YW 및 PK는 감사 PK (NRF-NRFF-2011-04 및 NRF2012NRF-CRP001-084)에게 수여 싱가포르 국립 연구 재단에서 자금 지원을 인정합니다. 우리는 또한 인프라 지원을위한 MBI 개방 실험실과 현미경의 핵심 시설을 주셔서 감사합니다.

<ol>
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저자는 공개 아무것도 없어.

도 1a에 도시 된 바와 같이 iPALM의 광학계는, 4 π 이중 대향 대물 설계에 기초한다. 표 1에 이전 (23) 설명 및 나열된 설정은 사용자 정의 가공 및 상업 모두 광학 기계 부품을 사용하여 구성된다. 우리의 설치뿐만 아니라, 하워드 휴즈 의학 연구소 (HHMI)는 Janelia 연구 캠퍼스에서 고급 이미지 센터에서 과학계에 액세스 할 수있는 시스템을 호스팅합니다. 전체 기계 도면, 제어 회로도 및 소프트웨어의 경우, 독자는 자세한 내용은 HHMI에서 랄드 헤스에 문의하는 것이 좋습니다. F 액틴을 시각화 iPALM뿐만 아니라 다른 단일 분자 파악 현미경 방법을 사용하는 주요 장점은 EM의 일반적 거친 샘플 처리를 필요로 기술 힘들고 준비, 숙련 된 실무자 3,23 비교 샘플 준비의 상대적 용이성 . AdditionallY는 형광 EM 어려운 남아 멀티 채널 영상에 자연스럽게 의무입니다. 추가로 후술되는 바와 같이 그럼에도 불구하고, 시료 준비 및 화상 형성 공정의 관점에서 모두 접근을위한 여러 전류 제한이있다. EM 샘플 준비를위한 경우와 같이 먼저,주의가 나노 수준에서 심각한 교란을 일으킬 수있는 해상도의 회절 제한 레벨에 적합한 프로토콜 이후, 시편 (35)의 양호한 미세 구조의 보존을 보장하기 위해주의해야한다. 액틴 이미징, 세포의 적절한 고정은 시편의 품질에 영향을 미치는 중요한 요소이다. 항체와 공동 얼룩의 경우, 에피토프 사이트가 영향을받을 수 있지만 그것은 아주 잘 세포 골격과 멤브레인을 유지하기 때문에 글루 타르 알데히드는 바람직한 정착입니다. 더 나은 항체 에피토프 부위를 보존하는 경향이 같은 경우에는, 파라 포름 알데히드가 허용 타협을 제공 할 수있다. 중요한 것은 단일 분자 현지화 현미경을 위해,이 고정 제는 경향이있다배경 형광도를 생성하는 단계; 따라서 보로 하이드 라이드에 의한 고정 이후 담금질, 특히 글루 타르 알데히드의 경우에 필요하다. 또한 형광 및 표지 전략의 적절한 선택은 성공적인 실험 중요한 요인들이다. 나이 퀴 스트 샘플링 정리 (36)에 의해 규정 된 때문에 F 액틴의 나노 사이즈로 라벨 고밀도는 기본적인 사상 네트워크를 포착해야한다. 액틴 들어 팔로이 딘은 여러 업체에서 제공 유기 형광의 넓은 범위로 매우 높은 밀도 라벨링 할 수있다. 팔로이 딘은 독성 때문에, 셀 및 permeabilization 정착이 필요하다. 라이브 셀 호환 굴지 라벨의 대체 전략은 단량체 굴지 또는 작은 액틴 결합 폴리 펩타이드와 중, 형광 단백질 (FP)의 융합을 사용하는 것을 포함한다. 그러나 이러한 (팔로이 딘에 비해 낮은 라벨 밀도를 산출하는 경향이 발견 <stroNG&gt; 그림 6). 우리는 정제 액틴가 직접 유기 형광 표지 및 전기 (37)에 의해 살아있는 세포에 도입하지만,이 방법은 많은 비용과 노동력이 될 것으로 예상 될 수 있다는 점에 유의하라. 형광의 측면에서, 알렉사 형석 647은 일반적으로 현지화 현미경 지속적으로 좋은 성능을 제공하는 것으로 확인되었습니다. 이는 단일 분자 피크와 형광 배경 (38) 사이의 콘트라스트 비 - 밝기 지수의 관점에서 설명 될 수있다. 배경이 누적 파악 정확성을 저하시킬 수 있기 때문에 높은 라벨 밀도, 높은 콘트라스트 비를 필요로한다. 이하 일관된 결과가 강력한 photoswitching 성능을에서 제공하는 알렉사 플 루어 647, 비교와 함께하지만, 우리의 경험에서, 이러한 ATTO488, ATTO520, 알렉사 플 루어 568 (그림 6B)와 같은 여러 가지 다른 형광,,,은 F - 굴지을 시각화하는 데 사용할 수 있습니다 영상 버퍼 광범위한 조건. 39. 이 동등하게 iPALM의 장점과 한계에 적용됩니다. 다른 3 차원 슈퍼 해상도 현미경 기술에 비해 iPALM는 X, Y 해상도 11보다 약 2 배 더 Z 축 해상도, 특히 z 축을 따라, 지금까지 최고 해결 광학 방식을 유지하고있다. 검량선은 주기적이기 때문에, 높은 해상도, Z 간섭계에 iPALM의 의존도는 또한 촬상 깊이에 대한 제한을 부과한다. 즉, Z 좌표마다 250 nm의 정도 (알렉사 형석 647 ~ 700 nm의 발광 대 파장)을 반복한다. 실제로, 이것은의 산장 깊이 내에서 여진 구역을 제한 TIRF 조명을 사용함으로써 해결 될 수있다 &lt;커브 글라스에서 200 나노 미터. 시편 흥분하지 않으며 눈에 보이지 않는 남아에 따라서, 깊은 형광. 그러나,이 또한 더 많은 내부 구조의 손이 닿지 않는 반면, 같은 초점 유착, 대뇌 피질의 세포 골격 및 복부 세포막과 커브 글라스에 근접들로 이미지화 할 수있는 생물학적 구조를 제한합니다. 고정 된 시료를 들어, 하나의 구제 (40) 냉동 절편 수행하는 것입니다. 그러나, 이러한 접근 방식은 매우 힘든이며, 일반적으로 액세스 할 수없는 전문 지식을 필요로한다. 대안 적으로, 3 차원 iPALM 폭풍에 사용 된 비 점수차 방식 - 디 포커스 (12)과 간섭을 결합하여 확장 범위 (Z)에 대해 적응 될 수있다. 이러한 접근 방식은 이중 Z 좌표 판독 고정밀하지만 주기적 간섭에 의해 상기 제, 덜 정확하지만, 비 주기적 점수차 디 포커스에 의한 제를 제공한다. 후자는 &quot;언 래핑&quot;또는 퇴화를 파괴하는 데 사용될 수간섭계 Z 좌표, 따라서 효과적으로 높은 NA 대물 렌즈의 초점 심도 극한 접근은 ~ 750 ㎚에 iPALM 촬상 깊이 배화를 가능하게한다. 위상 언 래핑으로 iPALM 인해 추가 40 디 포커싱을 3 차원으로 약간 감소 정밀도이라도 같은 미토콘드리아 깊은 샘플 내의 화상 구조에 사용되어왔다. iPALM의 다른 본질적인 한계는 촬상 속도이다. 프레임이 많은 수의 위치 파악 좌표의 높은 밀도를 얻기 위해 필요하기 때문에, 카메라의 속도 인수 속도에 제한이 일반적이다. 현재 EMCCD 카메라는 10에서 실행으로 - 20 MHz의 판독 속도, 분 수십 프레임의 충분한 수의 필요합니다. 이러한 긴 시간 규모는 표본이 고정되어야합니다. 여기서, 예 훨씬 빠르게 과학적 상보성 금속 산화물 반도체 (sCMOS) 카메라와 같은 카메라 기술의 최근 발전은, IM의 급격한 증가를 가능하게 할 수있다이 iPALM 아직 구현되지 않았지만, 속도 41 노화. 일부 단일 분자 파악 현미경 방식의 경우, 고밀도 단일 분자 필드 변형 알고리즘 (42)을 사용하여 분석 또는 43 감지 압축 될 수있다. 이러한 전략의 적응도, 따라서, 더 적은 프레임을 조밀 단일 분자 영상시킴으로써 iPALM 속도를 높이고있다. 공간 해상도의 속도 및 이미징 범위의 제한 및 강점으로, iPALM의 주요 응용 프로그램은 특정 단백질 14,15,44의 나노 조직을 공개하는 형광 라벨의 장점을 활용하는 미세 방법으로합니다. 또한 개선, 모두 광학 및 형광 기술의 관점에서, iPALM 공간 해상도에서 더 향상을 가능하게한다. 예를 들어, iPALM 이미지 처리는 현재 2 차원 가우스 함수에 의해 모델링 된 바와 같이 각각의 단일 분자와, 비교적 간단한 일차 근사 방법을 이용하는을로 소비되는 동위 원소 다이폴 평균. 이것은 상당히 공간 해상도를 향상시키는 점 - 확산 함수와 다이폴 방향 또는 시야각에 걸쳐 광학 수차의 명확한 치료보다 정확한 모델을 이용하는 최근의 방법으로 증강 될 수있다. 마찬가지로, photocaging는 크기 45의 주문에 의해 형광의 밝기를 향상하는,보고되었다. 자신의 미세 조직의 핵심 요소가 잘 특성화 된 이후 실제로는 F-액틴 세포 골격은 iPALM에 추가 방법론 개발을 테스트하기위한 매우 가치있는 모델 시스템이 될 수 있습니다. 사실 EM 수준의 해상력과 광학 현미경에 의해 단백질 조직을 분석 할 수있는 능력은 크게 세포의 구조와 기능의 무수한 측면에 대한 우리의 이해를 향상시킬 것으로 예상된다.

복잡한 세포 구조의 시각화는 오랫동안 생물 통찰력과 발견을 통합하고있다. 형광 현미경은, 그 해상력이 ~ 200 화상면에 나노 (X, Y, 또는 측 방향 치수) 및&gt; 500 nm의 광학 축 방향 (Z, 또는 축 방향 치수)에 회절 고분자 특이성 화상 세포 제한 할 수 있지만 1, 2. 따라서, 미세 기능 관찰 역사적 전자 현미경 (EM)로 제한되어왔다. 100 nm의 범위 1-6 - 다행히, 슈퍼 해상도 현미경의 최근 발전은 10에서 공간 해상도를 가능하게,이 제한을 회피했다. 특히, 슈퍼 해상도는 같은 PALM (광 활성화 현지화 현미경) 4, FPALM은 (형광 광 활성화 현지화 현미경) 5 (d)에 STORM (직접 확률 광학 재건 현미경) 6, 7, PAINT (같은 약어로 알려진 단일 분자 현지화에 기반 접근 방법 포이미징 나노 지형) 8 INT의 축적은, GSDIM (접지 상태 고갈 현미경 9) 개별 분자 반환 한 다음, 또는 SMACM (단일 분자 액티브 제어 현미경) (10),뿐만 아니라 그들의 3 차원 (3D) 구현, 간섭 PALM (iPALM) 11 3D-STORM (12)는 신경 세포의 축삭을, 다수의 생물학적 구조의 나노 조직에 새로운 통찰력을 드러내는 포함 귀중한되었습니다 및 초점 유착 14, 15, 세포 - 세포 접합 (16), 원자력이 17 모공, 13 시냅스 및 중심체 18 ~ 20 등이 있습니다. 슈퍼 해상도 현미경은 잠재적으로 유용하는 세포의 또 다른 미세 기능은 액틴 세포 골격이다. 셀 피질 필라멘트 (F) 액틴의 복잡한 그물 세공은 세포 형태 및 기계적 특성 (21)의 제어에 중요한 역할을한다. 조직 오F의 F-액틴 적극적 동적 강하게 중합, 가교, 회전율, 안정성 및 네트워크 토폴로지 (22)에 영향을 미치는 수많은 조절 단백질 비록 조절된다. 그러나, F - 굴지 그물 세공 구조의 특성은 세포 과정의 다양한 범위, 기존의 회절 제한 광학 현미경으로 자신의 관찰을 방해은 F-액틴 필라멘트의 작은 크기 (~ 8 ㎚)에 기계적인 통찰력에 대한 중요하지만, 따라서 액틴 미세 구조의 시각화 종래 독점적 EM에 의해 수행되었다. 여기서는, 접착 포유류 세포에서 액틴 세포 골격 F-시각화 3D 11,23에 매우 높은 정밀도 기능을 활용할 iPALM 초 해상 현미경 기술을 사용하는 프로토콜을 기술한다. iPALM 악기는 매우 전문 있지만, 이러한 장비를 설정하는 방법에 대한 지시는 최근 23 설명되어있는 호 주최 iPALM 현미경에 액세스하는 동안구 휴즈 의학 연구소는 또한 최소의 비용으로 연구 커뮤니티에 제공하고있다. 또한, 본원에 기재된 시료 제조 방법은 더 광범위하게 사용할 수있는 등의 점 확산 함수 (PSF)의 난시 디 포커싱 기반으로하는 다른 3D 슈퍼 해상도 방법, (12) 또는 이중면 검출부 (24)에 직접적으로 적용될 수있다. 우리는 일반적으로 단일 분자 현지화 기반의 초 고해상도 현미경을위한 필요한 성분이 충족되어야 단일 분자 현지화 기반의 초 고해상도 현미경을위한 세 가지 핵심 요구 사항을 수있는 광전 환성 형광 25이므로주의 : ⅰ) 높은 단일 분자 배경 신호의 밝기 및 대비 상대; ⅱ) 소정의 이미지 프레임 내의 단일 분자의 희박한 분포; 또한 나이 퀴 스트 - 샤로 알려진 기본 구조 (의 프로필을 캡처하기에 충분한 라벨 및 ⅲ) 높은 공간 밀도nnon 샘플링 기준) 26. 따라서, 만족스러운 결과를 위해, 형광체의 강조 photoswitching을 최적화하고 기본 미세 구조를 보존 할뿐만 아니라, 실험 기기 및 수집 측면하는 시험편의 적절한 준비를 모두 동등하게 배치되어야한다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>optical table</td>
			<td>Newport, CA&#160;</td>
			<td>RS4000</td>
			<td>iPALM, installed on 4 Newport Stabilizer vibration isolators&#160;</td>
		</tr>
		<tr>
			<td>vibration isolator for optical table</td>
			<td>Newport, CA&#160;</td>
			<td>S-2000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>laser-642</td>
			<td>Newport, CA&#160;</td>
			<td>1185055</td>
			<td>output power=100mw</td>
		</tr>
		<tr>
			<td>laser-561</td>
			<td>Newport, CA&#160;</td>
			<td>1168931</td>
			<td>output power=200mw</td>
		</tr>
		<tr>
			<td>laser-488</td>
			<td>Newport, CA&#160;</td>
			<td>1137970</td>
			<td>output power=200mw</td>
		</tr>
		<tr>
			<td>laser-405</td>
			<td>Newport, CA&#160;</td>
			<td>1142279</td>
			<td>output power=100mw</td>
		</tr>
		<tr>
			<td>broadband dielectric mirrors&#160;</td>
			<td>Thorlabs, NJ</td>
			<td>BB1-E02</td>
			<td>laser combiner</td>
		</tr>
		<tr>
			<td>dichroic beamsplitter&#160;</td>
			<td>Semrock, NY</td>
			<td>LM01-427-25</td>
			<td></td>
		</tr>
		<tr>
			<td>acousto-optic tunable filter&#160;</td>
			<td>AA Opto-Electronic, France</td>
			<td>AOTFnC-VIS-TN</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear polarizer</td>
			<td>Newport, CA&#160;</td>
			<td>05LP-VIS-B</td>
			<td></td>
		</tr>
		<tr>
			<td>baseplate</td>
			<td>local workshop</td>
			<td>customized</td>
			<td></td>
		</tr>
		<tr>
			<td>turning mirror (22.5&#176;)</td>
			<td>Reynard Corpporation, CA</td>
			<td>customized</td>
			<td>22.5&#176; mirror</td>
		</tr>
		<tr>
			<td>motorized optic mounts&#160;</td>
			<td>New Focus, CA</td>
			<td>8816</td>
			<td></td>
		</tr>
		<tr>
			<td>motorized XYZ translation stage</td>
			<td>Thorlabs, NJ</td>
			<td>MT3/M-Z6</td>
			<td>sample holder</td>
		</tr>
		<tr>
			<td>T-Cube DC servo motor controller</td>
			<td>Thorlabs, NJ</td>
			<td>TDC001</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezo Phase Shifter</td>
			<td>Physik Instrumente, Germany</td>
			<td>S-303.CD</td>
			<td></td>
		</tr>
		<tr>
			<td>objective lens</td>
			<td>Nikon, Japan</td>
			<td>MRD01691</td>
			<td>objective. Apo TIRF 60X/1.49oil</td>
		</tr>
		<tr>
			<td>translation stage</td>
			<td>New Focus, CA</td>
			<td>9062-COM-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pico Motor Actuator</td>
			<td>New Focus, CA</td>
			<td>8301</td>
			<td></td>
		</tr>
		<tr>
			<td>rotary Solenoid/Shutter</td>
			<td>DACO Instruments, CT</td>
			<td>5423-458</td>
			<td></td>
		</tr>
		<tr>
			<td>3-way beam splitter</td>
			<td>Rocky Mountain Instruments, CO</td>
			<td>customized</td>
			<td>beamsplitter</td>
		</tr>
		<tr>
			<td>Piezo Z/Tip/Tilt scanner</td>
			<td>Physik Instrumente, Germany</td>
			<td>S-316.10</td>
			<td></td>
		</tr>
		<tr>
			<td>motorized five-axis tilt aligner&#160;</td>
			<td>New Focus, CA</td>
			<td>8081</td>
			<td></td>
		</tr>
		<tr>
			<td>Picmotor ethernet controller</td>
			<td>New Focus, CA</td>
			<td>8752</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezo controllers/amplifier/digital operation module</td>
			<td>Physik Instrumente, Germany</td>
			<td>E-509/E-503/E-517</td>
			<td></td>
		</tr>
		<tr>
			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-523/20</td>
			<td>filters</td>
		</tr>
		<tr>
			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-588/21</td>
			<td></td>
		</tr>
		<tr>
			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-607/30</td>
			<td></td>
		</tr>
		<tr>
			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-676/37</td>
			<td></td>
		</tr>
		<tr>
			<td>notch filter</td>
			<td>Semrock, NY</td>
			<td>NF01-405/488/561/635</td>
			<td></td>
		</tr>
		<tr>
			<td>motorized filter wheel with controllter</td>
			<td>Thorlabs, NJ</td>
			<td>FW103H</td>
			<td></td>
		</tr>
		<tr>
			<td>EMCCD</td>
			<td>Andor, UK&#160;</td>
			<td>DU-897U-CSO-#BV</td>
			<td>3 sets</td>
		</tr>
		<tr>
			<td>Desktop computers for controlling cameras and synchronization</td>
			<td>Dell</td>
			<td>Precision T3500</td>
			<td>PC, 4 sets</td>
		</tr>
		<tr>
			<td>coverslips with fiducial</td>
			<td>Hestzig, VA</td>
			<td>600-100AuF</td>
			<td>sample preparation. fiducial marks with various density and spectra&#160; available</td>
		</tr>
		<tr>
			<td>fibronectin&#160;</td>
			<td>Millipore, MT</td>
			<td>FC010</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Electron Microscopy Sciences, PA</td>
			<td>15710</td>
			<td>fixation. 16%</td>
		</tr>
		<tr>
			<td>glutaraldehyde&#160;</td>
			<td>Electron Microscopy Sciences, PA</td>
			<td>16220</td>
			<td>25%</td>
		</tr>
		<tr>
			<td>triton X-100</td>
			<td>Sigma aldrich, MO</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>HUVEC cells</td>
			<td>Life Technologies, CA</td>
			<td>C-015-10C</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 200</td>
			<td>Life Technologies, CA</td>
			<td>M-200-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Vessel Endothelial Factors</td>
			<td>Life Technologies, CA</td>
			<td>A14608-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td></td>
			<td>14190367</td>
			<td></td>
		</tr>
		<tr>
			<td>Pennicillin/Streptomycin</td>
			<td></td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Life Technologies, CA</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma aldrich, MO</td>
			<td>P1851</td>
			<td>PHEM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>1st base, Malaysia</td>
			<td>BIO-1825</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma aldrich, MO</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Millipore, MT</td>
			<td>5985</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Alexa Fluor 647 Phalloidin</td>
			<td>Invitrogen, CA</td>
			<td>A22287</td>
			<td>staining</td>
		</tr>
		<tr>
			<td>sodium borohydride (NaBH4)&#160;</td>
			<td>Sigma aldrich, MO</td>
			<td>480886</td>
			<td>quenching</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>1st base, Malaysia</td>
			<td>BIO-1101</td>
			<td>imaging buffer</td>
		</tr>
		<tr>
			<td>glucose oxidase</td>
			<td>Sigma aldrich, MO</td>
			<td>G2133</td>
			<td></td>
		</tr>
		<tr>
			<td>catalase</td>
			<td>Sigma aldrich, MO</td>
			<td>C9322</td>
			<td></td>
		</tr>
		<tr>
			<td>cysteamine&#160;</td>
			<td>Sigma aldrich, MO</td>
			<td>30070</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Thorlabs, NJ</td>
			<td>G14250</td>
			<td></td>
		</tr>
		<tr>
			<td>vaseline</td>
			<td>Sigma aldrich, MO</td>
			<td>16415</td>
			<td>sample sealing</td>
		</tr>
		<tr>
			<td>lanolin</td>
			<td>Sigma aldrich, MO</td>
			<td>L7387</td>
			<td></td>
		</tr>
		<tr>
			<td>parafin wax</td>
			<td>Sigma aldrich, MO</td>
			<td>327204</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil&#160;</td>
			<td>Electron Microscopy Sciences, PA</td>
			<td>16915-04</td>
			<td>imaging. Cargille Type HF</td>
		</tr>
	</tbody>
</table>

three-dimensional super resolution microscopy of f-actin filaments by interferometric photoactivated localization microscopy (ipalm)

형광 현미경은 세포 내 특정 생체 분자를 직접 시각화 할 수 있습니다. 그러나, 종래의 형광 현미경, 공간 해상도는 광축을 따라 결상면과&gt; 내지 500nm에서 ~ 200 nm의 회절에 의해 제한된다. 결과적으로, 형광 현미경은 오랫동안 심각한 세포 내 미세 기능의 관찰에 한정되었다. 초 해상 현미경 방법의 최근 발전은 이러한 한계를 극복하고있다. 특히, 광전 환성 형광체의 출현 분자 길이 스케일에 접근 해상력을 제공하는 기반 지역화 초 해상 현미경을 가능하게한다. 여기서는 간섭계 광 활성화 지역화 현미경 (iPALM)이라는 단일 분자 파악 현미경 다상 간섭에 기초하여 입체 초 해상 현미경 법의 적용을 설명한다. 이 방법은에 거의 등방성 해상도를 제공세 가지 차원에서 20 나노 미터의 순서. iPALM 악기의 시료 준비 및 운영을 포함하여 사상 액틴 세포 골격을 시각화 프로토콜, 여기에 설명되어 있습니다. 이 프로토콜은 세포에서 다른 미세 구조 기능 연구에 쉽게 적응과 교훈입니다.

Watch this Scientific Journal Video about Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM at JoVE.com

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

우리는 접착 포유류 세포에서 액틴 세포 골격의 이미징에 간섭 광 활성화 지역화 현미경 (iPALM), 3 차원 단일 분자 지역화 초 해상 현미경 법의 적용 프로토콜을 제시한다. 이 방법은 다른 기존의 회절 제한 광학 현미경으로 해결되지 않은 남아있을 것입니다 나노 구조적 특징의 빛 기반 시각화 할 수 있습니다.

입체 슈퍼의 간섭 광 활성화 현지화 현미경으로 F-액틴 필라멘트의 해상도 현미경 (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

three-dimensional-super-resolution-microscopy-f-actin-filaments

Nanyang Technological University

Research

JoVE Journal

Biology

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

10.6K 조회수.  South University of Science and Technology of China, Shenzhen.  우리는 접착 포유류 세포에서 액틴 세포 골격의 이미징에 간섭 광 활성화 지역화 현미경 (iPALM), 3 차원 단일 분자 지역화 초 해상 현미경 법의 적용 프로토콜을 제시한다. 이 방법은 다른 기존의 회절 제한 광학 현미경으로 해결되지 않은 남아있을 것입니다 나노 구조적 특징의 빛 기반 시각화 할 수 있습니다. 

비디오: Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

형광등을 통해 F - 굴지 역학 라이브 셀 이미징은 현미경을 스페클 (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

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Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated 1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. 
Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated 4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. 
Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot 8-10. 
We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.

나노 FEM : 사진 활성화 된 현지화 현미경과 전자 현미경을 사용하여 단백질 현지화

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

최적화 STORM 슈퍼 해상도 현미경에 대한 테스트 샘플

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

단일 비리온 원자력 현미경 검사및 초분해성 형광 이미징을 위한 샘플 준비

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

고속 3D 구조 조명 현미경을 사용하여 라이브 박테리아의 Cytokinetic Z 반지의 슈퍼 해상도 이미징 (F3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

공 초점 및 다중 광자 현미경을 사용하여 다섯 형광 단백질에 의해 표시됨 조혈 줄기 및 전구 세포의 생체 내 클론 추적에서

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

VirusMapper 슈퍼 해상도 현미경을위한 오픈 소스 단일 입자 분석

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

고압 냉동, 전자 레인지를 이용한 콘트라스트 향상, 그리고 볼륨 이미지에 대 한 포함 하는 최소한의 수 지에 의해 생물학 견본 준비

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...