作者没有确认。

1，云母和准备幻灯片<ol><li>号广场1½（0.17毫米）到盖玻片染色架。 </li><li>超声处理在60℃下在2％洗涤剂30分钟</li><li>用去离子水冲洗20次。 </li><li>使用镊子去除幻灯片及吹干用压缩空气或氮气。 </li><li>切云母片成10×10mm的正方形片用剪刀或刀片。 </li><li>剪下每个云母片成用刀片2-3更薄传单。 注:此步骤需要使用锋利的刀片。 </li></ol> 2，云母大会和会议厅安装<ol><li>用乙醇清洁显微镜载玻片上。 </li><li>云母胶切成单张步骤1.6到玻片上用光学胶。低粘度粘合剂应更好地传播下降和胶水的云母。 </li><li>在紫外灯固化，10分钟。 注:粘合剂是由UV光具有最大吸收在350nm的范围内，以380nm的和推荐的能量固化的热曲IRED完全固化是4.5焦耳/厘米2。然而，不同的光源可用于本步骤（参见由粘合剂供应商提供的资料）。 </li><li>通过消除与透明胶带最初几层露出干净的云母表面。 </li><li>将光学胶小降（〜20微米）到云母表面。 注意:在这一步中，高粘度胶粘剂，低自发荧光水平建议。我们的经验表明，使用低（步骤2.2）和高粘度的粘合剂组合增加了显著成效云母分裂（步骤2.7）。 </li><li>轻轻地放在刚清洗干净的盖玻片上的粘合剂的下降，避免产生气泡，让解决1分钟。 </li><li>在紫外灯固化，10分钟。 注意:在此步骤之后，载玻片，云母和盖玻片的夹心可存放相当长的时间周期（长达数周）。继续下一个步骤只是实际SLB前的准备，以确保云母表面是新鲜的。 注:将粘合剂固化，UV光具有最大吸收在350的范围内，以380nm的和完全固化的推荐能量为4.5J/cm 2。然而，不同的光源可用于本步骤（参见由粘合剂供应商提供的资料）。 </li><li>使用exacto刀，轻轻地从一边载玻片下马盖玻片所示的视频。在大多数情况下，薄且平坦层云母将保持附着在盖玻片。云母附着于载玻片可重复使用（从步骤2.4重复）。 </li><li>检查表面质量用肉眼或在解剖显微镜下，以确保云母层仍粘在盖玻片和未​​分裂在步骤2.8中完全除去。做一个小的擦伤用钳子或解剖针将有助于其具有由云母可检测的不同浓度的粘合剂之间进行区分。 </li><li>从1.5 ml的小瓶瓶盖取下橡胶密封和采用光学倒挂胶帽表面胶或指甲油，并用固化UV灯，或让空气干燥10分钟，分别。 注意:如果样品准备同步光（TIRFM或共焦）与原子力显微镜成像，1.5 ml的小瓶帽可能太小，无法安装原子力显微镜头显微镜舞台上。在这种情况下，帽可以由任何塑料O形环具有更大的直径，适于AFM头安装被取代。在实验时，需要不损伤云母表面上除去帽的情况下，可以用来代替胶或指甲油硅脂。 </li></ol> 3，支持的脂质双分子层（SLB）的形成<ol><li>将新鲜制备的脂质体溶液倒入腔与表面。需要SLB形成的最小体积为〜30微升。 注:有关脂质体和SLB形成更多详细信息，请参阅发布协议，如如袋等 ，2014 15。 </li><li>请继续使用所需的协议负载均衡的形成。在孵化和成像，该腔室可以被放置在一个热块或加热的显微镜载物台上，以维持保持高于其熔融温度所用的脂质所需的温度。 </li></ol>

<ol>
	<li>Giocondi, M. -. C., 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=Surface+topography+of+membrane+domains.">Surface topography of membrane domains.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 1798, 703-718 (2010).</li><li>Garcia-Saez, A. J., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+analysis+of+membrane+dynamics.">Surface analysis of membrane dynamics.</a> Biochim Biophys Acta. 1798, 766-776 (2010).</li><li>Plochberger, 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=Cholesterol+slows+down+the+lateral+mobility+of+an+oxidized+phospholipid+in+a+supported+lipid+bilayer.">Cholesterol slows down the lateral mobility of an oxidized phospholipid in a supported lipid bilayer.</a> Langmuir. 26, 17322-17329 (2010).</li><li>El Kirat, K., Morandat, S., Dufr&#234;ne, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+analysis+of+supported+lipid+bilayers+using+atomic+force+microscopy.">Nanoscale analysis of supported lipid bilayers using atomic force microscopy.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 1798, 750-765 (2010).</li><li>Szmodis, A. W., Blanchette, C. D., Longo, M. L., Orme, C. A., Parikh, 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=Thermally+induced+phase+separation+in+supported+bilayers+of+glycosphingolipid+and+phospholipid+mixtures.">Thermally induced phase separation in supported bilayers of glycosphingolipid and phospholipid mixtures.</a> Biointerphases. 5, 120-130 (2010).</li><li>Strulson, M. K., Maurer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcontact+printing+for+creation+of+patterned+lipid+bilayers+on+tetraethylene+glycol+self-assembled+monolayers.">Microcontact printing for creation of patterned lipid bilayers on tetraethylene glycol self-assembled monolayers.</a> Langmuir. 27, 12052-12057 (2011).</li><li>Satriano, C., 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=Plasma+oxidized+polyhydroxymethylsiloxane--a+new+smooth+surface+for+supported+lipid+bilayer+formation.">Plasma oxidized polyhydroxymethylsiloxane--a new smooth surface for supported lipid bilayer formation.</a> Langmuir. 26, 5715-5725 (2010).</li><li>Bag, N., Sankaran, J., Paul, A., Kraut, R. S., Wohland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calibration+and+limits+of+camera-based+fluorescence+correlation+spectroscopy:+a+supported+lipid+bilayer+study.">Calibration and limits of camera-based fluorescence correlation spectroscopy: a supported lipid bilayer study.</a> Chemphyschem: a European Journal of Chemical Physics and Physical Chemistry. 13, 2784-2794 (2012).</li><li>Singh, S., Keller, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy+of+supported+planar+membrane+bilayers.">Atomic force microscopy of supported planar membrane bilayers.</a> Biophysical Journal. 60, 1401-1410 (1991).</li><li>Przybylo, M., 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=Lipid+Diffusion+in+Giant+Unilamellar+Vesicles+Is+More+than+2+Times+Faster+than+in+Supported+Phospholipid+Bilayers+under+Identical+Conditions.">Lipid Diffusion in Giant Unilamellar Vesicles Is More than 2 Times Faster than in Supported Phospholipid Bilayers under Identical Conditions.</a> Langmuir. 22, 9096-9099 (2006).</li><li>Scomparin, C., Lecuyer, S., Ferreira, M., Charitat, T., Tinland, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+in+supported+lipid+bilayers:+influence+of+substrate+and+preparation+technique+on+the+internal+dynamics.">Diffusion in supported lipid bilayers: influence of substrate and preparation technique on the internal dynamics.</a> Eur Phys J E Soft Matter. 28, 211-220 (2009).</li><li>Oreopoulos, J., Yip, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+scanning+probe+and+total+internal+reflection+fluorescence+microscopy.">Combined scanning probe and total internal reflection fluorescence microscopy.</a> Methods. 46, 2-10 (2008).</li><li>Shaw, J. E., Slade, A., Yip, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+in+situ+total+internal+reflectance+fluorescence/atomic+force+microscopy+studies+of+DPPC/dPOPC+microdomains+in+supported+planar+lipid+bilayers.">Simultaneous in situ total internal reflectance fluorescence/atomic force microscopy studies of DPPC/dPOPC microdomains in supported planar lipid bilayers.</a> J Am Chem Soc. 125, 11838-11839 (2003).</li><li>Skaug, M. J., Faller, R., Longo, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlating+anomalous+diffusion+with+lipid+bilayer+membrane+structure+using+single+molecule+tracking+and+atomic+force+microscopy.">Correlating anomalous diffusion with lipid bilayer membrane structure using single molecule tracking and atomic force microscopy.</a> J Chem Phys. 134, (2011).</li><li>Bag, N., Yap, D. H., Wohland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+dependence+of+diffusion+in+model+and+live+cell+membranes+characterized+by+imaging+fluorescence+correlation+spectroscopy.">Temperature dependence of diffusion in model and live cell membranes characterized by imaging fluorescence correlation spectroscopy.</a> Biochimica et Biophysica Acta. , (2013).</li><li>Sbalzarini, I. F., Koumoutsakos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feature+point+tracking+and+trajectory+analysis+for+video+imaging+in+cell+biology.">Feature point tracking and trajectory analysis for video imaging in cell biology.</a> J Struct Biol. 151, 182-195 (2005).</li><li>Linkert, M., 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=Metadata+matters:+access+to+image+data+in+the+real+world.">Metadata matters: access to image data in the real world.</a> J Cell Biol. 189, 777-782 (2010).</li><li>Schutz, G. J., Schindler, H., Schmidt, T. <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+microscopy+on+model+membranes+reveals+anomalous+diffusion.">Single-molecule microscopy on model membranes reveals anomalous diffusion.</a> Biophys J. 73, 1073-1080 (1997).</li><li>Matysik, A., Kraut, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TrackArt:+the+user+friendly+interface+for+single+molecule+tracking+data+analysis+and+simulation+applied+to+complex+diffusion+in+mica+supported+lipid+bilayers.">TrackArt: the user friendly interface for single molecule tracking data analysis and simulation applied to complex diffusion in mica supported lipid bilayers.</a> BMC Research Notes. 7, (2014).</li></ol>

作者什么都没有透露。

该协议描述了一种制备光滑而薄的云母表面的脂质双分子层沉积和高分辨率成像的方法。该技术只需要很少的手工技艺，大多限于仔细拆卸玻璃云母玻璃夹层（步骤2.8），这是获得高质量的云母表面至关重要的。总是需要新鲜剥离云母检查在这一点上，因为它有可能为云母，从光学粘合剂分离而没有解理，而使光学粘合剂的暴露区域。这可能导致在粘合剂而不是在云母上的双层的不必要的沉积。?...

本议定书的总体目标是展示了一种制备云母表面的云母支持的脂质双层（SLBs中）使用光学全内反射荧光显微镜（TIRFM）或共聚焦显微镜，它也可以用原子力结合高分辨率成像显微镜（AFM）。 SLBs中是一种广泛使用的模型为脂质聚类，相分离，双层的元件或多肽，蛋白质或其他化合物1-5及其相互作用的动力学的大量研究。不同底物可能用于SLB形成（ 即玻璃，云母，二氧化硅，聚合物）取决于研究4,6-8的性质。典型的膜研究依赖于显微镜为基础的成像技术，如TIRFM和原子力显微镜。因此，对于TIRFM成像，在玻璃表面是一个典型的选择，因为玻璃是透明的。是玻璃的制备比较容易，其结果的质量，主要是通过彻底的清洗表面前脂质囊泡的沉积来确定。原子力显微镜由于其较高的轴向分辨率要求云母表面。云母是一种硅酸盐矿物，具有接近完美的底面解理。因此，新鲜剥离的云母是原子级平整，允许甚至在亚纳米尺度9观察膜的高度差。 使用方法如荧光相关光谱（FCS），单分子跟踪（SMT），和光漂白（FRAP）后荧光恢复扩散的研究表明然而，该脂质膜动力学很大程度上依赖于表面在其上它们被沉积的类型，由此玻璃和云母能给广泛变化的结果10,11。这些不同之处包括该膜的探针不仅扩散系数，也是粒子具有不同的速率扩散的分离群体的检测，以及可能不同的状态之间进行切换。 因此，使用TIRFM和AFM技术获得的结果的直接比较是经常有问题，除非在同一表面（本例中的云母）被使用。虽然有一些研究，其中TIRFM和AFM双层成像于同云母表面12,13进行，云母很少使用，主要是因为处理问题的光学显微镜。云母编制要求切割手工成薄传单，然后用光学胶12粘在盖玻片。然而，这种方法需要一些练习，以达到满意的效果。此外，所得到的表面常常是波浪形而厚，使得它们难以与低的工作距离，高数值孔径的透镜使用。 如在此协议中所述制备云母表面非常薄（〜220微米，其中包括170微米盖玻片厚度）和极其平坦，从而避免"起伏"，这对于成功的高分辨率成像是至关重要的。它们可以被用来为TIRFM或共聚焦设置。此外，同样的样品可以被转移到的原子力显微镜，甚至同时成像的TIRFM /共聚焦和原子力显微镜。结合这两种技术可以与双层膜结构14扩散行为直接相关。由于云母表面的新鲜原木，他们是干净的，不需要费时，重复性差，并且有潜在危险的清洗程序（玻璃清洗协议通常包括化学物质，如Piranha溶液，硫酸，氢氧化钠/氢氧化钾）。安装一个小室中，在协议中还描述的，可减少所需的有效双层形成为小于50μl的小泡的体积。最后，表面组装的全过程是不费时（准备时间不超过30分钟），并且不需要手工技能的高度一样，传统的云母解理和胶合。

<table border="0" cellpadding="0" cellspacing="0" width="710">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Name of Material/ Equipment</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Bath Sonicator</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">FB15051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Coverslips 24 x 50mm - No H1.5</td>
			<td style="width:135px;">Marienfeld</td>
			<td style="width:119px;">102222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">DOPC</td>
			<td style="width:135px;">Avanti Polar Lipids</td>
			<td style="width:119px;">850357</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Hellmanex III (detergent)</td>
			<td style="width:135px;">Hellma Analytics</td>
			<td style="width:119px;">320.003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Mica V-1 Grade</td>
			<td style="width:135px;">SPI Suppliers</td>
			<td style="width:119px;">1872-CA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Optical Adhesive (high viscosity)</td>
			<td style="width:135px;">Norland Products</td>
			<td style="width:119px;">NOA63</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Optical Adhesive (low viscosity)</td>
			<td style="width:135px;">Norland Products</td>
			<td style="width:119px;">NOA60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sphingomyelin-ATTO647N</td>
			<td style="width:135px;">AttoTec</td>
			<td>AD 647N-171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">UV lamp</td>
			<td style="width:135px;">Synoptics Ltd.</td>
			<td style="width:119px;">GelVue GVM20</td>
			<td>The lamp was set to 100% power</td>
		</tr>
	</tbody>
</table>

preparation of mica supported lipid bilayers for high resolution optical microscopy imaging

支持的脂质双层（SLBs中）被广泛用作模型用于研究膜性能（相分离，聚类，动力学）及其与其它化合物，例如药物或肽的相互作用。然而SLB特性不同，取决于所使用的支持。 常用的技术为SLB成像和测量的单分子荧光显微镜，FCS和原子力显微镜（AFM）。因为大多数光学成像研究，进行了在玻璃支撑体，而AFM需要一个非常平坦的表面（通常是云母），从这些技术的结果不能直接相比，由于这些材料的电荷和平滑特性强烈影响扩散。遗憾的是，所需的切割和粘合薄片云母至载玻片手巧的高电平代表一个障碍，以常规使用的云母为SLB制剂。虽然这将是选择的方法，例如制备云母表面往往最终会被不均匀（波浪），不易形象，尤其是小的工作距离，高数值孔径透镜。在这里，我们提出了制备薄，平的云母表面的脂质囊泡沉积和SLB准备一个简单的和可重复的方法。此外，我们的定制室只需要非常小的体积小泡的SLB形成。整个过程结果的高效，简单和廉价的生产高品质的脂质双分子层表面可直接媲美AFM研究使用。

在SLBs中的荧光脂质探针的扩散行为是根据不同的衬底上。 TIRFM结合的SMT技术是可视化的粒子运动并提取它们的扩散系数的一种有价值的方法。一个鞘磷脂-ATTO647N探针扩散在DOPC（1,2 -二油酰-sn-甘油基-3 -磷酸胆碱）支承于玻璃和云母双层的单分子的信号显示在附加的动画人物。在云母表面被根据这里提出的方案制备。为了估计光学像差，半高（FWHM）全宽进行测量，并利用马赛克二维PSF尺寸Ima...

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Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

我们提出了制备云母支持的脂质双层膜为高分辨率显微镜的方法。云母是在原子尺度透明且平坦，但很少采用，因为处理困难成像;我们的制剂导致的云母薄片甚至沉积，并且降低了在双分子层制备中使用的材料。

支持的脂双层云母制备的高分辨率光学显微成像

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

preparation-mica-supported-lipid-bilayers-for-high-resolution-optical

Research

JoVE Journal

Bioengineering

13.3K 次观看.  Nanyang Technological University. 我们提出了制备云母支持的脂质双层膜为高分辨率显微镜的方法。云母是在原子尺度透明且平坦，但很少采用，因为处理困难成像;我们的制剂导致的云母薄片甚至沉积，并且降低了在双分子层制备中使用的材料。 

视频: Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

刀口扫描显微镜的样品制备，图像和分析协议

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

科研

生物工程

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

宽带超声医疗成像应用，光学检测仪

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

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

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

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., ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

原子力显微镜成像和支持的脂质双层力谱

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

在支撑脂双层配体纳米簇阵列

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

可切换声光学分辨率光声显微镜<em&gt;体内</em&gt;小动物血液血管成像

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.

人类胰岛神经网络的高分辨率3D 成像

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

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Subtitles

支持的脂双层云母制备的高分辨率光学显微成像