAC'ta Dahlberg Araştırma Grubu</td><td></td><td>https://github.com/pdahlb/CLEM_software_suite.git</td></tr><tr><td>Yusufçuk 2022.1</td><td>Nesne Araştırma Sistemleri</td><td></td><td>https://dragonfly.comet.tech/</td></tr><tr><td>ENZEL</td><td>Delmic B. V.</td><td></td><td>ENZEL sistemi özelleştirilmiş bir enstrüman olup şu anda ticari olarak mevcut değildir</td></tr><tr><td>IMOD 4.12</td><td>Colorado Üniversitesi</td><td></td><td>https://bio3d.colorado.edu/imod/</td></tr><tr><td>LD4 sıvı azot dewarı</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>Pin kapağı manipülatör aracı</td><td>Nanosoft</td><td></td><td>https://www.nanosoftmaterials.com/product-page/pin-lid-manipulator-tool</td></tr><tr><td>Python toolkit</td><td>Stanford/SLAC'ta Dahlberg Araştırma Grubu</td><td></td><td>https://github.com/pdahlb/trico_softwaresuite</td></tr><tr><td>Quantifoil R2/2, LF, 200 ağ, Au EM ızgara</td><td>Elektron Mikroskopi Bilimleri</td><td></td><td>https://www.emsdiasum.com/quantifoil-r-22-lf-200-mesh-gold</td></tr><tr><td>Seri EM</td><td>Colorado Üniversitesi Boulder</td><td></td><td>https://bio3d.colorado.edu/SerialEM/</td></tr><tr><td>Spearlab standart gemisi (transfer dewar)</td><td>Nanosoft</td><td></td><td>https://www.nanosoftmaterials.com/product-page/spearlab-standard-vessel</td></tr><tr><td>Cizbit</td><td>Elektron Mikroskopi Bilimleri</td><td></td><td>https://www.emsdiasum.com/style-3sa</td></tr></tbody></table>

hücrelerin hedeflenen frezelenmesi için kriyojenik tesadüfi floresan, elektron ve İyon işını mikroskobu kullanan İş akışı

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

Hücrelerin Hedeflenen Frezelenmesi için Kriyojenik Tesadüfi Floresan, Elektron ve İyon Işını Mikroskobu Kullanan İş Akışı

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

Biyoloji

Yerli yakın eyalette bakteri hücrelerinin ultrastrüktür çalışma elektron cryotomography (EKT) nasıl kullanılacağı "makromoleküler" (~ 4 nm) çözünürlük, burada göstermektedir.

Bakteriyel Hücre Elektron Cryotomography

Cryo elektron mikroskopları, Tarama (SEM) veya İletim (TEM), ya çok yüksek su içeriği ile 1 biyolojik örneklerin veya diğer malzemelerin karakterizasyonu için kullanılır. A SEM / Odaklı İyon Işın (FİB) örneklerinde ilgi özelliklerini belirlemek ve cryo-TEM transfer için ince, elektron-saydam tabakalara ayıklamak için kullanılır.

Odaklanmış iyon demeti Of Cryo-elektron Mikroskopi Numune Hazırlama By Means

Burada, bir kriyojenik sıcaklıkta katı hal öğütme ile memeli hücrelerin bozulması Elde edilen hücre tozu, bir hücre, ekstrenin üretilmesi ve antikor-bağlı mikron çaplı paramanyetik taneler üzerine afinite yakalama ile ilgili protein kompleksleri izole etmek için protokoller tarif eder.

Cryomilled memeli hücrelerinden protein kompleksi olan ele geçirme kapasitesini

Burada silikon nitrür membranlar ve dalma-donma bir senkrotron kriyojenik X-ışını nanoprobun ile X-ışını floresan görüntüleme öncesinde hücre kültürü için bir protokoldür. Sadece oda sıcaklığında nano-analiz yapıldığında, dondurulmuş numuneler daha da dondurularak kurutulabilir. Bunlar hücre içi element bileşimi hakkında bilgi edinmek için kritik adımlardır.

Silikon Nitrür Membranları ve Synchrotron X-ışını Floresan Nano-Analizi için Kriyopreyeüzerine Hücre Kültürü

Kriyojenik Odaklı İyon Işını (FIB) ve Taramalı Elektron Mikroskobu (SEM) teknikleri, bozulmamış katı-sıvı arayüzlerinin kimyası ve morfolojisi hakkında önemli bilgiler sağlayabilir. Bu tür arayüzlerin yüksek kaliteli Enerji Dağıtıcı X-ışını (EDX) spektroskopik haritalarını hazırlama yöntemleri, enerji depolama cihazlarına odaklanarak detaylandırılmıştır.

Kriyo-Odaklı İyon Işını Frezelemeyi Taramalı Elektron Mikroskobu ve Spektroskopi ile Birleştirerek Sıvı-Katı Arayüzlerinin Nano Ölçekli Karakterizasyonu

Burada sunulan protokol, sıvı kriyojende vitrifikasyondan önce en az 90 ms karıştırılan kendi kendine fitillenen nanotel ızgaraya iki ilgi örneği sunmak için yeni bir robotik sistem olan Spotiton'un kullanımını açıklamaktadır.

Yeni Bir Robotik Sistem Kullanarak Zaman Çözümlenen Çalışmalar için Kriyo-Elektron Mikroskobik Izgara Hazırlığı, Spotiton

Bu protokol, kriyo yapısına sahip aydınlatma mikroskopisi kullanılarak biyolojik kriyo ile korunmuş örneklerin nasıl görüntülenerek görüntülenlerin ortaya koyduğunu göstermektedir. U2OS hücrelerinin sitoskeletonlarını görüntüleyerek metodolojiyi gösteriyoruz.

Kriyojenik Olarak Korunmuş Hücrelerden Kriyo-Yapılandırılmış Aydınlatma Mikroskobik Veri Toplama

Kriyo-elektron tomografisi için dalma dondurulmuş biyolojik örneklerden vitreus ızgara üzerinde lamel üretmek için odaklanmış iyon ışını frezeleme kullanımı.

Kriyo-Elektron Tomografisi için Çift Işınlı Taramalı Elektron Mikroskobu Kullanarak Vitreus Biyolojik Numunelerinden Lamel Hazırlanması

Kriyo-elektron tomografisi ile in situ makromoleküllerin yüksek çözünürlüklü yapısal çalışmaları için kriyo odaklı iyon ışını mikromakine ile dalma donmuş biyolojik örneklerin lamel hazırlanması için bir protokol sunuyoruz. Sunulan protokol, Saccharomyces cerevisiaeiçindeki makromoleküllerin yapısal karakterizasyonu için yüksek tekrarlanabilirlik ile yüksek kaliteli lamel hazırlanması için yönergeler sağlar.

Kriyo-Elektron Tomografisi için Saccharomyces cerevisiae'nin Hazırlanması ve Kriyo-FIB mikromakinesi

Burada, kriyo-elektron tomografisi için hücresel numunelerin hazırlanmasına rehberlik etmek için 3D bağıntılı odaklanmış iyon ışını frezeleme için bir boru hattı sunuyoruz. İlgilenilen floresan etiketli proteinlerin 3 boyutlu konumu önce kriyo-floresan mikroskobu ile belirlenir ve daha sonra öğütme için hedeflenir. Protokol memeli, maya ve bakteri hücreleri için uygundur.

Yüksek Çözünürlüklü Kriyo-Elektron Tomografisi için 3D Korbağıntılı Odaklanmış İyon Işını Frezeleme ile Numune Hazırlama

Bu protokol, kalın dokular için kriyo-elektron tomografisini (kriyo-ET) standartlaştırır; yüksek basınçlı dondurma (HPF) ile waffle yöntemi, kriyojenik odaklı ion ışını (kriyo-FIB) kaldırma ve doz-simetrik, paralel alım ile entegre edilir; böylece fare hipokampusunda tekrarlanabilir in situ analizi için uniform ince lameller ve vitrifikasyon örnekler elde edilir.

Fare Hipokampusunda Kalın Dokular İçin Kriyo-Elektron Tomografi İş Akışı Gösterildi

Keşfedin kriyojenik lamella üretimi için floresan mikroskopi, odaklanmış iyon demesi frezeleme ve taramalı elektron mikroskobu entegrasyonunu. Bu yöntem, floresanla etiketlenmiş hücresel yapıların hassas hedeflenmesini ve kriyojenik elektron tomografisi ile ayrıntılı analizini mümkün kılar.

Hücrelerin Hedeflenen Frezelenmesi için Kriyojenik Tesadüfi Floresan, Elektron ve İyon Işını Mikroskobu Kullanan İş Akışı

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