이 작업은 R.E.D. (15SDG25560035)에 AHA에서 교부 금에 의해 지원 되었다. 저자 박사 페르난도 산타나 그의 Leica SR GSD 3D 현미경의 사용을 위해 친절 하 게 제공 FP1 항 체에 대 한 박사 요하네스 지옥 인정 하 고 싶습니다.

<ol>
	<li>Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R., 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=STED+microscopy+reveals+that+synaptotagmin+remains+clustered+after+synaptic+vesicle+exocytosis.">STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis.</a> Nature. 440 (7086), 935-939 (2006).</li><li>Gustafsson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+structured-illumination+microscopy:+wide-field+fluorescence+imaging+with+theoretically+unlimited+resolution.">Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution.</a> Proc Natl Acad Sci U S A. 102 (37), 13081-13086 (2005).</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 (5793), 1642-1645 (2006).</li><li>Hell, S. W., Kroug, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ground-state-depletion+fluorscence+microscopy:+A+concept+for+breaking+the+diffraction+resolution+limit.">Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit.</a> JAppl Phys B. 60 (5), 495-497 (1995).</li><li>Bretschneider, 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=Breaking+the+diffraction+barrier+in+fluorescence+microscopy+by+optical+shelving.">Breaking the diffraction barrier in fluorescence microscopy by optical shelving.</a> Phys Rev Lett. 98 (21), 218103 (2007).</li><li>Flynn, J. M., Santana, L. F., Melov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+transcriptional+profiling+of+adult+mouse+cardiomyocytes.">Single cell transcriptional profiling of adult mouse cardiomyocytes.</a> J Vis Exp. (58), e3302 (2011).</li><li>Davare, M. A., Horne, M. C., Hell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+phosphatase+2A+is+associated+with+class+C+L-type+calcium+channels+(Cav1.2)+and+antagonizes+channel+phosphorylation+by+cAMP-dependent+protein+kinase.">Protein phosphatase 2A is associated with class C L-type calcium channels (Cav1.2) and antagonizes channel phosphorylation by cAMP-dependent protein kinase.</a> J Biol Chem. 275 (50), 39710-39717 (2000).</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 (12), 1027-1036 (2011).</li><li>Chozinski, T. J., Gagnon, L. A., Vaughan, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twinkle,+twinkle+little+star:+photoswitchable+fluorophores+for+super-resolution+imaging.">Twinkle, twinkle little star: photoswitchable fluorophores for super-resolution imaging.</a> FEBS Lett. 588 (19), 3603-3612 (2014).</li><li>Dempsey, G. T., 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=Photoswitching+mechanism+of+cyanine+dyes.">Photoswitching mechanism of cyanine dyes.</a> J Am Chem Soc. 131 (51), 18192-18193 (2009).</li><li>Nahidiazar, L., Agronskaia, A. V., Broertjes, J., van den Broek, B., Jalink, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+Imaging+Conditions+for+Demanding+Multi-Color+Super+Resolution+Localization+Microscopy.">Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy.</a> PLoS One. 11 (7), e0158884 (2016).</li><li>Ovesny, M., Krizek, P., Borkovec, J., Svindrych, Z., Hagen, G. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+optical+reconstruction+microscopy-based+relative+localization+analysis+(STORM-RLA)+for+quantitative+nanoscale+assessment+of+spatial+protein+organization.">Stochastic optical reconstruction microscopy-based relative localization analysis (STORM-RLA) for quantitative nanoscale assessment of spatial protein organization.</a> Mol Biol Cell. 27 (22), 3583-3590 (2016).</li><li>Ries, J., Kaplan, C., Platonova, E., Eghlidi, H., Ewers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+versatile+method+for+GFP-based+super-resolution+microscopy+via+nanobodies.">A simple, versatile method for GFP-based super-resolution microscopy via nanobodies.</a> Nat Methods. 9 (6), 582-584 (2012).</li><li>Dixon, R. 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=Graded+Ca2+/calmodulin-dependent+coupling+of+voltage-gated+CaV1.2+channels.">Graded Ca2+/calmodulin-dependent coupling of voltage-gated CaV1.2 channels.</a> Elife. 4, (2015).</li><li>Annibale, P., Vanni, S., Scarselli, M., Rothlisberger, U., Radenovic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+clustering+artifacts+in+photoactivated+localization+microscopy.">Identification of clustering artifacts in photoactivated localization microscopy.</a> Nat Methods. 8 (7), 527-528 (2011).</li><li>Nan, X., 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-molecule+superresolution+imaging+allows+quantitative+analysis+of+RAF+multimer+formation+and+signaling.">Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling.</a> Proc Natl Acad Sci U S A. 110 (46), 18519-18524 (2013).</li><li>Fricke, F., Beaudouin, J., Eils, R., Heilemann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One,+two+or+three?+Probing+the+stoichiometry+of+membrane+proteins+by+single-molecule+localization+microscopy.">One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy.</a> Sci Rep. 5, 14072 (2015).</li></ol>

저자는 공개 없습니다 경쟁 관심사 있다.

회절 한계를 넘어 이미지를 허용 하는 기술의 최근 폭발 공간과 시간에서 신호 하는 포유류 세포의 복잡성에 새로운 윈도우를 제공 했다. 이러한 기술에는 폭풍, 호텔 위치, 팜, GSD, 시뮬레이션, 및 그들의 이체 (예를 들어, dSTORM, FPALM) 포함 됩니다. 이러한 기술 뒤에 과학자의 독창성 빛의 회절을 경 세 하는 물리학의 법칙에 의해 부과 된 한계를 우회 하 것을 허용 했다. 이 거 대 한 성취에도...

셀룰러 신호 반응 번역 세포질 응답을 시작 하는 내부 및 외부 환경 변화. 그들은 인간의 생리, 호르몬 및 신경 전달 물질 방출, 심장 박동, 시각, 수정, 및 인지 기능에 대 한 기초로 제공의 모든 측면을 통제 한다. 이러한 신호 폭포의 병 태 생리 조건 암, 알 츠 하이 머 병, 파 킨 슨 병, 등의 형태로 심각한 결과 가질 수 있습니다. 수십 년 동안, 생물학과 의학 조사자 이용 형광 단백질, 프로브, 바이오 센서는 형광 현미경 기본 도구로 결합 세포 이들의 정확한 공간과 일시적인 조직 이해 하 신호입니다.
Epifluorescence, confocal, 또는 총 내부 반사 형광 (TIRF) 현미경 등의 광학 기술의 힘은 그들의 감도, 속도, 및 라이브 셀 이미징, 주요 한계는 호환 그들의 회절 제한 된 해상도, 의미 구조 또는 200-250 nm 보다 작은 단백질 복합물은 확인할 수 없습니다. 결정적 슈퍼 해상도의 이론적이 고 실용적인 개발 (예를 들면, 자극된 방출 소모 현미경 (호텔 위치1), 구조화 조명 현미경 (SIM2) 또는 확률론 슈퍼 해상도 (예: , photoactivated 지역화 현미경 (팜3), 또는 바닥 상태 고갈 (GSD4,5)), 측면 및 축 해상도 형광 현미경 검사 법의 순서를 회절 방 벽을 넘어 확장 되었습니다 나노미터의 수 만입니다. 따라서, 조사자는 지금 시각화 하 고 단백질 역학과 조직 근처 분자 수준에서 기능을 변환 하는 방법을 이해 탁월한 기능이 있다.
바닥 상태 고갈 현미경 개별 분자 반환 뒤 (GSDIM), 또는 단순히 GSD로 알려져 있다 circumvents 회절 제한 fluorophores4,5를 동시에 방출의 수를 줄임으로써. 높은 에너지 레이저 빛 fluorophore 분류 샘플, 광자와 전자를 폭격 하 고 확률 그들은 ' 스핀 플립 '을 받게 되며4상태 삼중 항 또는 ' 어두운 상태 '를 입력 증가 자극 하는 데 사용 됩니다. 이 효과적으로 접지 상태를 고갈 시킨다, 그러므로 이름 '그라운드 상태 고갈'. Triplet 상태에 fluorophores 광자를 방출 하지 않는 하 고 샘플 주차 나타납니다. 그러나, 이러한 fluorophores 확률론 바닥 상태를 반환 하 고 triplet 상태에 반환 하기 전에 접지 흥분 상태 전환을 방출 하는 여러 광자를 통해 갈 수 있다. 적은 fluorophores 방출 어떤 주어진된 시간에, 함께 포 톤 버스트 fluorophores 이웃 공간 및 일시적으로 구분 되는 개별 fluorophores에서 방출. 광자의 버스트 가우스 기능, 렌즈, 사용 하는 빛의 파장의 수 가늠 구멍 (NA)에 종속 되는 지역화 정밀 fluorophore의 위치에 해당 하는 계산된 중심으로 들어갈 수 있는 여기 고 결정적인, fluorophore 당 방출 된 광자의 수. GSD에의 한 제한, fluorophores의 하위 집합만 적극적으로 언제 든 지 방출, 이후 이미지 수천을 수집 해야 합니다 완전 한 현지화 지도를 만들려고 몇 분 동안 이다. 높은 레이저 전원 요구와 함께 긴 수집 시간 GSD 라이브 샘플 보다 고정에 적합 한 더 나은 의미 합니다.
이 문서에서는 설명 합니다 막 및 바인딩과 그물 (ER)의 슈퍼 해상도 현미경 이미징에 대 한 고정된 샘플의 준비-상주 단백질 (필요한 소모품 및 시 약 참조 테이블의 재료의 목록)에 대 한. 어떻게이 프로토콜 쉽게 계량 크기와 L 유형 전압-문이 캘리포니아2 + 채널 (Cav1.2) 심장 myocytes의 sarcolemma에서의 클러스터링의 정도에 적응 하거나 수는 응급실의 세포 분포를 시각화 하는 데 사용의 예 시연 됩니다. 개시, 번역, 그리고 궁극적으로 많은 캘리포니아2 +의 기능 이해에 매우 중요 하다는 유통 및 이러한 세포 구성 성분의 이해-종속 신호 폭포. 예를 들어 Cav1.2 채널은 흥분 수축 연결, 수용 체-중재 Ca2 + 출시는 응급실에서 아마도 포유류 세포에서 가장 유비 쿼터 스 신호 폭포는 근본적으로 중요 합니다.

<table>
	<tbody>
		<tr>
			<td>KOH</td>
			<td>Thermo Scientific</td>
			<td>P250-500</td>
			<td>To clean coverglass</td>
		</tr>
		<tr>
			<td>#1.5 coverglass 18 x 18 mm</td>
			<td>Marienfeld Superior</td>
			<td>0107032)</td>
			<td>To grow/process/image cells</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Thermo Scientific</td>
			<td>BP3994</td>
			<td>Dilute to 1X with de-ionized water</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma</td>
			<td>P4832</td>
			<td>Aids with cell adhesion to cover glass</td>
		</tr>
		<tr>
			<td>laminin</td>
			<td>Sigma</td>
			<td>114956-81-9</td>
			<td>Aids with cell adhesion to cover glass</td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Thermo Scientific</td>
			<td>11150-059</td>
			<td>Ventricular myocyte culture media</td>
		</tr>
		<tr>
			<td>DMEM 11995</td>
			<td>Gibco</td>
			<td>11995</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>Fetal bovine Serum (FBS)</td>
			<td>Thermo Scientific</td>
			<td>10437028</td>
			<td>Media supplement</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td>Media supplement</td>
		</tr>
		<tr>
			<td>0.05% trypsin-EDTA</td>
			<td>Corning</td>
			<td>25-052-CL</td>
			<td>Cell culture solution</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td>Transient transfection reagent</td>
		</tr>
		<tr>
			<td>Ca2+-free PBS</td>
			<td>Gibco</td>
			<td>1419-144</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>100 % Methanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>A414-4</td>
			<td>Cell Fixation</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Cell Fixation</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma Aldrich</td>
			<td>SLBR6504V</td>
			<td>Cell Fixation</td>
		</tr>
		<tr>
			<td>SEAblock</td>
			<td>Thermo Scientific</td>
			<td>37527</td>
			<td>BSA or other blocking solution alternatives exist</td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td>Detergent to permeabilize cells</td>
		</tr>
		<tr>
			<td>Rabbit anti-CaV1.2 (FP1)</td>
			<td>Gift</td>
			<td>N/A</td>
			<td>Commercial anti-CaV1.2 antibodies exist such as Alomone Labs Rb anti-CaV1.2 (ACC-003)</td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-RFP</td>
			<td>Rockland Inc.</td>
			<td>200-301-379</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 donket anti-rabbit IgG (H+L)</td>
			<td>Invitrogen (Thermo Scientific)</td>
			<td>A31573</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 goat anti-mouse IgG (H+L)</td>
			<td>Invitrogen (Thermo Scientific)</td>
			<td>A11031</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td>Prevents microbial growth for long term storage of samples</td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma</td>
			<td>C40</td>
			<td>Photoswitching buffer ingredient</td>
		</tr>
		<tr>
			<td>Glucose oxidase</td>
			<td>Sigma</td>
			<td>G2133</td>
			<td>Photoswitching buffer ingredient</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma</td>
			<td>T6066</td>
			<td>Photoswitching buffer ingredient</td>
		</tr>
		<tr>
			<td>beta-Mercaptoethylamin hydrochloride</td>
			<td>Fisher</td>
			<td>BP2664100</td>
			<td>Photoswitching buffer ingredient</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma</td>
			<td>63689</td>
			<td>Photoswitching buffer ingredient</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>S271-3</td>
			<td>Photoswitching buffer ingredient</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher</td>
			<td>D14-212</td>
			<td>Photoswitching buffer ingredient</td>
		</tr>
		<tr>
			<td>Glass Depression slides</td>
			<td>Neolab</td>
			<td>1 &#8211; 6293</td>
			<td>To mount samples</td>
		</tr>
		<tr>
			<td>Twinsil</td>
			<td>Picodent</td>
			<td>13001000</td>
			<td>To seal coverglass</td>
		</tr>
		<tr>
			<td>sec61&#946;-mCherry plasmid</td>
			<td>Addgene</td>
			<td>49155</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SR GSD 3D microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Washing block solution</td>
			<td></td>
			<td></td>
			<td>20 % SEAblock in PBS</td>
		</tr>
		<tr>
			<td>Primary antibody incubation solution</td>
			<td></td>
			<td></td>
			<td>0.5 % Triton-X100, 20 % SEAblock, in PBS</td>
		</tr>
		<tr>
			<td>Secondary antibody incubation solution</td>
			<td></td>
			<td></td>
			<td>1:1000 in PBS</td>
		</tr>
	</tbody>
</table>

ground state depletion super-resolution imaging in mammalian cells

형광 현미경 검사 법 및 세포 생물학에서 발전은 밀접 하 게 연관는 향상 된 셀룰러 이벤트 자주 하는 방법에 대 한 우리의 이해에 극적인 도약 선도 시각화 하는 기능 세포 기능. 개발 및 슈퍼 해상도 현미경의 상당 하 게 확장 했다 광학 해상도의 한계 ~ 250-20에서 nm. 생물학은 colocalization 회절 제한 된 지역 내에서 분자 상호 작용을 설명 하는 제한 된 더 이상, 오히려 그것은 이제 개별 분자의 동적 상호 작용을 시각화 수 있습니다. 여기, 우리 바닥 상태 고갈 고정된 셀 이미징에 대 한 현미경 검사 법에 의해 시각화 및 세포 단백질의 정량화에 대 한 프로토콜을 설명합니다. 우리는 두 개의 서로 다른 막 단백질, 바인딩과 그물 translocon, sec61β, 그리고 원형질 막 지역화 전압 개폐 L 유형 캘리포니아2 + 채널 (CaV1.2)의 요소에서 예제를 제공합니다. 논의 특정 현미경 매개 변수, 고정 방법, 사진 스위칭 버퍼 정립, 그리고 함정 및 이미지 처리의 과제.

소개에 명시 된 여러 다른 슈퍼 해상도 현미경 이미징 형식 있습니다. 이 프로토콜은 GSD 슈퍼 해상도 영상에 집중 한다. 대표 이미지와 지역화 지도 그림 2 및 그림 3에 나와 있습니다.
그림 2 와 ER 단백질, mCherry-Sec61β, 위에서 설명한 메서드를 사용 하 여 처리 ?...

Watch this Scientific Journal Video about Ground State Depletion Super-resolution Imaging in Mammalian Cells at JoVE.com

Ground State Depletion Super-resolution Imaging in Mammalian Cells

이 문서는 하나 이상의 플라즈마 포유류 세포에 있는 바닥 상태 고갈 (GSD) 슈퍼 해상도 현미경을 사용 하 여 세포 막 단백질의 검출에 대 한 프로토콜을 설명. 여기, 우리 토론 혜택과 시각화 및 세포 단백질의 정량화에 대 한 이러한 접근을 사용 하 여 고려 사항.

포유류 세포에 있는 바닥 상태 고갈 슈퍼 해상도 영상

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

ground-state-depletion-super-resolution-imaging-in-mammalian-cells

Research

JoVE Journal

Biology

7.1K 조회수.  University of California School of Medicine, Davis. 이 문서는 하나 이상의 플라즈마 포유류 세포에 있는 바닥 상태 고갈 (GSD) 슈퍼 해상도 현미경을 사용 하 여 세포 막 단백질의 검출에 대 한 프로토콜을 설명. 여기, 우리 토론 혜택과 시각화 및 세포 단백질의 정량화에 대 한 이러한 접근을 사용 하 여 고려 사항.

비디오: Ground State Depletion Super-resolution Imaging in Mammalian Cells

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

와이드 필드 현미경을 사용하여 장기 이미징 포유 동물 세포

연구

생물학

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

세균 본부 기계의 슈퍼 해상도 이미징

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

모기 내장 Metagenomic RNA-seq에 대한 Ribosomal RNA의 고갈

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

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 ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

이미징 로컬 CA<sup&gt; 2 +</sup&gt; 배양 된 포유 동물 세포에서 신호

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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 ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

동적 세포 돌출부에서 소프트웨어를 이용한 단백질 농도 추적을위한 그래픽 사용자 인터페이스

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

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 ...