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，它允许电力和温控。这是最好的修复直接安装到光学平台，以最大限度地提高被动散热，从而减少二极管失灵的危险，由于温度控制器故障。 </li><li> PC / APC光纤连接器安装的光束准直光学。这是关键，以保证准直光束有最小的发散，所以是最有用的可调光纤端口。确保所选择的光纤端口匹配二极管尾纤光纤的模场直径。如果光束是使用声光偏转器（AOD）或电光偏转器的光栅（EOD）的准直激光束腰身也必须是略小于偏转器孔径的大小。 </li><li>以足够的距离，从显微镜的准直适配器固定在光学平台上，以允许束路由，扩展和其他所需的组件的位置。调整纤维港口整体束路径显微镜相媲美的距离，以确保一致的束腰。 </li><li>安装在图1中所示的反射镜。从显微镜中取出目标，并用反射镜将光束通过客观安装阶段中的孔中。如果优选，分色镜DM1和DM3的放置可以被省略，直到后来。 DM2和DM3而都shortpass透过可见光，同时反射近红外及以上。 </li><li>是有帮助的暂时安装，对准显微镜的光轴上的红色激光指针代替的客观。一个自定义的的机械适配器是必要的，以确保中心定位的激光指针。从激光指示器的可见光束，然后可以回路由光纤端口的光圈的中心，然后，可以使用安装的透镜（见下文）。 </li><li>安装980 nm的扩束（L8和L9）在适当的距离的光纤端口，允许未来插入转向组件（AOD或EOD）为必要1。扩束必须稍微装得过满后焦距光圈的客观。 （在这里，125毫米和60毫米焦距的透镜是在约两倍的光束束腰的开普勒安排）。可见激光指针梁（参见1.6节），以确保适当的镜头的位置和粗调。 </li><li>安装l为980纳米转向镜头（L2和L3）望远镜安排在（这里都有60毫米焦距）1。 L3被安装在一个平面上的共轭物镜的后焦平面。贴装，L3上的一个精确的XYZ定位阶段，以允许光束转向。它是有帮助的XYZ阶段有其微米的数字指标，允许重复定位和重新定位的陷阱。 0.5"范围内的旅行通常是足够的，但是更长的旅行沿光轴L3定位可能会有所帮助。使用可见的激光笔光束（参见1.6节），以确保适当的镜头的位置和粗调。 </li></ol> 2。激光探测器的安装<ol><li>安装DM3，冷凝器的二向色反射镜，如在图1中示出。通常需要一个自定义安装。四光电二极管（QPD）或位置敏感探测器（PSD）8固定侧的冷凝器组件和ENSURE 980 nm激光束反射的DM3击中它大致的中心。当使用QPD，确保它被安装在一个小的XY载物台以允许上的激光束的传感器的精确的定心。 </li><li>安装L1（通常是30毫米镜头）DM3和传感器之间。位置L1，以将光束聚焦到一个点上的传感器。 </li><li>安装之前L1阻止的1064 nm的光束以及从显微镜任何杂散的可见光反射照明器和环境照明的陷波滤波器。 </li></ol> 3。安装的1,064 nm波长全息陷阱<ol><li>全息设置的一部分是围绕市售硬件/软件包，这个软件包中使用的全息反射镜被评为5或10瓦/厘米2的最大入射功率。单模式TEM00光束在这个功率范围内，可以很容易地来源于一个1,064 nm波长的DPSS激光器。 </li><li>在高架安装1,064 nm激光平台，以大致为980线（参见第1）的光束路径的高度相匹配。 </li><li>如果不是直接控制，可手动调整激光功率，通过安装一个半波片（HWP）和偏振器（PBS）后激光输出孔。是有帮助的偏振器安装在一个旋转的阶段，能够匹配光束偏振全息镜要求。 </li><li>安装1,064 nm的扩束（L6和L7）。必须扩大相匹配的对角尺寸的全息镜的激光光束束腰。对于较大的膨胀比（大于10倍），它可能是一个关注的保持膨胀​​机的大小小。因此，它可能是可取的，与异常小的焦距（这里使用镜头:16毫米和175毫米）。 </li><li>安装其他镜像1,064纳米束直接通过目标。 <ol><li>安全的DM1分色（45°的入射角）运动的安装和装配在980纳米光束路径，以便，它允许undiminishe的ð传输，光束。 </li><li>启动雷射光束。 DM1镜应反映可见光的足够量的，适当地定位该光束的路径中的空间光调制器（SLM）。在SLM也需要的角度，​​从而使输入和输出的激光束的正常发生率尽可能接近。然而，入射角必须足够大，以确保激光束由透镜轴承，及其他光学部件不剪切。 5°的角度应该是容易实现的，足够小。最后的距离DM1到SLM必须进行精确测量，从而使插入的透镜L4和L5（见下文3.6）共轭的的SLM反射镜平面和物镜的后焦平面。 </li><li>安装镜子直接从1,064 nm的扩束光的SLM。确保雷射光束扩束击中孔径中心。 </li></ol></li><li>安装镜头L4和L5（这里:12镜头5毫米和200毫米）。此望远镜对共轭物的SLM反射镜平面的物镜的后焦平面，也减少了只是稍微过满的目标回光圈的光束束腰。我们选择了长焦距的镜头空间的SLM远离DM1。这不仅清除第二激光线的空间，但也往往使取向变得更容易。 </li><li>拆下激光指针。离开安装适配器作为粗对准光圈。 </li></ol> 4。系统安装和校准附注<ol><li>透镜L3和SLM必须定位成光学共轭的后焦面的客观。 L4和L5的共同焦点的样品平面光学共轭的，如果的光俘获束显微镜的无限空间注入。 </li><li>唱IR卡浏览器在980 nm的光束对准一起去激光指针适配器中的孔的中心轴线。 </li><li>使用红外感应卡考勤确保1064 nm的光束击中DM1，L2和L3和1064 nm的光束沿着激光指针适配器中的孔的中心轴线的相同点为980 nm的光束。 </li><li>更换一个客观的激光指针安装适配器。高数值孔径的油或水的目标，是典型的。 </li><li> 9中所描述的"行走"的径向对称的干涉图案的激光束，直到在相机上看到的980 nm的陷阱对齐。 </li><li>随着全息镜像关闭（ 即作为一个被动的镜子）使用SLM和DM1"走"未衍射1,064 nm的光束对准1064nm的陷阱。 </li><li>在SLM产生了显着的未衍射光束产生一个强有力的不可移动的激光陷阱中的视场。对应的，这是非常有用的，但可能是不理想的实验。为了阻止这个陷阱，一个可以插入一个小的不透明的对象未衍射的光的路径中，在样品平面共轭的位置（ 例如，第É共同的焦点，L4和L5）。此中央点阻滞剂的大小需要是稍微大于艾里斑的直径为聚焦光（100-300微米直径的所描述的系统中的阻断剂）。 </li><li> djust 1,064纳米束使用偏光镜的偏振，到SLM方向相匹配。旋转的半波片，然后随意设定光束的输出功率。 </li><li>如果需要，插入到980 nm激光线AOD或排爆光束转向元素。确保适当结合这些元素的后焦平面的目标，并重新调整的陷阱。安装在方向盘测角舞台上的元素是很有帮助的。 </li></ol>

<ol>
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什么都没有透露。

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目标，?...

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><tbody><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&quot; 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;amp; DM3); 45 &amp;deg; AOI</td><td>Chroma Technology</td><td>t750spxrxt</td><td>Bellows Falls, VT</td></tr><tr><td>dichroic mirror (DM1); 45 &amp;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&quot; 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?...

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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

High resolution microscope systems with optical traps allow for precise manipulation of various refractive objects, such as dielectric beads 1 or cellular ...

construction-high-resolution-microscope-with-conventional-holographic

Universidad de Cartagena UDEC

Research

JoVE Journal

Engineering

12.1K 次观看.  University of Utah. 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。

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

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

科研

免疫与感染

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

lensfree芯片断层用人多角度的光照和像素超高分辨率的显微镜

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

生物工程

Compact Lens-less Digital Holographic Microscope for MEMS Inspection and Characterization

A micro-electro-mechanical-system (MEMS) is a widely used component in many industries, including energy, biotechnology, medical, communications, and automotive. However, effective inspection and characterization metrology systems are needed to ensure the functional reliability of MEMS. This study presents a system based on digital holography as a tool for MEMS metrology. Digital holography has gained increasing attention in the past 20 years. With the fast development and decreasing cost of sensor arrays, resolution of such systems has increased broadening potential applications. Thus, it has attracted attention from both research and industry sides as a potential reliable tool for industrial metrology. Indeed, by recording the interference pattern between an object beam (which contains sample height information) and a reference beam on a CCD camera, one can retrieve the quantitative phase information of an object. However, most of digital holographic systems are bulky and thus not easy to implement on industry production lines. The novelty of the system presented is that it is lens-less and thus very compact. In this study, it is shown that the Compact Digital Holographic Microscope (CDHM) can be used to evaluate several characteristics typically consider as criteria in MEMS inspections. The surface profiles of MEMS in both static and dynamic conditions are presented. Comparison with AFM is investigated to validate the accuracy of the CDHM.

We present a compact reflection digital holographic system (CDHM) for inspection and characterization of MEMS devices. A lens-less design using a diverging input wave providing natural geometrical magnification is demonstrated. Both static and dynamic studies are presented.

紧凑的镜头少数字全息显微镜的MEMS检测与表征

A micro-electro-mechanical-system (MEMS) is a widely used component in many industries, including energy, biotechnology, medical, communications, and ...

工程学

Demonstration of a Hyperlens-integrated Microscope and Super-resolution Imaging

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and nanotechnology. Although near-field scanning microscopy and superlenses have improved the resolution in the near-field region, far-field imaging in real-time remains a significant challenge. Recently, the hyperlens, which magnifies and converts evanescent waves into propagating waves, has emerged as a novel approach to far-field imaging. Here, we report the fabrication of a spherical hyperlens composed of alternating silver (Ag) and titanium oxide (TiO2) thin layers. Unlike a conventional cylindrical hyperlens, the spherical hyperlens allows for two-dimensional magnification. Thus, incorporation into conventional microscopy is straightforward. A new optical system integrated with the hyperlens is proposed, allowing for a sub-wavelength image to be obtained in the far-field region in real time. In this study, the fabrication and imaging setup methods are explained in detail. This work also describes the accessibility and possibility of the hyperlens, as well as practical applications of real-time imaging in living cells, which can lead to a revolution in biology and nanotechnology.

The use of a hyperlens has been regarded as a novel super-resolution imaging technique due to its advantages in real-time imaging and its simple implementation with conventional optics. Here, we present a protocol describing the fabrication and imaging applications of a spherical hyperlens.

Hyperlens 综合显微镜和超分辨率成像的演示

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and ...

Construction and Operation of a Light-driven Gold Nanorod Rotary Motor System

The possibility to generate and measure rotation and torque at the nanoscale is of fundamental interest to the study and application of biological and artificial nanomotors and may provide new routes towards single cell analysis, studies of non-equilibrium thermodynamics, and mechanical actuation of nanoscale systems. A facile way to drive rotation is to use focused circularly polarized laser light in optical tweezers. Using this approach, metallic nanoparticles can be operated as highly efficient scattering-driven rotary motors spinning at unprecedented rotation frequencies in water.
In this protocol, we outline the construction and operation of circularly-polarized optical tweezers for nanoparticle rotation and describe the instrumentation needed for recording the Brownian dynamics and Rayleigh scattering of the trapped particle. The rotational motion and the scattering spectra provides independent information on the properties of the nanoparticle and its immediate environment. The experimental platform has proven useful as a nanoscopic gauge of viscosity and local temperature, for tracking morphological changes of nanorods and molecular coatings, and as a transducer and probe of photothermal and thermodynamic processes.

Plasmonic gold nanorods can be trapped in liquids and rotated at kHz frequencies using circularly-polarized optical tweezers. Introducing tools for Brownian dynamics analysis and light scatteringspectroscopy leads to a powerful system for research and application in numerous fields of science.

轻型金纳米旋转电机系统的施工与运行

The possibility to generate and measure rotation and torque at the nanoscale is of fundamental interest to the study and application of biological and ...

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts&#39; rheology.

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo.

无珠光高音喇叭在食子胚胎中的细胞力学探测

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...

发育生物学

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

使用双光镊和微流体进行单分子研究

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the ...

生物化学

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

使用光学镊子测量禁闭中的亚细胞力学的直接力

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain large field-of-view, high-contrast images in thick cells and tissues. Here, we introduce highly inclined swept tile (HIST) microscopy that overcomes this problem. A pair of cylindrical lenses was implemented to generate an elongated excitation beam that was scanned over a large imaging area via a fast galvo mirror. A 4f configuration was used to position optical components. A scientific complementary metal-oxide semiconductor camera detected the fluorescence signal and blocked the out-of-focus background with a dynamic confocal slit synchronized with the beam sweeping. We present a step-by-step instruction on building the HIST microscope with all basic components.

A detailed instruction is described on how to build a highly inclined swept tile (HIST) microscope and its usage for single-molecule imaging.

构建用于扩展视场单分子成像的高倾角扫描磁贴显微镜的指南

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain ...

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 &#181;m&#160;x 200 &#181;m&#160;x 200 &#181;m using LED illumination, to 5 mm&#160;x 5 mm&#160;x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.

Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object.

的弱散射主题内嵌数字全息显微镜（DIHM）

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of ...