Name: construction of a high resolution microscope with conventional and holographic optical trapping capabilities
Uploaded: 2013-04-22T13:00:00
Description: Watch this Scientific Journal Video about Construction of a High Resolution Microscope with Conventional and Holographic Optical Trapping Capabilities at JoVE.com

经费是由美国犹他州大学。我们要感谢徐坚博士（加州大学默塞德）和的BJN雷迪博士（加州大学欧文分校）有用的讨论。

1。安装980 nm波长单光阱<ol><li>在980纳米波长的光学俘获往往是最佳的生物物理学实验和廉价的激光二极管输出功率高达300毫瓦都是现成的。这是优选的公知的模场直径的单模光纤，其与保偏尾纤的二极管激光器。需要足够长，以作为模式过滤器的纤维，通常是与任何一个FC / PC或FC / APC连接终止。其中，FC / APC是可取的，以尽量减少反射光不稳定和潜在的反馈。 </li><li> 980 nm激光二极管固定在山上...

<ol>
	<li>Svoboda, K., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+applications+of+optical+forces.">Biological applications of optical forces.</a> Annu. Rev. Biophys. Biomol. Struct. 23, 247-285 (1994).</li><li>Ashkin, A., Schutze, K., Dziedzic, J. M., Euteneuer, U., Schliwa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+generation+of+organelle+transport+measured+in+vivo+by+an+infrared+laser+trap.">Force generation of organelle transport measured in vivo by an infrared laser trap.</a> Nature. 348, 346-348 (1990).</li><li>Shubeita, G. T., Tran, S. 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=Consequences+of+motor+copy+number+on+the+intracellular+transport+of+kinesin-1-driven+lipid+droplets.">Consequences of motor copy number on the intracellular transport of kinesin-1-driven lipid droplets.</a> Cell. 135, 1098-1107 (2008).</li><li>Polin, M., Ladavac, K., Lee, S. H., Roichman, Y., Grier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+holographic+optical+traps.">Optimized holographic optical traps.</a> Opt Express. 13, 5831-5845 (2005).</li><li>Sun, B., Roichman, Y., Grier, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+holographic+optical+trapping.">Theory of holographic optical trapping.</a> Opt. Express. 16, 15765-15776 (2008).</li><li>Moffitt, J. R., Chemla, Y. R., Smith, S. B., Bustamante, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+optical+tweezers.">Recent advances in optical tweezers.</a> Annu. Rev. Biochem. 77, 205-228 (2008).</li><li>Grier, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revolution+in+optical+manipulation.">A revolution in optical manipulation.</a> Nature. 424, 810-816 (2003).</li><li>Neuman, K. C., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping.">Optical trapping.</a> Rev. Sci. Instrum. 75, 2787-2809 (2004).</li><li>Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+tweezers+in+cell+biology.">Laser tweezers in cell biology.</a> Introduction. Methods Cell Biol. 55, xi-xii (1998).</li><li>Spudich, J. A., Rice, S. E., Rock, R. S., Purcell, T. J., Warrick, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+traps+to+study+properties+of+molecular+motors.">Optical traps to study properties of molecular motors.</a> Cold Spring Harb. Protoc. 2011, 1305-1318 (2011).</li><li>Visscher, K., Gross, S. P., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+multiple-beam+optical+traps+with+nanometer-resolution+position+sensing.+Selected+Topics+in+Quantum+Electronics.">Construction of multiple-beam optical traps with nanometer-resolution position sensing. Selected Topics in Quantum Electronics.</a> IEEE Journal of. 2, 1066-1076 (1996).</li><li>Valentine, M. T., Guydosh, N. R., 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=Precision+steering+of+an+optical+trap+by+electro-optic+deflection.">Precision steering of an optical trap by electro-optic deflection.</a> Opt Lett. 33, 599-601 (2008).</li></ol>

我们已经构建了一个仪器，结合了两种不同类型的光学陷阱（ 图1），以提供独立的诱捕设施为对象的操作和测量。 980纳米激光二极管围绕“常规”光陷阱。被扩展时，该光束转向，然后注射到我们的倒置显微镜（“浅红色”束在图1中）。是围绕一个1,064 nm的DPSS激光全息光学陷阱。束被扩展以适应大小的空间光调制器（SLM）的SLM，减少在低入射角稍微过满的后焦光圈的?...

光学俘获生物物理学6中的关键技术之一。一个关键的进步一直在光学诱捕的发展全息陷阱允许创建三维俘获模式，而不是传统的点陷阱7。这种全息陷阱具有定位屈光对象的通用性的优点。然而，传统的陷阱，可以很容易地对准更对称比市售全息套件。他们还允许被困物体的快速精确跟踪。这里，我们描述的系统（ 图1）相结合的两个捕集方法在一台仪器中，并允许用户利用的好处，同时以适当的。 建设光陷阱（基于单个或多个激光束）的一般考虑别处8-10详细讨论。在这里，我们勾勒出具体考虑我们的Setup并提供细节，我们的校准程序。比如，已经描述了两束光俘获系统之前（ 例如，参考文献11），用于捕集的折射率的对象去耦读出捕获的对象的位置和使用的其他（故意低功率波束），通常使用一个激光束。然而，在这里，两个激光束的高功率（300 mW或更高），因为两者都是用于俘获。对于生物系统的测量，激光器用于诱捕最佳应该属于一个特定的波长的近红外窗口，以尽量减少光诱导蛋白降解1。在这里，我们选择了使用980 nm的二极管和1,064 nm的DPSS激光器，因为其低成本，高可用性和易于操作。 我们也选择了使用空间光调制器（SLM）创建和操纵多个陷阱，同时在实时4,5。这些设备是市售的但是他们整合成一个完整的设置提出了独特的挑战。在这里，我们描述了一个切实可行的办法，解决这些潜在的困难，并提供了一​​个高度灵活的工具。我们提供了一个明确的例子具体描述的设置，可以用来作为一个指南的修改设计。

<table border="1">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Optical table</td>
			<td>Newport corporation</td>
			<td>ST-UT2-56-8</td>
			<td>Irvine, CA</td>
		</tr>
		<tr>
			<td>Microscope, Inverted, Eclipse Ti</td>
			<td>Nikon USA</td>
			<td>MEA53220</td>
			<td>Melville, NY</td>
		</tr>
		<tr>
			<td>Plan apo 100X oil objective (1.4 NA)</td>
			<td>Nikon USA</td>
			<td>MRD01901</td>
			<td>Melville, NY</td>
		</tr>
		<tr>
			<td>Oil condenser Lens 1.4 NA</td>
			<td>Nikon USA</td>
			<td>MEL41410</td>
			<td>Melville, NY</td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor technology USA</td>
			<td>Ixon DU897</td>
			<td>South Windsor, CT</td>
		</tr>
		<tr>
			<td>1/3" CCD IEEE1394 camera</td>
			<td>NET USA Inc</td>
			<td>Foculus FO124SC</td>
			<td>Highland IN</td>
		</tr>
		<tr>
			<td>Laser, TEM00, SLM, 1,064 nm wavelength</td>
			<td>Klastech Laser Technologies</td>
			<td>Senza-1064-1000</td>
			<td>Dortmund; Germany</td>
		</tr>
		<tr>
			<td>laser diode, TEM00, SLM, 980 nm</td>
			<td>Axcel Photonics</td>
			<td>BF-979-0300-P5A</td>
			<td>Marlborough, MA</td>
		</tr>
		<tr>
			<td>laser diode mount</td>
			<td>ILX Lightwave</td>
			<td>LDX-3545, LDT-5525, and LDM-4984</td>
			<td>Bozeman, MT</td>
		</tr>
		<tr>
			<td>adjustable fiber ports</td>
			<td>Thorlabs</td>
			<td>PAF-X-11-B</td>
			<td>Newton, NJ</td>
		</tr>
		<tr>
			<td>holographic system</td>
			<td>Arryx</td>
			<td>HOTKIT-ADV-1064</td>
			<td>Chicago, IL</td>
		</tr>
		<tr>
			<td>holographic mirror</td>
			<td>Boulder Non-linear Systems</td>
			<td>this is a part of HOTKIT-ADV-1064</td>
			<td>Lafayette, CO</td>
		</tr>
		<tr>
			<td>Calcite polarizer</td>
			<td>Thorlabs</td>
			<td>GL10-B</td>
			<td>Newton, NJ</td>
		</tr>
		<tr>
			<td>half-wave plate</td>
			<td>Thorlabs</td>
			<td>WPH05M-1064</td>
			<td>Newton, NJ</td>
		</tr>
		<tr>
			<td>Polarizer rotation mount</td>
			<td>Thorlabs</td>
			<td>PRM1</td>
			<td>Newton, NJ</td>
		</tr>
		<tr>
			<td>half-wave plate rotation mount</td>
			<td>Thorlabs</td>
			<td>RSP1</td>
			<td>Newton, NJ</td>
		</tr>
		<tr>
			<td>Shutter</td>
			<td>Thorlabs</td>
			<td>SH05</td>
			<td>Newton, NJ</td>
		</tr>
		<tr>
			<td>dichroic mirrors (DM2 &amp; DM3); 45&deg; AOI</td>
			<td>Chroma Technology</td>
			<td>t750spxrxt</td>
			<td>Bellows Falls, VT</td>
		</tr>
		<tr>
			<td>dichroic mirror (DM1); 45&deg; AOI</td>
			<td>Thorlabs</td>
			<td>DMSP1000R</td>
			<td>Newton, NJ</td>
		</tr>
		<tr>
			<td>custom mechanical adapter</td>
			<td>Thorlabs</td>
			<td>SM1A11 and AD12F with enlarged inner bore</td>
			<td>Newton, NJ</td>
		</tr>
		<tr>
			<td>notch filter</td>
			<td>Semrock</td>
			<td>FF01-850/310-25</td>
			<td>Rochester, NY</td>
		</tr>
		<tr>
			<td>Acousto-Optic deflector (2-axis)</td>
			<td>intraAction</td>
			<td>DTD-584CA28</td>
			<td>Bellwood, IL</td>
		</tr>
		<tr>
			<td>goniometric stage</td>
			<td>New Focus</td>
			<td>9081</td>
			<td>Santa Clara, CA</td>
		</tr>
		<tr>
			<td>60 mm steering lenses</td>
			<td>Thorlabs</td>
			<td>LA1134-B</td>
			<td>Newton, NJ</td>
		</tr>
		<tr>
			<td>16 mm aspherical expander lens</td>
			<td>Thorlabs</td>
			<td>AC080-016-C</td>
			<td>Newton, NJ</td>
		</tr>
		<tr>
			<td>175 mm expander lens</td>
			<td>Thorlabs</td>
			<td>LA1229-C</td>
			<td>Newton, NJ</td>
		</tr>
		<tr>
			<td>Spot blocker (cabron-steel sphere)</td>
			<td>Bal-Tec</td>
			<td>0.0100" diameter</td>
			<td>Los Angeles, CA</td>
		</tr>
		<tr>
			<td>Microspheres (Carboxyl-polystyrene)</td>
			<td>Spherotech</td>
			<td>CP-45-10</td>
			<td>Lake Forest, IL</td>
		</tr>
	</tbody>
</table>

construction of a high resolution microscope with conventional and holographic optical trapping capabilities

光阱的高清晰度显微镜系统允许精确地操纵各种折射对象，如电介质有孔玻璃珠1或细胞器2,3，以及对高时空分辨率读出他们的相对位置的陷阱中心。本文描述的系统有一个这样的“传统的”陷阱在980 nm处的作业。另外还提供了一个第二光学捕获系统，采用市售的全息包，同时在图4,5在波长为1064 nm的显微镜领域的创建和操作复杂的俘获模式。这两个系统的组合允许在同一时间的多个折射对象的操纵的同时进行高速和高分辨率的测量，运动和力的生产纳米和微微牛顿规模。

组设置，使操作员可以实时捕获多个折射对象，并将其放置在所有三个维度中，在视场之内。我们说明了仪器的性能后的全息，捕集微球11（ 图2）。限制每个对象的陷阱手动重新定位时捕获最后的安排，犹他州立大学的这个实验进行描绘的标志。合的功能的全息和常规陷阱如图3所示。传统的陷阱中央珠移动速度越来越快（1.3，10和82微米/秒的速度陷阱），而全息定义的?...

Watch this Scientific Journal Video about Construction of a High Resolution Microscope with Conventional and Holographic Optical Trapping Capabilities at JoVE.com

Construction of a High Resolution Microscope with Conventional and Holographic Optical Trapping Capabilities

本文介绍的系统采用传统的光陷阱，以及一个独立的全息光阱线，能够创建和操作多个陷阱。这使得创建复杂的几何安排的折光颗粒，同时还允许同时高速，高分辨率测量生物酶的活性。

高分辨率显微镜与常规和全息光学捕获能力建设

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