Name: 단일 분자 국소화 현미경을 사용한 세포 핵의 절대 DNA 밀도 매핑 (동영상) | JoVE Journal
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Description: 배우세요 단일 분자 국소화 현미경과 Voronoi tessellation을 활용한 세포 핵 내 절대 DNA 밀도 측정 기법을. 이 방법은 염색질 구조의 공간적 제약을 정밀하게 분석하여 세포 주기 단계와 유전 정보를 연결합니다.

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5 minutes

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</ol>

<table><tbody><tr><td><강>세포 배양</강></td><td></td><td></td><td></td></tr><tr><td>&micro;-디쉬 35mm, 하이 그리드-500 유리 바닥</td><td>이비디</td><td>81168</td><td></td></tr><tr><td>C3H 10T1/2</td><td>IMB (니어스 연구실)</td><td></td><td></td></tr><tr><td>DMEM</td><td>서모피셔</td><td>12320032</td><td></td></tr><tr><td>dPBS</td><td>서모피셔</td><td>14190144</td><td></td></tr><tr><td>FBS</td><td>라이프 테크놀로지</td><td>16000-044</td><td></td></tr><tr><td>헬러</td><td>현미경 코어 시설(IMB)</td><td></td><td></td></tr><tr><td>HFB</td><td>IMB (쿠벤 연구소)</td><td></td><td></td></tr><tr><td>L-글루타민</td><td>시그마-올드리치</td><td>G7513</td><td></td></tr><tr><td>HFB에 대한 비필수 아미노산과 비타민</td><td></td><td></td><td></td></tr><tr><td>피루브산나트륨</td><td>S8636</td><td></td><td></td></tr><tr><td><강>샘플 준비</강></td><td></td><td></td><td></td></tr><tr><td>카탈라제</td><td>머 크</td><td>2593710</td><td></td></tr><tr><td>포도당</td><td>서모피셔</td><td>241922500</td><td></td></tr><tr><td>포도당 산화효소</td><td>머 크</td><td>49180</td><td></td></tr><tr><td>파라포름알데히드</td><td>시그마-올드리치</td><td>158127</td><td></td></tr><tr><td>RNase 칵테일</td><td>서모피셔</td><td>AM2286</td><td></td></tr><tr><td>시톡스 오렌지</td><td>서모피셔</td><td>S11368</td><td></td></tr><tr><td>테트라스펙 형광 미세구 샘플러 키트</td><td>서모피셔</td><td>T7284</td><td></td></tr><tr><td>트리톤 X-100</td><td>서모피셔</td><td>327372500</td><td></td></tr><tr><td>Software</td><td></td><td></td><td></td></tr><tr><td>바이오포맷</td><td>OpenMicroscopy.org</td><td></td><td>오픈 소스 소프트웨어 https://www.openmicroscopy.org/bio-formats/</td></tr><tr><td>셀프로파일러 v4.2.1</td><td>CellProfiler.org</td><td></td><td>오픈 소스 소프트웨어 https://cellprofiler.org</td></tr><tr><td>피지</td><td>nih.gov</td><td></td><td>오픈 소스 소프트웨어 https://imagej.net/software/fiji/?Downloads</td></tr><tr><td>땅</td><td>nih.gov</td><td></td><td>오픈 소스 소프트웨어 https://github.com/Jan-NM/LAND</td></tr><tr><td>매트랩 2021</td><td>수학 저작</td><td></td><td>상업용 소프트웨어 - "이미지 처리 도구 상자"가 필요하다</td></tr><tr><td>R 4권.. 0.3</td><td>r-project.org</td><td></td><td>오픈 소스 소프트웨어 https://www.r-project.org</td></tr><tr><td>썬더스톰 v1.3</td><td></td><td></td><td>오픈 소스 소프트웨어 https://zitmen.github.io/thunderstorm/</td></tr><tr><td><스트롱>현미경:</스트롱></td><td></td><td></td><td></td></tr><tr><td>AF 7000</td><td>라이카</td><td></td><td></td></tr><tr><td>라이카 GSD</td><td>라이카</td><td></td><td></td></tr><tr><td>SMLM 현미경</td><td>크레머 연구소</td><td></td><td>Dr. S-Y가 맞춤 제작했습니다. 첸</td></tr></tbody></table>

단일 분자 국소화 현미경을 사용한 세포 핵의 절대 dna 밀도 매핑

Mapping Absolute DNA Density in Cell Nuclei using Single-molecule Localization Microscopy

단일 분자 국소화 현미경을 사용한 세포 핵의 절대 DNA 밀도 매핑

Within the cell nucleus, silent genes are generally located in chromatin areas of high density called heterochromatin, whereas active genes can be mostly ...

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Research

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JoVE Journal

Biology

생물학

단일 분자 현미경은 핵 수송에 새로운 통찰력을 제공 approch.

원자력 교통 단일 분자 이미징

우리가 직접 라벨 형광 프로브를 사용하여 3D DNA 물고기에 의해 핵 구조를 분석하기위한 강력하고 다양한 프로토콜을 설명합니다.

직접 레이블이 프로브를 사용하여 강력한 3D DNA 물고기

단일 분자 추적과 함께 Photoactivated 현지화 현미경 (PALM)은 허용 라이브 대장균 세포에서 단백질-DNA 상호 작용의 직접 관찰 및 정량화.

Photoactivated 단일 분자 추적을 사용하여 라이브 박테리아 세포에있는 단백질-DNA 상호 작용을 시각화

형광 표지 된 세포의 다중 이미지 기반 분석을 가능하게하는 유연한 정보학 워크 플로우입니다 발표. 워크 플로는 핵 및 세포질 마커를 정량화하고 이러한 구획 사이에 마커 전위를 계산한다. 절차 96- 웰 포맷으로 간접 면역 형광에 의해 마커 검출 용 siRNA를 안정적인 방법을 사용하여 세포의 섭동 제공된다.

높은 콘텐츠, 오픈 소스 소프트웨어에 의해 지원되는 범용 현미경 데이터에서 형광 마커, 개별 셀 정량화를위한​​ 워크 플로우

5-methylcytosine (oxi mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5 fC)의 산화 형태를 새로 발견 하 고 5-carboxylcytosine (5caC) 독특한 기능 역할을 가진 고유한 DNA 수정 나타낼 수 있습니다. 여기 oxi mCs의 공간 배급, 신호 강도 프로 파일링 및 colocalization의 시각화를 위한 반 정량 워크플로 설명 되어 있습니다.

DNA 수정에 대 한 Immunostaining: 공초점 이미지의 계산 분석

3D 단일 분자 국소화 현미경 검사법은 살아있는 세균 세포에서 형광표지된 단백질의 공간 위치 및 운동 궤적을 조사하는 데 사용됩니다. 본원에 기재된 실험 및 데이터 분석 프로토콜은 풀화된 단일 분자 궤적에 기초하여 세포성 단백질의 널리 퍼진 확산 거동을 결정한다.

단일 분자 추적 현미경 검사법 - 세포화 분자의 확산 상태를 결정하는 도구

3D 다색 DNA FISH는 3D 보존 핵 내에서 다중 게놈 궤시를 시각화하는 도구를 나타내며, 단일 세포 수준에서 핵 공간 내에서 상호 상호 작용 및 국소화를 명확하게 정의합니다. 여기서, 단계별 프로토콜은 인간 1차 세포의 광범위한 스펙트럼에 대해 기재된다.

인간의 1 차 세포에서 핵 아키텍처를 연구하는 3D 다색 DNA FISH 도구

이 프로토콜은 원형질막 단백질의 나노스케일 공간 조직을 해명하기 위한 단일 항체 표지(SAL)를 설명합니다. 단일 분자 수준에서 누적된 항체-에피토프 상호작용을 활용하여 막 SAL(mSAL)은 국소 에피토프 분포를 지도화하는 동시에 천연 세포 환경에서 항체 결합 행동을 포착합니다.

단일 항체 표지를 이용한 막 단백질의 단분자 위치 현미경

우리는 유크로마틴, 헤테로크로마틴, RNAP II의 재현 가능한 지도화를 가능하게 하는 3색 염색질 단분자 위치 현미경(SMLM) 염색 및 분석 프로토콜을 제시합니다. 이 프로토콜은 크로마틴 관련 표적을 포함한 고밀도 핵 환경에서 효율적인 다색 라벨링을 가능하게 하여 신뢰할 수 있는 동시 검출을 가능하게 합니다.

밀집된 핵 환경에서 크로마틴 연구를 위한 다중 표지 단일 분자 위치 현미경 프로토콜

이 프로토콜은 DNA 고유 형광 스펙트럼의 샘플 준비, 데이터 수집 및 분석에 대한 상세한 작업을 제공합니다.

내재 대비를 이용한 DNA 분광학적 초해상도 영상

이 프로토콜은 최적화된 분리, 고정, 표지를 활용하여 분리된 애기장대 핵의 단일 분자 위치 현미경 영상을 효율적으로 수행하여 크로마틴과 RNA 중합효소 II의 신뢰성 있는 나노 규모 시각화를 달성합니다. 이 접근법은 고해상도 영상을 위한 다른 염색질 관련 단백질이나 히스톤 변형에도 쉽게 적용할 수 있습니다.

애기장대 핵에서 단일 분자 위치 현미경을 위한 간소화된 프로토콜

배우세요 단일 분자 국소화 현미경과 Voronoi tessellation을 활용한 세포 핵 내 절대 DNA 밀도 측정 기법을. 이 방법은 염색질 구조의 공간적 제약을 정밀하게 분석하여 세포 주기 단계와 유전 정보를 연결합니다.

단일 분자 국소화 현미경을 사용한 세포 핵의 절대 DNA 밀도 매핑

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