Name: ground state depletion super-resolution imaging in mammalian cells
Uploaded: 2017-11-05T08:30:00
Description: Watch this Scientific Journal Video about Ground State Depletion Super-resolution Imaging in Mammalian Cells at JoVE.com

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.

1. 세척 유리 Coverslips <ol><li>샘플 처리 하기 전에 하루 1 시간에 대 한 코의 1 M 솔루션에서 sonicating 여 #1.5 유리 coverslips를 청소. 이것은 coverslips에 있을 수 있는 어떤 형광 오염 물질을 제거 합니다.
	<ol><li>드 이온된 수와 coverslips에서 코를 철저 하 게 린스.</li>
		<li>일단 코에 청소, 70% 에탄올 사용 하 여 준비까지 있는 coverslips 저장.</li>
	</ol></li></ol> 2. 코팅 유리 Coversli...

<ol>
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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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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>

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 ...

The overall goal of this fluorescent imaging technique is to visualize and quantify cellular proteins in fixed cells at subdiffraction-limited resolution using a form of super-resolution microscopy called ground state depletion microscopy and individual molecule return. This method helps answer key questions in the field of cell biology and membrane biophysics, as it circumvents diffraction-limited resolution, which helps investigators more precisely define, visualize and quantify the distribution of cellular proteins. The main advantage of this protocol is that it enables the acquisition of images with an approximate ten-fold improvement in resolution over conventional fluorescence microscopy imaging approaches, such as confocal microscopy.Demonstrating the procedure will be Oscar Vivas, a post-doc in my laboratory, and Karen Hannigan, a post-doc in Rose Dixon's laboratory. To begin, prepare the cover slips and cells as described in the accompanying text protocol. Then fix the samples and perform immunocytochemistry in order to label the plasma membrane and/or intracellular membrane proteins of interest.Next, prepare the oxygen scavenging Glock solution by first adding 12.5 microliters of a catalase stock solution, 3.5 milligrams of glucose oxidase, and 0.5 microliters of one molar Tris to 49.5 microliters of PBS in a 1.5 milliliter microcentrifuge tube. Close the tube and briefly vortex it to dissolve the components. Then centrifuge the solution for three minutes.Now, prepare the photo switching inducing thial solution by dissolving beta-mercaptoethylamine solution into PBS to a concentration of 100 millimolar and modify the pH to 8.0. Aliquot and store the thial solution in the freezer for up to one week. Immediately before imaging, mix the thial and oxygen scavenging components together with a saline buffer and store the solution on ice.Using 100 microliters of the prepared photo switching buffer, mount cover slips containing stained cells onto the depression slides. Next, seal the edges of the cover slip with a silicone glue to prevent rapid oxidation of the imaging buffer. Wait several minutes until the silicone glue has fully cured, prior to imaging.Otherwise, the cover slip will drift. To begin, select an appropriate laser to illuminate the sample, based on the chosen fluorophore. For an Alexa 647 fluorophore, use a 642 nanometer laser.Next, chose the appropriate dichroic imaging cube and objective lens. Open the imaging software. Select the 2D acquisition mode then set the exposure time to 11 milliseconds.Also, set the EM Gain to 300 and select an appropriate detection threshold. Next, turn on Auto Event Control and set the number of Events Per Images to eight. Enter the Pixel Size as 20 nanometers.In the GSD Tools tab, go to the High Resolution Image Calculation options and ensure that Histogram Mode is selected. To image proteins at the plasma membrane, select TIRF mode and modify the incidence angle to determine the penetration depth. Then, set the laser intensity to 100%in order to send electrons to the dark state.Next, select Single Molecule Detection while pumping and set to acquire automatically when the frame correlation is 0.20. For acquisition, set laser intensity to 50%and the number of acquisition frames to 60, 000. Then begin taking images.To quantify protein cluster size, open the image file of a 10 nanometer histogram single molecule localization map in ImageJ. In the Image menu, go to Adjust and use the Auto option to optimize the brightness and contrast of the image. Then, under the Type menu, choose 8-bit.Next, open the Analyze menu, click Set Scale and enter the values shown here. Then, in the Analyze menu, select Set Measurements and check the box beside the Area option. Back in the Image menu, select Adjust, Threshold, and then select Over/Under.Move the maximum value to 255, the minimum value to one and click Apply. Finally, open the Analyze menu and select Analyze Particles. Place a check mark to select Pixel units, Display results and Summarize.Then set the size to four to infinity, select the Bare Outlines option and click OK.The cell imaged here was transfected with the endoplasmic reticulum protein, mCherry-Sec61 beta, and imaged using both defraction-limited TIRF microscopy and super-resolution GSD microscopy in TIRF mode. These curves represent the diameter of the ER tubules. The image on the right shows an improvement in the lateral resolution and represents a more accurate representation of the structure of ER tubules.The improvement in resolution offered by super-resolution GSD imaging is further demonstrated in these images, showing a labeled cardiac myocyte with an anti-CAV 1.2 antibody. Using GSD mode, the improvement in the lateral resolution is prominent and separate clusters of channels are easier to identify. Once mastered, this protocol can be completed in less than three days.The super-resolution imaging of a single cell takes 10 to 30 minutes, depending on the number of frames required. Following this procedure, other methods like electromicroscopy can be performed in order to answer addition questions like how the fluorescent protein of interest interacts with the complex cellular environment. After watching this video, you should have a good understanding of how to prepare, acquire and analyze samples to visualize one or more cellular proteins for super-resolution microscopy using ground state depletion followed by individual molecule return.

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

Research

JoVE Journal

Biology

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

와이드 필드 현미경을 사용하여 장기 이미징 포유 동물 세포

연구

생물학

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

포유 동물 세포에서 RNAi 심사를위한 선교 esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

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 ...

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 ...