Name: 세포의 표적 밀링을 위한 극저온 일치 형광, 전자 및 이온빔 현미경을 사용한 워크플로 (동영상) | JoVE Journal
Uploaded: 2025-10-17T13:50:00.000+00:00
Duration: 8 min 29 s
Description: 학습하세요 극저온 라멜라 제작을 위한 형광 현미경과 FIB-SEM 통합 워크플로우를. 실시간 형광 피드백으로 정확한 밀링이 가능하며, 센트리올과 같은 희귀 세포 구조를 보존하여 저온 전자 단층촬영 분석을 가능하게 합니다.

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5 minutes

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<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>아퀼로스 2 냉동 FIB</td><td>서모피셔 사이언티픽</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<브레르/> & gad_source=1& gad_campaignid=12 764255901& gbraid=0AAAAADxi_GS s7OvZmg4cgCUm0PjSiq9X9& gclid= Cj0KCQjwh5vFBhCyARIsAHBx2w xlIfYHMvqY3sSWu6oEAdUynCSf9 KRg9Jhjp7l0Dot9qTl6GyQaEo8aAiy 2EALw_wcB</td></tr><tr><td>오토그리드 호환 크라이오 그리드 박스</td><td>전자현미경 과학</td><td></td><td>https://www.emsdiasum.com/autogrid<브레르/> -compatible-cryo-grid-box?srsltid= AfmBOorQAd3esD8fLmnDDO2TvrNk cj4UINXFnPSMgrpEmkWd0H7ImUq7</td></tr><tr><td>오토그리드 링과 C-클립</td><td>나노소프트</td><td></td><td>https://www.nanosoftmaterials.com/product-page/100-pack-autogrid-rings-c-clips</td></tr><tr><td>CLEM 소프트웨어 스위트</td><td>스탠포드/SLAC의 달버그 연구 그룹</td><td></td><td>https://github.com/pdahlb/CLEM_software_suite.git</td></tr><tr><td>드래곤플라이 2022.1</td><td>대상 연구 시스템</td><td></td><td>https://dragonfly.comet.tech/</td></tr><tr><td>엔젤</td><td>델믹 B. V.</td><td></td><td>ENZEL 시스템은 맞춤형 기기이며 현재 상업적으로 제공되지 않습니다</td></tr><tr><td>IMOD 4.12</td><td>콜로라도 대학교</td><td></td><td>https://bio3d.colorado.edu/imod/</td></tr><tr><td>LD4 액체질소 듀어(LD4)</td><td>나노소프트</td><td></td><td>https://www.nanosoftmaterials.com/product-page/ld4-liquid-dewar-4-liters</td></tr><tr><td>MATLAB 2023</td><td>수학 작품</td><td></td><td>https://www.mathworks.com/products/matlab.html</td></tr><tr><td>핀 뚜껑 조작기 도구</td><td>나노소프트</td><td></td><td>https://www.nanosoftmaterials.com/product-page/pin-lid-manipulator-tool</td></tr><tr><td>파이썬 툴킷</td><td>스탠포드/SLAC의 달버그 연구 그룹</td><td></td><td>https://github.com/pdahlb/trico_softwaresuite</td></tr><tr><td>Quantifoil R2/2, LF, 200 메쉬, Au EM 그리드</td><td>전자현미경 과학</td><td></td><td>https://www.emsdiasum.com/quantifoil-r-22-lf-200-mesh-gold</td></tr><tr><td>시리얼 EM</td><td>콜로라도 대학교 볼더 캠퍼스</td><td></td><td>https://bio3d.colorado.edu/SerialEM/</td></tr><tr><td>스피어랩 표준 선박 (이송 듀어)</td><td>나노소프트</td><td></td><td>https://www.nanosoftmaterials.com/product-page/spearlab-standard-vessel</td></tr><tr><td>핀셋</td><td>전자현미경 과학</td><td></td><td>https://www.emsdiasum.com/style-3sa</td></tr></tbody></table>

세포의 표적 밀링을 위한 극저온 일치 형광, 전자 및 이온빔 현미경을 사용한 워크플로

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

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

연구

JoVE Journal

Biology

생물학

가까운 원시 상태에서 세균 세포의 ultrastructure 연구를 전자 cryotomography (요법)를 사용하는 방법을 우리는 "macromolecular"(~ 4 NM) 해상도, 여기를 보여줍니다.

세균성 세포의 전자 Cryotomography

극저온 전자 현미경은 스캐닝 (SEM) 또는 전송 (TEM) 중 하나는, 널리 높은 수분 함량 1 생체 시료 또는 기타 자료의 특성에 사용됩니다. SEM / 집중 이온 빔 (FIB)는 샘플에 대한 관심의 기능을 식별하고 극저온 TEM에 전송을위한 얇은 전자 투명한 얇은 판을 추출하는 데 사용됩니다.

집중 이온 빔의 극저온 전자 현미경 표본 준비하여 의미

여기에서는, 극저온 온도에서 고체 밀링에 의해 포유류 세포를 파괴 얻어진 세포 분말로부터 세포 추출물을 제조하고, 항체의 결합 마이크론 스케일 상자성 비드에 친 화성 포획하여 관심 단백질 복합체를 분리하는 프로토콜을 기술한다.

Cryomilled 포유 동물 세포에서 단백질 단지 선호도 캡처

여기에 제시된 것은 싱크로트론 극저온 X선 나노프로브를 사용한 X선 형광 이미징 이전에 실리콘 질화물 멤브레인 및 플런지 동결에 대한 세포 배양 프로토콜이다. 실온 나노 분석만 이 제공되면, 동결된 시료는 더욱 동결건조될 수 있다. 이들은 세포내 원소 조성물에 대한 정보를 얻기 위한 중요한 단계이다.

싱크로트론 X선 형광 나노 분석을 위한 실리콘 질화물 멤브레인의 세포 배양 및 저온 준비

극저온 집중 이온 빔 (FIB) 및 주사 전자 현미경 (SEM) 기술은 손상되지 않은 고액 계면의 화학 및 형태학에 대한 주요 통찰력을 제공 할 수 있습니다. 이러한 인터페이스의 고품질 에너지 분산 X선(EDX) 분광지도를 준비하는 방법은 에너지 저장 장치에 중점을 두고 상세히 설명되어 있습니다.

Cryo-Focused Ion Beam Milling과 주사 전자 현미경 및 분광법을 결합하여 액체-고체 인터페이스의 나노스케일 특성화

여기에 제시된 프로토콜은 새로운 로봇 시스템인 Spotiton의 사용을 설명하며, 액체 저온기에서 진동하기 전에 최소 90ms를 혼합하는 나노 와이어 그리드에 관심 있는 두 샘플을 전달합니다.

새로운 로봇 시스템, 스포티폰을 사용하여 시간 해결 연구를 위한 극저온 전자 현미경 그리드 준비

이 프로토콜은 극저온 구조조명 현미경을 사용하여 생물학적 냉동 보존 샘플을 이미지화하는 방법을 보여줍니다. 우리는 U2OS 세포의 세포 골격을 화상 진찰하여 방법론을 보여줍니다.

극저온 보존 된 세포에서 극저온 구조 조명 현미경 데이터 수집

집속 이온빔 밀링을 사용하여 극저온 전자 단층 촬영을 위한 급락 동결 생물학적 샘플에서 유리질 그리드 상의 라멜라를 생성합니다.

극저온 전자 단층 촬영을 위한 이중 빔 주사 전자 현미경을 사용하여 유리체 생물학적 샘플에서 라멜라 준비

우리는 극저온 전자 단층 촬영을 통해 대용량 분자의 고해상도 구조 연구를 위해 극저온 중심이온 빔 마이크로머시닝에 의한 급락 냉동 생물학적 표본의 라멜라 제제를 위한 프로토콜을 제시합니다. 제시된 프로토콜은 사카로미세스 세레비시아에내부의 거대 분자의 구조적 특성화를 위한 높은 재현성을 가진 고품질 라멜라의 제조를 위한 지침을 제공한다.

냉동 전자 단층 촬영을위한 사카로미세스 cerevisiae의 준비 및 Cryo-FIB 마이크로 매칭

여기에서는 초저온 전자 단층 촬영을 위한 세포 샘플 준비를 안내하는 3D 상관 집속 이온 빔 밀링을 위한 파이프라인을 제시합니다. 형광 태그가 부착된 관심 단백질의 3D 위치는 먼저 극저온 형광 현미경으로 측정한 다음 밀링을 대상으로 합니다. 이 프로토콜은 포유류, 효모 및 박테리아 세포에 적합합니다.

고분해능 극저온 전자 단층촬영을 위한 3D-Correlative Focused Ion Beam Milling을 이용한 시료 전처리

이 프로토콜은 고압 냉동(HPF)과 와플 방법, 극저온 집중 이온 빔(cryo-FIB) 리프트아웃, 선량 대칭 병렬 채취를 통합하여 두꺼운 조직에 대한 냉동 전자 단층촬영(cryo-ET)을 표준화하여, 쥐 해마에서 재 현 가능한 현장 분석을 위한 균일하게 얇은 층상과 유리화 표본을 제공합니다.

쥐 해마에서 입증된 두꺼운 조직을 위한 냉동 전자 단층촬영 워크플로우

학습하세요 극저온 라멜라 제작을 위한 형광 현미경과 FIB-SEM 통합 워크플로우를. 실시간 형광 피드백으로 정확한 밀링이 가능하며, 센트리올과 같은 희귀 세포 구조를 보존하여 저온 전자 단층촬영 분석을 가능하게 합니다.

세포의 표적 밀링을 위한 극저온 일치 형광, 전자 및 이온빔 현미경을 사용한 워크플로

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