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>
	<li>Kanchanawong, P., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization-based+super-resolution+imaging+of+cellular+structures.">Localization-based super-resolution imaging of cellular structures.</a> Methods Mol Biol. 1046, 59-84 (2013).</li><li>Bertocchi, C., Goh, W. I., Zhang, Z., Kanchanawong, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+imaging+by+super+resolution+fluorescence+microscopy+and+its+emerging+applications+in+biomedical+research.">Nanoscale imaging by super resolution fluorescence microscopy and its emerging applications in biomedical research.</a> Crit Rev Biomed Eng. 41, 281-308 (2013).</li><li>Kanchanawong, P., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+light-based+imaging+of+three-dimensional+cellular+ultrastructure.">Advances in light-based imaging of three-dimensional cellular ultrastructure.</a> Curr Opin Cell Biol. 24, 125-133 (2012).</li><li>Betzig, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313, 1642-1645 (2006).</li><li>Hess, S. T., Girirajan, T. P., Mason, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high+resolution+imaging+by+fluorescence+photoactivation+localization+microscopy.">Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.</a> Biophys J. 91, 4258-4272 (2006).</li><li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nat Methods. 3, 793-795 (2006).</li><li>Heilemann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subdiffraction-resolution+fluorescence+imaging+with+conventional+fluorescent+probes.">Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes.</a> Angew Chem Int Edit. 47, 6172-6176 (2008).</li><li>Sharonov, A., Hochstrasser, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wide-field+subdiffraction+imaging+by+accumulated+binding+of+diffusing+probes.">Wide-field subdiffraction imaging by accumulated binding of diffusing probes.</a> P Natl Acad Sci USA. 103, 18911-18916 (2006).</li><li>F&#246;lling, J., Bossi, M., Bock, H., Medda, R., Wurm, C. A., Hein, B., Jakobs, S., Eggeling, C., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+nanoscopy+by+ground-state+depletion+and+single-molecule+return.">Fluorescence nanoscopy by ground-state depletion and single-molecule return.</a> Nat Methods. 5, 943-945 (2008).</li><li>Biteen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-moldecule+active-control+microscopy+(SMACM)+with+photo-reactivable+EYFP+for+imaging+biophysical+processes+in+live+cells.">Single-moldecule active-control microscopy (SMACM) with photo-reactivable EYFP for imaging biophysical processes in live cells.</a> Nat Methods. 5, 947-949 (2008).</li><li>Shtengel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferometric+fluorescent+super-resolution+microscopy+resolves+3D+cellular+ultrastructure.">Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure.</a> P Natl Acad Sci USA. 106, 3125-3130 (2009).</li><li>Huang, B., Wang, W., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+super-resolution+imaging+by+stochastic+optical+reconstruction+microscopy.">Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.</a> Science. 319, 810-813 (2008).</li><li>Dani, A., Huang, B., Bergan, J., Dulac, C., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super+resolution+imaging+of+chemical+synapses+in+the+brain.">Super resolution imaging of chemical synapses in the brain.</a> Neuron. 68, 843-856 (2010).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Talin+determines+the+nanoscale+architecture+of+focal+adhesions.">Talin determines the nanoscale architecture of focal adhesions.</a> P Natl Acad Sci USA. 112 (35), E4864-E4873 (2015).</li><li>Kanchanawong, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+architecture+of+integrin-based+cell+adhesions.">Nanoscale architecture of integrin-based cell adhesions.</a> Nature. 468, 580-584 (2010).</li><li>Wu, Y., Kanchanawong, P., Zaidel-Bar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin-delimited+adhesion-independent+clustering+of+e-cadherin+forms+the+nanoscale+building+blocks+of+adherens+junctions.">Actin-delimited adhesion-independent clustering of e-cadherin forms the nanoscale building blocks of adherens junctions.</a> Dev Cell. 32 (2), 139-154 (2015).</li><li>Szymborska, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+Pore+Scaffold+Structure+Analyzed+by+Super-Resolution+Microscopy+and+Particle+Averaging.">Nuclear Pore Scaffold Structure Analyzed by Super-Resolution Microscopy and Particle Averaging.</a> Science. 341, 655-658 (2013).</li><li>Lawo, S., Hasegan, M., Gupta, G. D., Pelletier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subdiffraction+imaging+of+centrosomes+reveals+higher-order+organizational+features+of+pericentriolar+material.">Subdiffraction imaging of centrosomes reveals higher-order organizational features of pericentriolar material.</a> Nat Cell Biol. 14, 1148-1158 (2012).</li><li>Mennella, V., Agard, D. A., Huang, B., Pelletier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amorphous+no+more:+subdiffraction+view+of+the+pericentriolar+material+architecture.">Amorphous no more: subdiffraction view of the pericentriolar material architecture.</a> Trends Cell Biol. 24, 188-197 (2014).</li><li>Mennella, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subdiffraction-resolution+fluorescence+microscopy+reveals+a+domain+of+the+centrosome+critical+for+pericentriolar+material+organization.">Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization.</a> Nat Cell Biology. 14, 1159-1168 (2012).</li><li>Fletcher, D. A., Mullins, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+mechanics+and+the+cytoskeleton.">Cell mechanics and the cytoskeleton.</a> Nature. 463, 485-492 (2010).</li><li>Salbreux, G., Charras, G., Paluch, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+cortex+mechanics+and+cellular+morphogenesis.">Actin cortex mechanics and cellular morphogenesis.</a> Trends Cell Biol. 22, 536-545 (2012).</li><li>Shtengel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cellular+ultrastructure+by+PALM,+iPALM,+and+correlative+iPALM-EM.">Imaging cellular ultrastructure by PALM, iPALM, and correlative iPALM-EM.</a> Method Cell Biol. 123, 273-294 (2014).</li><li>Juette, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+sub-100+nm+resolution+fluorescence+microscopy+of+thick+samples.">Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples.</a> Nat Methods. 5, 527-529 (2008).</li><li>Lippincott-Schwartz, J., Patterson, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+fluorescent+proteins+for+diffraction-limited+and+super-resolution+imaging.">Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging.</a> Trends Cell Biol. 19, 555-565 (2009).</li><li>Shannon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Communication+in+the+presence+of+noise.">Communication in the presence of noise.</a> Proc. IRE. 37, 10-21 (1949).</li><li>Good, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+ion+buffers+for+biological+research.">Hydrogen ion buffers for biological research.</a> Biochemistry. 5, 467-477 (1966).</li><li>Aitken, C. E., Marshall, R. A., Puglisi, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+oxygen+scavenging+system+for+improvement+of+dye+stability+in+single-molecule+fluorescence+experiments.">An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments.</a> Biophys J. 94, 1826-1835 (2008).</li><li>Shin, W. D., Goldman, R. D., Swedlow, J. R., Spector, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Live Cell Imaging: A Laboratory Manual. , (2010).</li><li>Aquino, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-color+nanoscopy+of+three-dimensional+volumes+by+4Pi+detection+of+stochastically+switched+fluorophores.">Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores.</a> Nat Methods. 8, 353-359 (2011).</li><li>Dempsey, G. T., Vaughan, J. C., Chen, K. H., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+fluorophores+for+optimal+performance+in+localization-based+super-resolution+imaging.">Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging.</a> Nat Methods. 8, 1027-1036 (2011).</li><li>Pertsinidis, A., Zhang, Y., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subnanometre+single-molecule+localization,+registration+and+distance+measurements.">Subnanometre single-molecule localization, registration and distance measurements.</a> Nature. 466, 647-651 (2010).</li><li>Baddeley, D., Cannell, M. B., Soeller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Localization+Microscopy+Data.">Visualization of Localization Microscopy Data.</a> Microsc Microanal. 16, 64-72 (2010).</li><li>El Beheiry, M., Dahan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ViSP:+representing+single-particle+localizations+in+three+dimensions.">ViSP: representing single-particle localizations in three dimensions.</a> Nat Methods. 10, 689-690 (2013).</li><li>Schnell, U., Dijk, F., Sjollema, K. A., Giepmans, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolabeling+artifacts+and+the+need+for+live-cell+imaging.">Immunolabeling artifacts and the need for live-cell imaging.</a> Nat Methods. 9, 152-158 (2012).</li><li>Shroff, H., Galbraith, C. G., Galbraith, J. A., Betzig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+photoactivated+localization+microscopy+of+nanoscale+adhesion+dynamics.">Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics.</a> Nat Methods. 5, 417-423 (2008).</li><li>Yamashiro, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+single-molecule+speckle+microscopy+reveals+modification+of+the+retrograde+actin+flow+by+focal+adhesions+at+nanometer+scales.">New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales.</a> Mol Biol Cell. 25, 1010-1024 (2014).</li><li>Shroff, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-color+super+resolution+imaging+of+genetically+expressed+probes+within+individual+adhesion+complexes.">Dual-color super resolution imaging of genetically expressed probes within individual adhesion complexes.</a> P Natl Acad Sci USA. 104, 20308-20313 (2007).</li><li>Chen, B. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lattice+light-sheet+microscopy:+imaging+molecules+to+embryos+at+high+spatiotemporal+resolution.">Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.</a> Science. 346, (2014).</li><li>Brown, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super+resolution+fluorescence+imaging+of+mitochondrial+nucleoids+reveals+their+spatial+range,+limits,+and+membrane+interaction.">Super resolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction.</a> Mol Cell Biol. 31, 4994-5010 (2011).</li><li>Huang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video-rate+nanoscopy+using+sCMOS+camera-specific+single-molecule+localization+algorithms.">Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms.</a> Nat Methods. 10, 653-658 (2013).</li><li>Daostorm, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAOSTORM:+an+algorithm+for+high-density+super-resolution+microscopy.">DAOSTORM: an algorithm for high-density super-resolution microscopy.</a> Nat methods. 8, 279 (2011).</li><li>Zhu, L., Zhang, W., Elnatan, D., Huang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Faster+STORM+using+compressed+sensing.">Faster STORM using compressed sensing.</a> Nat Methods. 9, 721-723 (2012).</li><li>Van Engelenburg, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+ESCRT+machinery+at+HIV+assembly+sites+reveals+virus+scaffolding+of+ESCRT+subunits.">Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits.</a> Science. 343, 653-656 (2014).</li><li>Vaughan, J. C., Jia, S., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrabright+photoactivatable+fluorophores+created+by+reductive+caging.">Ultrabright photoactivatable fluorophores created by reductive caging.</a> Nat Methods. 9, 1181-1184 (2012).</li></ol>

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. ?...

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.

iPALM 중요 요건은 광학 시스템의 정렬, 등록 및 교정된다. 추출 Z 좌표에 대한 이들 3 방향 빔 스플리터 내에서 필요한 적절한 간섭을 보장하기 위해 필요하다. 지속적인 모니터링을 사용하려면, 형광의 일정 지점 소스가 필요합니다. 이것은 형광 금 또는 그 포토 루미 국소 표면 플라즈몬 공명 (LSPR)에서 발생하는 이중 금속 나노 입자 (23)를 사용하여 달성 될 수?...

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

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 ...

연구

생물학

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 ...