Name: conducting multiple imaging modes with one fluorescence microscope
Uploaded: 2018-10-28T13:00:00
Description: Watch this Scientific Journal Video about Conducting Multiple Imaging Modes with One Fluorescence Microscope at JoVE.com

樱承认塞尔学者计划和 NIH 主任的新创新奖的支持。作者承认保罗 Selvin 的实验室 (伊利诺伊大学香槟分校) 为定位3维透镜提供了有用的建议。

1. 显微镜设计和组装
<ol><li>
激励路径 注: 激励路径包括激光器、差分干涉对比度 (DIC) 分量、显微镜本体及其照明臂。<ol>
<li>准备一个隔振的光学表。例如, 结构阻尼表为 48 x 96 x 12 "为所有组件提供足够的空间。 注: 在具有温度控制的房间内建立设置 (例如, 21.4 ±0.55 摄氏度)。温度稳定性对于保持光学对准至关重要。</li>
		<li>安装一个显微镜机身, 配有用于光纤连接的照明?...

<ol>
	<li>Lipson, S. G., Lipson, H., Tannhauser, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optical physics. , (1995).</li><li>T&#246;r&#246;k, P., Wilson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rigorous+theory+for+axial+resolution+in+confocal+microscopes.">Rigorous theory for axial resolution in confocal microscopes.</a> Optics Communications. 137 (1-3), 127-135 (1997).</li><li>Klar, T. A., 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=Subdiffraction+resolution+in+far-field+fluorescence+microscopy.">Subdiffraction resolution in far-field fluorescence microscopy.</a> Optics Letters. 24 (14), 954-956 (1999).</li><li>Hell, S. W., Wichmann, J. <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+resolution+limit+by+stimulated+emission:+stimulated-emission-depletion+fluorescence+microscopy.">Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy.</a> Optics Letters. 19 (11), 780-782 (1994).</li><li>Gustafsson, M. G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surpassing+the+lateral+resolution+limit+by+a+factor+of+two+using+structured+illumination+microscopy.">Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy.</a> Journal of Microscopy. 198 (2), 82-87 (2000).</li><li>Gustafsson, M. G. 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=Three-dimensional+resolution+doubling+in+wide-field+fluorescence+microscopy+by+structured+illumination.">Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination.</a> Biophysical Journal. 94 (12), 4957-4970 (2008).</li><li>Schermelleh, 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=Subdiffraction+multicolor+imaging+of+the+nuclear+periphery+with+3D+structured+illumination+microscopy.">Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy.</a> Science. 320 (5881), 1332-1336 (2008).</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> Nature Methods. 3 (10), 793-795 (2006).</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>Balzarotti, 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=Nanometer+resolution+imaging+and+tracking+of+fluorescent+molecules+with+minimal+photon+fluxes.">Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes.</a> Science. 355 (6325), 606-612 (2017).</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> Nature Methods. 9 (12), 1181-1184 (2012).</li><li>Ha, 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=Probing+the+interaction+between+two+single+molecules:+fluorescence+resonance+energy+transfer+between+a+single+donor+and+a+single+acceptor.">Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (13), 6264-6268 (1996).</li><li>Roy, R., Hohng, S., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+single-molecule+FRET.">A practical guide to single-molecule FRET.</a> Nature Methods. 5 (6), 507-516 (2008).</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 (5864), 810-813 (2008).</li><li>Hua, 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=An+improved+surface+passivation+method+for+single-molecule+studies.">An improved surface passivation method for single-molecule studies.</a> Nature Methods. 11 (12), 1233-1236 (2014).</li><li>Fei, 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=RNA+biochemistry.+Determination+of+in+vivo+target+search+kinetics+of+regulatory+noncoding+RNA.">RNA biochemistry. Determination of in vivo target search kinetics of regulatory noncoding RNA.</a> Science. 347 (6228), 1371-1374 (2015).</li><li>Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+individual+mRNA+molecules+using+multiple+singly+labeled+probes.">Imaging individual mRNA molecules using multiple singly labeled probes.</a> Nature Methods. 5 (10), 877-879 (2008).</li><li>Stracy, M., Kapanidis, A. N. <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+and+super-resolution+imaging+of+transcription+in+living+bacteria.">Single-molecule and super-resolution imaging of transcription in living bacteria.</a> Methods. 120, 103-114 (2017).</li><li>Wang, S., Moffitt, J. R., Dempsey, G. T., Xie, X. 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=Characterization+and+development+of+photoactivatable+fluorescent+proteins+for+single-molecule-based+superresolution+imaging.">Characterization and development of photoactivatable fluorescent proteins for single-molecule-based superresolution imaging.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (23), 8452-8457 (2014).</li><li>Sekar, R. B., Periasamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+resonance+energy+transfer+(FRET)+microscopy+imaging+of+live+cell+protein+localizations.">Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations.</a> The Journal of Cell Biology. 160 (5), 629-633 (2003).</li><li>Shi, 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=Super-resolution+microscopy+reveals+that+disruption+of+ciliary+transition-zone+architecture+causes+Joubert+syndrome.">Super-resolution microscopy reveals that disruption of ciliary transition-zone architecture causes Joubert syndrome.</a> Nature Cell Biology. 19 (10), 1178-1188 (2017).</li><li>Youn, Y., Ishitsuka, Y., Jin, C., Selvin, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+nanoimprint+lithography+for+drift+correction+in+super-resolution+fluorescence+microscopy.">Thermal nanoimprint lithography for drift correction in super-resolution fluorescence microscopy.</a> Optics Express. 26 (2), 1670-1680 (2018).</li></ol>

这种混合显微镜系统无需购买多台显微镜。所有零件的总成本, 包括光学表、表安装人工、软件和工作站, 约为23万美元。定制加工部件, 包括 mag 透镜和3维透镜, 成本约为700美元 (成本取决于不同机构的实际费用)。用于基于单分子检测的 SR 显微显微镜的典型商业可用集成系统的成本超过30万美元 ~ 40万, 且不容易 smFRET 测量, 或在不使用白光的情况下为合理的视野提供 epi 成像。源。因此, 此处介绍的...

荧光显微镜是现代生物科学研究的重要工具, 在许多生物实验室中都经常进行荧光成像。通过用荧光基团标记感兴趣的生物分子, 我们可以在显微镜下直接可视化它们, 并记录在体内或体外的定位、构象、相互作用和装配状态的时间依赖性变化。传统的荧光显微镜具有衍射有限的空间分辨率, 在横向方向上约为 200-300 nm, 在轴向1、2中为 500-700 nm, 因此仅限于100s 的成像。纳米到微米级。为了揭示分子组装或组织中更精细的细节, 开发了各种能打破衍射极限的 SR microscopies。用于实现 SR 的策略包括非线性光学效应, 如受激发射损耗 (STED) 显微镜3、4和结构化照明显微镜 (SIM)5、6、 7、随机检测单个分子, 如随机光学重建显微术 (风暴)8和光活化定位显微镜 (PALM)9, 以及两者的组合, 如 MINFLUX10。在这些 sr microscopies 中, 基于单分子检测的 sr 显微镜可以相对容易地从单分子显微镜设置中进行修改。通过重复激活和成像光敏荧光蛋白 (FPs) 或在感兴趣的生物分子标记的光交换染料, 空间分辨率可以达到 10-20 nm11。要获得实时的分子相互作用和构象动力学信息, 需要埃到纳米分辨率。smFRET12,13是实现这一决议的一种方法。通常, 根据感兴趣的生物学问题, 需要不同空间分辨率的成像方法。
通常, 对于每种类型的成像, 都需要特定的激励和/或发射光学配置。例如, 单分子检测最常用的照明方法之一是通过全内部反射 (TIR), 在这种情况下, 需要通过棱镜或通过物镜实现特定的激励角度。对于 smFRET 检测, 供体和受体染料的排放需要在空间上分离, 并定向到电子乘法、电荷耦合器件 (EMCCD) 的不同部分, 这可以通过一组反射镜和双色光束分配器实现。放置在排放路径中。对于三维 (3-D) SR 成像, 需要在发射路径中产生散光效应的光学元件, 如圆柱透镜14。因此, 自制或商业上可用的集成显微镜通常在功能上专门用于每种类型的成像方法, 并且在相同的设置下不灵活地在不同的成像方法之间切换。在这里, 我们提出了一个经济高效的混合系统, 提供三种不同成像方法之间的可调节和重复的开关: 传统的 epi-荧光成像, 衍射有限分辨率, 单分子检测 SR。成像和多色单分子检测, 包括 smFRET 成像 (图 1A)。具体而言, 此处提供的设置包含多色激励的光纤耦合输入激光器和激励路径中的商业照明臂, 允许对激励角度进行编程控制, 以便在 epi 模式和 TIR 模式之间切换。在发射路径中, 可拆卸的自制圆柱透镜盒放置在显微镜机身内, 用于3维 SR 成像, 在 EMCCD 摄像机前放置一个商用光束分配器, 可选择性地启用检测多个发射通道同时。

<table>
	<tbody>
		<tr>
			<td>Nikon Ti-E microscope stand</td>
			<td>Nikon</td>
			<td>Ti-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Nikon</td>
			<td>100X NA 1.49 CFI HP TIRF</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopy imaging software</td>
			<td>Nikon</td>
			<td>NIS-Elements Advanced Research/HC</td>
			<td>HC includes &#34;JOBS&#34; module, the programmed acquisition module being used for SR imaging.</td>
		</tr>
		<tr>
			<td>The illumination arm</td>
			<td>Nikon</td>
			<td>Ti-TIRF-EM Motorized Illuminator Unit M</td>
			<td>This arm has a slot for a magnification lens</td>
		</tr>
		<tr>
			<td>Analyze block</td>
			<td>Nikon</td>
			<td>Ti-A</td>
			<td>This is installed in the filter turret.</td>
		</tr>
		<tr>
			<td>Z-drift correction system</td>
			<td>Nikon</td>
			<td>PFS</td>
			<td>This system is composed by the stepmotor on the objective nosepiece, IR LED, and a detector.</td>
		</tr>
		<tr>
			<td>Optical table top</td>
			<td>TMC</td>
			<td>783-655-02R</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical table bases</td>
			<td>TMC</td>
			<td>14-426-35</td>
			<td></td>
		</tr>
		<tr>
			<td>647 nm laser</td>
			<td>Cobolt</td>
			<td>90346 (0647-06-01-0120-100)</td>
			<td>Modulated Laser Diode 647nm 120mW incl. laser head, CDRH control box, USB cable and PSU (Power Supply Unit)</td>
		</tr>
		<tr>
			<td>561 nm laser</td>
			<td>Coherent</td>
			<td>1280721</td>
			<td>OBIS 561nm LS 150mW Laser System</td>
		</tr>
		<tr>
			<td>488 nm laser</td>
			<td>Cobolt</td>
			<td>90308 (0488-06-01-0060-100)</td>
			<td>Modulated Laser Diode 488nm 60mW incl. laser head, CDRH control box, USB cable and PSU (Power Supply Unit)</td>
		</tr>
		<tr>
			<td>405 nm laser</td>
			<td>Crystalaser</td>
			<td>DL405-025-O</td>
			<td>405 (+/-5)nm, 25mW, Circular , M2 &#60;1.3, Low Noise, CW, TTL up to 20MHz. 2 BNC connectors for TTL &#38; Analog adjust</td>
		</tr>
		<tr>
			<td>Heat sink</td>
			<td>Cobolt</td>
			<td>11658 (HS-03)</td>
			<td>Two units, Heat sink without fan HS-03, Heat sink for 647 nm and 488 nm lasers</td>
		</tr>
		<tr>
			<td>Heat sink</td>
			<td>Coherent</td>
			<td>1193289</td>
			<td>Obis heat sink with fan, 165 x 50 x 50 mm for the 561 nm laser</td>
		</tr>
		<tr>
			<td>CAB-USB-miniUSB</td>
			<td>Cobolt</td>
			<td>10908</td>
			<td>Two units, communication cable for 647 nm and 488 nm lasers</td>
		</tr>
		<tr>
			<td>aluminum for height adjustment</td>
			<td>McMaster-Carr</td>
			<td>9146T35</td>
			<td>Multipurpose 6061 Aluminum, Rectangular Bar, 4MM X 40MM, 1&#39; Long for raising 561 nm laser</td>
		</tr>
		<tr>
			<td>aluminum for height adjustment</td>
			<td>McMaster-Carr</td>
			<td>8975K248</td>
			<td>Multipurpose 6061 Aluminum, 1-1/4&#34; Thick X 3&#34; Width X 1&#39; Length for raising 405 nm laser</td>
		</tr>
		<tr>
			<td>BNC cable</td>
			<td>L-com</td>
			<td>CC58C-6</td>
			<td>RG58C Coaxial Cable, BNC Male / Male, 6.0 ft</td>
		</tr>
		<tr>
			<td>BNC adapter</td>
			<td>L-com</td>
			<td>BA1087</td>
			<td>Coaxial Adapter, BNC Bulkhead, Grounded</td>
		</tr>
		<tr>
			<td>SMA to BNC Adapter</td>
			<td>HOD</td>
			<td>SMA-870</td>
			<td>Cobolt MLD lasers have SMA interface, so this adapter is used for BNC connection.</td>
		</tr>
		<tr>
			<td>SMB to BNC Adapter</td>
			<td>Fairview Microwave</td>
			<td>FMC1638316-12</td>
			<td>SMB Plug to BNC Female Bulkhead Cable RG316 Coax in 12 Inch for Coherent Obis lasers</td>
		</tr>
		<tr>
			<td>Data Acquisition Card</td>
			<td>National Instruments</td>
			<td>PCI-6723</td>
			<td>13-Bit, 32 Channels, 800 kS/s Analog Output Device for controlling lasers, DIC LED, and etc</td>
		</tr>
		<tr>
			<td>Barrier Filter Wheel controller</td>
			<td>Sutter Instrument</td>
			<td>Lambda 10-B</td>
			<td>Optical Filter Changer</td>
		</tr>
		<tr>
			<td>Emission Splitter</td>
			<td>Cairn</td>
			<td>OptoSplit III</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichroic beamsplitter</td>
			<td>Chroma</td>
			<td>T640LPXR-UF2</td>
			<td>Dichroic beamsplitter separating red emission from green emission in OptoSplit III</td>
		</tr>
		<tr>
			<td>Dichroic beamsplitter</td>
			<td>Chroma</td>
			<td>T565LPXR-UF2</td>
			<td>Dichroic beamsplitter separating green &#38; red emission from blue emission in OptoSplit III</td>
		</tr>
		<tr>
			<td>Emission filter</td>
			<td>Chroma</td>
			<td>ET700/75M</td>
			<td>Two units, Emission filter for red emission (like Alexa Fluor 647) in OptoSplit III as well as in the Barrier filter wheel</td>
		</tr>
		<tr>
			<td>Emission filter</td>
			<td>Chroma</td>
			<td>ET595/50M</td>
			<td>Two units, Emission filter for yellow/green emission (like Cy3B) in OptoSplit III as well as in the Barrier filter wheel</td>
		</tr>
		<tr>
			<td>Emission filter</td>
			<td>Chroma</td>
			<td>ET525/50M</td>
			<td>Two units, Emission filter for blue emission(like Alexa Fluor 488/GFP) in OptoSplit III as well as in the Barrier filter wheel</td>
		</tr>
		<tr>
			<td>Emission filter</td>
			<td>Semrock</td>
			<td>FF02-447/60-25</td>
			<td>Emission filter for violet emission (like DAPI/Alexa Fluor 405), installed in the Barrier filter wheel</td>
		</tr>
		<tr>
			<td>Dichroic beamsplitter</td>
			<td>Chroma</td>
			<td>zt405/488/561/647/752rpc-UF3</td>
			<td>Multiband dichroic beam splitter for 647, 561, 488, and 405 nm laser excitations inside of the microscope body</td>
		</tr>
		<tr>
			<td>DAPI Filter set</td>
			<td>Chroma</td>
			<td>49000</td>
			<td>installed in the microscope body</td>
		</tr>
		<tr>
			<td>Nikon laser/TIRF filtercube</td>
			<td>Chroma</td>
			<td>91032</td>
			<td></td>
		</tr>
		<tr>
			<td>590 long pass filter</td>
			<td>Chroma</td>
			<td>T590LPXR-UF1</td>
			<td>for combining 647 nm laser and 561 nm laser</td>
		</tr>
		<tr>
			<td>525 long pass filter</td>
			<td>Chroma</td>
			<td>T525LPXR-UF1</td>
			<td>for combining already combined 647 nm and 561nm lasers with 488 nm laser</td>
		</tr>
		<tr>
			<td>470 long pass filter</td>
			<td>Chroma</td>
			<td>T470LPXR-UF1</td>
			<td>for combining already combined 647 nm, 561 nm and 488 nm lasers with 405 nm laser</td>
		</tr>
		<tr>
			<td>Laser clean-up filter (647)</td>
			<td>Chroma</td>
			<td>zet640/20x</td>
			<td>for cleaning up other wavelengths from the 647 nm laser</td>
		</tr>
		<tr>
			<td>Laser clean up filter (488)</td>
			<td>Semrock</td>
			<td>LL01-488-25</td>
			<td>for cleaning up other wavelengths from the 488 nm laser</td>
		</tr>
		<tr>
			<td>LED light source</td>
			<td>Excelitas</td>
			<td>X-Cite120LED</td>
			<td>used only for DAPI imaging</td>
		</tr>
		<tr>
			<td>Mirror mount</td>
			<td>Newport</td>
			<td>SU100-F3K</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical posts</td>
			<td>Newport</td>
			<td>PS-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamping fork</td>
			<td>Newport</td>
			<td>PS-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Power Meter</td>
			<td>Newport</td>
			<td>PMKIT</td>
			<td>For measuring laser power</td>
		</tr>
		<tr>
			<td>Dichroic beamcombiner mount</td>
			<td>Edmund Optics</td>
			<td>58-872</td>
			<td>C-Mount Kinematic Mount, for holding dichroic beamcombiners in the laser excitation assembly</td>
		</tr>
		<tr>
			<td>Retaining ring</td>
			<td>Thorlabs</td>
			<td>CMRR</td>
			<td>used for dichroic beamcombiner mounts</td>
		</tr>
		<tr>
			<td>Fiber Adapter Plate</td>
			<td>Thorlabs</td>
			<td>SM1FC</td>
			<td>FC/PC Fiber Adapter Plate with External SM1 (1.035&#34;-40) Thread</td>
		</tr>
		<tr>
			<td>Z-axis translational mount</td>
			<td>Thorlabs</td>
			<td>SM1Z</td>
			<td>Z-Axis Translation Mount, 30 mm Cage Compatible</td>
		</tr>
		<tr>
			<td>Achromatic Doublet lens</td>
			<td>Thorlabs</td>
			<td>AC050-008-A-ML</td>
			<td>&#216;5 mm, Mounted Achromatic Doublets, AR Coated: 400 - 700 nm</td>
		</tr>
		<tr>
			<td>Cage Plate</td>
			<td>Thorlabs</td>
			<td>CP1TM09</td>
			<td>30 mm Cage Plate with M9 x 0.5 Internal Threads, 8-32 Tap</td>
		</tr>
		<tr>
			<td>Cage Assembly Rod</td>
			<td>Thorlabs</td>
			<td>ER4</td>
			<td>Cage Assembly Rod, 4&#34; Long, &#216;6 mm</td>
		</tr>
		<tr>
			<td>Cage Mounting Bracket</td>
			<td>Thorlabs</td>
			<td>CP02B</td>
			<td>30 mm Cage Mounting Bracket</td>
		</tr>
		<tr>
			<td>Single mode optical fiber</td>
			<td>Thorlabs</td>
			<td>P5-405BPM-FC-2</td>
			<td>Patch Cable, PM, FC/PC to FC/APC, 405 nm, Panda, 2 m</td>
		</tr>
		<tr>
			<td>Multi mode optical fiber</td>
			<td>Thorlabs</td>
			<td>M42L01</td>
			<td>&#216;50 &#181;m, 0.22 NA, FC/PC-FC/PC Fiber Patch Cable, 1 m</td>
		</tr>
		<tr>
			<td>Achromatic Doublet lens (mag lens)</td>
			<td>Thorlabs</td>
			<td>ACN127-025-A</td>
			<td>ACN127-025-A - f=-25.0 mm, &#216;1/2&#34; Achromatic Doublet, ARC: 400-700 nm , a concave lens in the &#34;mag lens&#34;</td>
		</tr>
		<tr>
			<td>Achromatic Doublet lens (mag lens)</td>
			<td>Thorlabs</td>
			<td>AC127-050-A</td>
			<td>f=50.0 mm, &#216;1/2&#34; Achromatic Doublet, ARC: 400-700 nm, a convex lens in the &#34;mag lens&#34;</td>
		</tr>
		<tr>
			<td>Retaining ring</td>
			<td>Thorlabs</td>
			<td>SM05PRR</td>
			<td>SM05 Plastic Retaining Ring for &#216;1/2&#34; Lens Tubes and Mounts, for &#34;mag lens&#34;</td>
		</tr>
		<tr>
			<td>Nylon-tipped screw</td>
			<td>Thorlabs</td>
			<td>SS3MN6</td>
			<td>M3 x 0.5 Nylon-Tipped Setscrew, 6 mm Long, for holding &#34;3D lens&#34;</td>
		</tr>
		<tr>
			<td>3D lens</td>
			<td>CVI Laser Optics</td>
			<td>RCX-25.4-50.8-5000.0-C-415-700</td>
			<td>f=10 m, rectangular cylindrical lens</td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor</td>
			<td>iXon Ultra 888</td>
			<td></td>
		</tr>
		<tr>
			<td>100 nm multichannel beads</td>
			<td>Thermo</td>
			<td>T7279, TetraSpeck microspheres</td>
			<td></td>
		</tr>
		<tr>
			<td>red dye</td>
			<td>Thermo</td>
			<td>Alexa Fluor 647</td>
			<td></td>
		</tr>
		<tr>
			<td>yellow-green dye</td>
			<td>GE Healthcare</td>
			<td>Cy3</td>
			<td></td>
		</tr>
		<tr>
			<td>green dye</td>
			<td>GE Healthcare</td>
			<td>Cy3B</td>
			<td></td>
		</tr>
		<tr>
			<td>blue dye</td>
			<td>Thermo</td>
			<td>Alexa Fluor 488</td>
			<td></td>
		</tr>
	</tbody>
</table>

conducting multiple imaging modes with one fluorescence microscope

荧光显微术是检测生物分子原位和实时监测其动力学和相互作用的有力工具。除了常规的免疫荧光显微镜外, 还开发了各种成像技术来实现特定的实验目标。一些广泛使用的技术包括单分子荧光共振能量转移 (smFRET), 它可以报告构象变化和分子相互作用与埃分辨率, 和单分子检测基于超分辨率 (SR) 成像, 与衍射有限显微镜相比, 它可以提高约十到多倍的空间分辨率。在这里, 我们提出了一个客户设计的集成系统, 它在一个显微镜中合并多种成像方法, 包括常规的 epi 荧光成像、基于单分子检测的 SR 成像和多色单分子检测,包括 smFRET 成像。通过切换光学元件, 可以轻松、可重复地实现不同的成像方法。这种设置很容易通过任何生物科学的研究实验室, 需要进行例行和多样化的成像实验, 以降低成本和空间相对于建立单独的显微镜为个人目的。

这种显微镜允许在不同的成像方法之间进行灵活和可重复的切换。这里我们展示了每个成像模块收集的样本图像。
图 5D显示了 SR 采集过程中闪烁的分子的 PSF。数以千计的这样的图像被重建, 以生成最终的 SR 图像 (图 5E)。图 5E显示了与图 7A</stro...

Watch this Scientific Journal Video about Conducting Multiple Imaging Modes with One Fluorescence Microscope at JoVE.com

Conducting Multiple Imaging Modes with One Fluorescence Microscope

使用一个荧光显微镜进行多种成像模式

在这里, 我们提出了一个实用的指南, 建立一个综合显微镜系统, 合并传统的 epi 荧光成像, 单分子检测的超分辨率成像和多色单分子检测, 包括单分子荧光共振能量转移成像, 成一种以经济高效的方式进行设置。

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

此方法可以帮助回答光学显微镜领域的关键问题，例如如何使用同一显微镜拍摄超分辨率图像和 FRET 图像。该技术的主要优点是，我们可以将三个不同的成像模块组合成一台显微镜，从而显著降低总体成本。该方法的可视化演示至关重要，因为激励路径的组装很难学习。它们需要正确选择零件和敏感的光学校准。首先，通过 PCI 接口安装数据采集卡，并用它来将激光器连接到计算机。通过晶体管-晶体管逻辑输出控制激光的开、关行为，通过该卡的模拟输出控制激光的功率调整。接下来，准备一个振动隔离光学表与反射镜和分束器，如这里所示，并随附文本协议中所述。通过将光纤适配器板首先安装在 z 轴转换支架中，将激光束组合成单模光纤。接下来，在笼板中安装无色双片镜头。使用延长杆连接适配器和镜头以形成保持架。然后，使用一英寸厚的光学柱将笼子安装在光学桌子上。通过调整元件来对齐 647 纳米激光器，通过光纤获得最大的激光功率输出。第一个激光器的对齐完成后，临时安装一对虹膜，然后一个一个地对齐其余的激光器。使用功率计检查每个激光器的对齐效率。请务必在适配器板前面留一个光圈，以减少激光的反射。接下来，设计和安装放大镜，如随附的文本协议中所述。为了创造提取每个分子的 z 坐标所必需的散光效果，请将具有 10 米焦距的 3D 镜头放入盒式磁带中，并将其插入发射光束路径中。为了最大限度地减少连续多色荧光成像期间的振动，请使用放置在显微镜旁边的屏障滤镜轮中的发射过滤器。在单分子 FRET 实验中，为了同时进行多色检测，请将另一个滤波器集放在发射分路器中。要使用光能激发设置衍射受限成像，首先将激发激光器的偶发角调整为照明臂的光显模式。接下来，分离 3D 镜头，并将旁路立方体插入发射分路器。然后，插入磁镜，以扩大照明。设置完成后，根据预期结果拍摄样品的多通道、z 堆栈和/或时间失效的图像。要建立表面固定分子的多色单分子检测，首先将滤芯滚轮移动到空位置。这将允许激光通过。然后，将激发激光的杂角调整到草皮角度，并分离磁光和 3D 镜头。接下来，首先将旁路立方体替换为校准立方体，使所有光线都通过所有通道，从而在发射分路器中使用三通道模式。然后，在 DIC 下打开相机，并调整发射分路器的光圈，直到屏幕上出现三个完全分离的通道。转动发射分路器上的垂直水平调节控制旋钮，大致对齐三个通道。接下来，关闭相机，将校准立方体替换为三立方。放置100纳米多通道珠的样品。在488纳米的激发下，100纳米多通道珠发出不同波长的光，实现三通道对齐。然后，打开相机和 488 纳米激光器，放大其中一个明亮的磁珠，最后通过再次转动调节控制旋钮来对齐三个通道。现在样品就位后，使用弱激光，导航到具有合理点密度的区域，并调整激光功率和曝光时间，以实现可接受的信噪和光漂白水平。然后使用成像软件拍摄延时图像。对于超高分辨率成像，请首先插入 3D 镜头并移除磁镜镜头。然后确定最佳激发激光的偶发角为草皮角度。为了找到SR成像的正确目标高度，使用DIC成像来查找细胞的中间平面。按单元格变为透明的高度确定平面。确定所需的焦平面后，开始超高分辨率成像。在成像时，改变 405 纳米激光的功率，以保持闪烁点的合理密度。无需紫罗兰激光电源即可开始成像。计算特定时间段的闪烁点数，并增加紫罗兰激光功率，使闪烁点的数量保持在视野中用户定义的计数阈值以上。通过检测成像帧中每个点的质心来分析数据，并从 X 和 Y 宽度提取每个点的 z 值。构建重建的图像，并在 3D 中可视化对象。这种显微镜设置允许在不同的成像方法之间灵活且可重复切换，包括传统的荧光成像、基于单分子检测的超分辨率成像和多色单分子检测。为了在分子组合中揭示更精细的细节，超分辨率显微镜结合了数千个图像，如这个图像。然后重建这些图像以生成最终的超分辨率图像。超分辨率技术允许高空间分辨率，提供其他技术无法看到的细节。这突出显示在此处显示的两个图像中。超分辨率图像显示与荧光图像相同的细菌调节 RNase，但允许单分子检测。SmFRET 是另一种能够对纳米分辨率产生焦虑的方法。在这里，折叠的RNA分子被标记为绿色供体染料和红色接受染料。荧光强度轨迹可以从单个单个分子中提取，从而产生F FRET效率作为时间函数。不要忘记，使用激光可能很危险，在执行此过程时，应始终采取佩戴眼罩或降低激光功率等预防措施。

Research

JoVE Journal

Bioengineering

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

在活细胞中的荧光寿命成像分子转子

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

科研

生物工程

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

光频域成像<em&gt;体外</em肺切除标本：获得一对一的图像病理组织学的相关性

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

时间推移荧光成像拟南芥根系生长的根环境的快速操作使用RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

一分钟，小一瓦光热消融肿瘤使用Porphysomes，内在多功能纳米囊泡

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a ...

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

定量的光学显微镜：测量细胞生物物理特性与标准光学显微镜

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

3d轨道跟踪的改进双光子显微镜之应用：细胞内的囊泡的跟踪

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

指南结构照明显微镜TIRF在高速，多种颜色

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular ...