This work is supported by Ministry of Science and Technology under NSC-101-2923-M-002-001-MY3 and NSC-102-2112-M-002-018-MY3. This research is also supported by the Japan Society for the Promotion of Science (JSPS) through the &#8220;Funding Program for Next Generation World-Leading Researchers (NEXT Program),&#8221; initiated by the Council for Science and Technology Policy (CSTP) and JSPS Asian CORE Program.

1. GNS样品制备<ol><li>前在大约40千赫制备样品，声处理1 ml的GNS胶体溶液至少15分钟，以防止颗粒聚集，这可能会导致LSPR峰偏移。 </li><li>降100-200微升的GNS胶体在载玻片与商业硅酸镁铝（MAS）的涂层，以固定的GNSS。 </li><li>后至少1分钟，通过用蒸馏水冲洗除去多余的胶体。的等待时间取决于GNSS的所需分布密度。典型地，1-3分钟结果在合适的密度，使粒子能够容易地识别，因为他们最被彼此隔离。如果等待时间过长，可能会出现显著聚集。 </li><li>通过用氮气吹扫干燥样品。 </li><li> （可选的）要映射GNSS在玻璃上具有高分辨率，在此阶段23进行扫描电子显微镜（SEM）。一个例子的图像中FIGUR提供E 1，显示出全球导航卫星系统的特征密度。使用场发射SEM以获取图像。一旦油加入到样品（下一步骤），这将是很难除去的油，并观察用SEM样品。 </li><li>加一滴油具有相同的折射率到样品，以覆盖GNSS和消除从玻璃基板的强反射。 </li><li>放置的盖玻璃上的样品的顶部，并与指甲油密封。 </li><li>等待至少5分钟，直到指甲油干了。样品现已准备就绪。 </li></ol> 2.对准自制的共聚焦显微镜<ol><li> 图 2所示为安装的方案。对准显微镜体本身的白光照明的道路。打开显微镜的卤素光源，并按照显微镜制造商的手册，实现了科勒照明条件。以使白色卤素光束几乎平行于背面物镜的光圈，部分由50/50分束器反射，然后向激光传播。 </li><li>打开电流计反射镜，以确保它们保持在正确的初始位置，即，在扫描范围的中心。 </li><li>放置至少两个目标，由薄的纸，它同心环板制成，沿卤素光路，并与卤光束对齐。 </li><li>为了进行成像，选择532 nm激光。要执行光谱测量，选择超连续激光器。在对准中，激光的功率应小于10微瓦在物镜的后孔，以避免非线性。然后，准直入射激光束对面的向即将离任的卤素束的两个目标的援助。当这个过程完成后，将激光束的粗对准已经实现。 </li><li>透过物镜的后孔的中心对准的激光束。通常情况下，使用OIL-浸泡的目的。添加的油的油浸物镜与GNS样品之间的下降。使用的光电倍增管（PMT）作为检测器来收集GNSS的散射信号。 </li><li>放置在PMT的前面20微米直径的针孔来阻挡焦散射信号。打开电流计反射镜和PMT（经由家庭建造软件），调节样品台以最大化GNSS的反向散射信号的针孔位置和高度，然后观察个体GNS在计算机屏幕上。全球导航卫星系统与正确对准的样品的xy图像示于图2B。 </li><li>稍微改变样品台的高度，以检查聚焦的同心度。如果不是同心的，调整与扫描器直到GNS的中心的前部的两个反射镜的光束保持在同一位置，而试料台的高度被改变。确保该PSF的XZ图像类似于图2C以确保正确的光束对准。处理这两个图像与低通和高斯平滑滤波器。 </li></ol>散射非线性的3表征<ol><li>在低激发强度（小于10 4瓦/厘米2），通过以下方案获得2.6金纳米颗粒的图像。 </li><li>打开ImageJ的图像（或任何其它图像分析软件）。绘制横跨GNSS的图像中的一个的线（参见图2B），并使用分析- &gt;的ImageJ的工具绘图信息检索散射强度分布。适合所选择的PSF由一个高斯函数的轮廓: <img alt="公式（2）" src="/files/ftp_upload/53338/53338eq2.jpg" /> 其中 y是PMT读出，y值0是背景值（如果有的话），A是峰值幅度，w是宽度，x是空间坐标，而x c是中心coordinatE中的高斯函数。相应PSF的FWHM为（½ln2） 瓦特基于所述物镜的数值孔径（NA），共焦PSF的FWHM的理论可以被估计为近似0.43升/ NA，其中λ是激励波长。比较这两个数来检查成像系统的对准。 </li><li>增加激发强度通过手动改变中性密度（ND）滤光器在图2A中，并在每个强度电平记录后向散射图像。采取从每个GNS在不同激发强度的中心的散射信号的值，并绘制散射信号与激励强度的曲线。检查前几个点，这应当表现出一种线性关系时的激发强度是足够低的线性度。绘制基于第一几个点的线性拟合的线。如果随后的点散射强度下降低于该线性趋势，saturatioN有发生。 </li><li>观察饱和散射后，逐渐减小低于饱和阈值的强度，和图像相同GNSS再次确保了非线性行为的可逆性。 </li></ol> 4.测量单个金纳米球的散射光谱<ol><li>为了测量从一个单一的GNS的散射光谱，使用超连续激光器作为激光源。激光器的初始波长范围为450纳米至1750纳米。以除去可能会损坏样品和光学部件的过量红外线功率，放置一个或两个反射镜的超连续激光后的权利，以反映可见光，并用光束转储收集过量的红外光。 </li><li>按照第2对准程序直接超连续激光在激光扫描共聚焦显微镜。使用宽带50/50 BS，以确保在整个可见光范围光谱覆盖范围。 </li><li>获得一个像玻璃的全球导航卫星系统。定位单个GNS的图像中，并固定在颗粒上的入射光的宽带光的焦点。 </li><li>使用翻转镜在PMT以引导反向散射信号朝向分光计，其配备了一个电荷耦合器件的前方，然后取所选择的单一的GNS的频谱。请注意，这里的频谱是GNS散射和背景的混合由于来自其他表面的反射。 </li><li>切换回PMT检测，并拍摄图像，以确定粒子的位置没有改变。然后，转移焦点，以在这一点，没有粒子存在。切换回光谱仪，走多了一个频谱，它代表了背景。 </li><li>从频谱从步骤4.4减去4.5步的背景光谱，以获得单个GNS一个明确的散射光谱。 </li></ol> 5.对准SAX显微镜<ol><li> 参见图3用于该SAX显微镜，其中，从拍频获得的两个声光调制器（AOM）之间的理想正弦瞬时调制的方案。首先，调整激光的光束尺寸满足随后的声光调制器的需要。通过使用50/50分束器分裂532nm的激光光分成两个光束。 </li><li>通过两个声光调制器引导两束，其中一个光束穿过各AOM。两个声光调制器的调制频率必须是不同的。例如，一种可能是在40.000兆赫，另一个在40.010兆赫，得到10千赫的频率差。这个差频将是用于该SAX信号的基本调制频率 f 米 。 </li><li>取第一级衍射光束从两个声光调制器，并且通过使用另一个50/50的束分离器结合两个光束。该声光调制器之后调整镜子使准直两个光束。 </li><li>加，其连接到示波器的光电检测器来监视颞modulatioñ。分割用滑动玻璃的激光的一小部分，并将其发送到光检测器， 如图3。随着正确的调制和光束重叠，观察正弦强度调制的主频率 f 米 ，类似于所示的波形的在图4中。 注意:调制的背景需要尽可能低，以获得最大的调制深度。此外，使用示波器的傅立叶分析函数来检查所述调制的谐波失真减少。为了实现成功的SAX实现，保证了完美的正弦激励强度调制以最小的初始非线性。 </li><li>断开光检测器的电输出从示波器并连接到锁定放大器的参考输入。 </li><li> 如图3，对准激光束分成共焦系统按照前面的协议。 HERE，PMT的电输出连接到锁定放大器作为信号输入。 </li><li>使用空白盖玻璃作为样品，并检查电检测系统的线性度，通过逐渐增加励磁电源,, 如图5，其中所述检测器是低于1-V的读出值的线性。在所有的后续测量，小心抑制读数低于此值。 </li><li>设置锁定放大器的输出导出的电压信号的绝对幅度。通过改变高次谐波分量在参考信道设置，获取SAX信号，A 1，A 2的振幅，并依此类推。 </li><li>从锁定放大器的线性和非线性的信号输出到一个数据采集卡，还接收的光栅扫描电流反射镜的驱动电压信号。用一个定制的Labview程序的帮助下，同步锁定放大器和的信号电流镜以形成图像。 </li><li>为了优化信噪比中的图像中，选择适当的锁定放大器的像素获取和积分时间。例如，当激励的主调制频率 f m为 10千赫，也就是说，当周期为100微秒，设置锁定放大器的积分时间为比周期长至少三次。加入检测镜移动时，采集速度被设定在与SAX摄像模式每秒1500个像素。 </li></ol>

<ol>
	<li>Fang, N., Lee, H., Sun, C., Zhang, 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-limited+optical+imaging+with+a+silver+superlens.">Sub-diffraction-limited optical imaging with a silver superlens.</a> Science. 308 (5721), 534-537 (2005).</li><li>Lee, B., Kim, S., Kim, H., Lim, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+plasmonics+in+light+beaming+and+focusing.">The use of plasmonics in light beaming and focusing.</a> 34 (2), 47-87 (2010).</li><li>Lal, S., Link, S., Halas, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-optics+from+sensing+to+waveguiding.">Nano-optics from sensing to waveguiding.</a> Nature Photon. 1 (11), 641-648 (2007).</li><li>Kawata, S., Inouye, Y., Verma, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmonics+for+near-field+nano-imaging+and+superlensing.">Plasmonics for near-field nano-imaging and superlensing.</a> Nature Photon. 3 (7), 388-394 (2009).</li><li>Homola, J., Yee, S. S., Gauglitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+plasmon+resonance+sensors:+review.">Surface plasmon resonance sensors: review.</a> Sensor. Actuat. B-Chem. 54 ((1-2)), 3-15 (1999).</li><li>Nie, S., Emory, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+single+molecules+and+single+nanoparticles+by+surface-enhanced+Raman+scattering.">Probing single molecules and single nanoparticles by surface-enhanced Raman scattering.</a> Science. 275 (5303), 1102-1106 (1997).</li><li>Hache, F., Ricard, D., Flytzanis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+nonlinearities+of+small+metal+particles+-+surface-mediated+resonance+and+quantum+size+effects.">Optical nonlinearities of small metal particles - surface-mediated resonance and quantum size effects.</a> J. Opt. Soc. Am. B. 3 (12), 1647-1655 (1986).</li><li>Balamurugan, B., Maruyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+an+enhanced+interband+absorption+in+Au+nanoparticles:+Size-dependent+electronic+structure+and+optical+properties.">Evidence of an enhanced interband absorption in Au nanoparticles: Size-dependent electronic structure and optical properties.</a> Applied Physics Letters. 87 (14), 143105 (2005).</li><li>Link, S., El-Sayed, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+and+temperature+dependence+of+the+plasmon+absorption+of+colloidal+gold+nanoparticles.">Size and temperature dependence of the plasmon absorption of colloidal gold nanoparticles.</a> J. Phys. Chem. B. 103 (21), 4212-4217 (1999).</li><li>Kelly, K. L., Coronado, E., Zhao, L. L., Schatz, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+optical+properties+of+metal+nanoparticles:+The+influence+of+size,+shape,+and+dielectric+environment.">The optical properties of metal nanoparticles: The influence of size, shape, and dielectric environment.</a> J. Phys. Chem B. 107 (3), 668-677 (2003).</li><li>Kauranen, M., Zayats, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+plasmonics.">Nonlinear plasmonics.</a> Nature Photon. 6 (11), 737-748 (2012).</li><li>Homola, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Present+and+future+of+surface+plasmon+resonance+biosensors.">Present and future of surface plasmon resonance biosensors.</a> Anal Bioanal Chem. 377 (3), 528-539 (2003).</li><li>Jain, P. K., El-Sayed, I. H., El-Sayed, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Au+nanoparticles+target+cancer.">Au nanoparticles target cancer.</a> Nano Today. 2 (1), 18-29 (2007).</li><li>Wada, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femtosecond+all-optical+devices+for+ultrafast+communication+and+signal+processing.">Femtosecond all-optical devices for ultrafast communication and signal processing.</a> New J. Phys. 6 (183), .</li><li>Elim, H. I., Yang, J., Lee, J. Y., Mi, J., Ji, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+saturable+and+reverse-saturable+absorption+at+longitudinal+surface+plasmon+resonance+in+gold+nanorods.">Observation of saturable and reverse-saturable absorption at longitudinal surface plasmon resonance in gold nanorods.</a> Appl. Phys. Lett. 88 (8), 083107 (2006).</li><li>Ros, I., Schiavuta, P., Bello, V., Mattei, G., Bozio, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femtosecond+nonlinear+absorption+of+gold+nanoshells+at+surface+plasmon+resonance.">Femtosecond nonlinear absorption of gold nanoshells at surface plasmon resonance.</a> Phys. Chem. Chem. Phys. 12 (41), 13692-13698 (2010).</li><li>De Boni, L., Wood, E. L., Toro, C., Hernandez, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Saturable+Absorption+in+Gold+Nanoparticles.">Optical Saturable Absorption in Gold Nanoparticles.</a> Plasmonics. 3 (4), 171-176 (2008).</li><li>Gurudas, U., 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=Saturable+and+reverse+saturable+absorption+in+silver+nanodots+at+532+nm+using+picosecond+laser+pulses.">Saturable and reverse saturable absorption in silver nanodots at 532 nm using picosecond laser pulses.</a> J. Appl. Phys. 104 (7), 073107 (2008).</li><li>Bohren, C. F., Huffman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Absorption and scattering of light by small particles. , (1983).</li><li>Chu, S. W., 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=Saturation+and+reverse+saturation+of+scattering+in+a+single+plasmonic+nanoparticle.">Saturation and reverse saturation of scattering in a single plasmonic nanoparticle.</a> ACS Photon. 1 (1), 32-37 (2014).</li><li>Chu, S. W., 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=Measurement+of+a+saturated+emission+of+optical+radiation+from+gold+nanoparticles:+application+to+an+ultrahigh+resolution+microscope.">Measurement of a saturated emission of optical radiation from gold nanoparticles: application to an ultrahigh resolution microscope.</a> Phys. Rev. Lett. 112 (1), 017402 (2014).</li><li>Fujita, K., Kobayashi, M., Kawano, S., Yamanaka, M., Kawata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+confocal+microscopy+by+saturated+excitation+of+fluorescence.">High-resolution confocal microscopy by saturated excitation of fluorescence.</a> Phys. Rev. Lett. 99 (22), 228105 (2007).</li><li>Smith, K. C. A., Oatley, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+scanning+electron+microscope+and+its+fields+of+application.">The scanning electron microscope and its fields of application.</a> Brit. J.Appl. Phys. 6 (11), (1955).</li><li>Yu, J. Y., 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=A+diffraction-limited+scanning+system+providing+broad+spectral+range+for+laser+scanning+microscopy.">A diffraction-limited scanning system providing broad spectral range for laser scanning microscopy.</a> Rev. Sci. Instru. 80 (11), 113704 (2009).</li><li>Lee, H., 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=Point+spread+function+analysis+with+saturable+and+reverse+saturable+scattering.">Point spread function analysis with saturable and reverse saturable scattering.</a> Opt. Express. 22 (21), 26016-26022 (2014).</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=Nonlinear+structured-illumination+microscopy:+Wide-field+fluorescence+imaging+with+theoretically+unlimited+resolution.">Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution.</a> Proc. Natl. Acad. Sci. U. S. A. 102 (37), 13081-13086 (2005).</li><li>Yonemaru, Y., Yamanaka, M., Smith, N. I., Kawata, S., Fujita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saturated+Excitation+Microscopy+with+Optimized+Excitation+Modulation.">Saturated Excitation Microscopy with Optimized Excitation Modulation.</a> ChemPhysChem. 15 (4), 743-749 (2014).</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>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=Far-field+optical+nanoscopy.">Far-field optical nanoscopy.</a> Science. 316 (5828), 1153-1158 (2007).</li><li>Huang, B., Wang, W. Q., Bates, M., Zhuang, X. W. <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>Heintzmann, R., Jovin, T. M., Cremer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saturated+patterned+excitation+microscopy+-+a+concept+for+optical+resolution+improvement.">Saturated patterned excitation microscopy - a concept for optical resolution improvement.</a> J. Opt. Soc. Am. A. 19 (8), 1599-1609 (2002).</li><li>Tzang, O., Pevzner, A., Marvel, R. E., Haglund, R. F., Cheshnovsky, O. <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+in+Label-Free+Photomodulated+Reflectivity.">Super-Resolution in Label-Free Photomodulated Reflectivity.</a> Nano Lett. 15 (2), 1362-1367 (2015).</li></ol>

The authors declare that they have no competing financial interests.

在协议中，有几个关键步骤。首先，制备样品时，纳米颗粒的密度不应太高，以避免颗粒之间电浆耦合。如果两个或多个颗粒非常接近彼此，耦合导致LSPR波长偏移到更长的波长，从而显著降低非线性。然而，这种成像技术实际上映射，代替微粒本身电浆模式的分布。因此，可以预期，与适当的激发波长中，耦合电浆模式也可以显示强散射的非线性，并且可以具有增强的分辨率进行成像。第二，这...

等离子体的研究已经引起了极大的兴趣，因为它在许多不同的领域1-4的应用。一种在等离子体中研究最多的领域是表面等离子体激元，其中的传导电子夫妇在金属和电介质之间的界面的外部电磁波的集体振荡。表面等离子体激元已探索其在亚波长光学，生物光子学，以及显微镜5,6潜在的应用。在由于局部表面等离子共振（LSPR）金属纳米颗粒的超小体积的强场增强吸引，不仅是因为它的优异的敏感性，颗粒大小，颗粒形状，以及周围介质 7的介电性质的广泛关注，因为它能够提高固有的弱非线性光学效应11 -10的能力，而且还。 LSPR的特殊敏感性是生物传感和近外商投资企业有价值LD成像技术12,13。另一方面，电浆结构的增强非线性可以利用在光子集成电路中的应用，如光交换和全光信号处理14,15。众所周知的电浆吸收线性正比于在低强度电平的激发强度。当激励足够强，吸收达到饱和。有趣的是，在较高的强度，在吸收再次增大。这些非线性效应被称为饱和吸收（SA），15-17和反向可饱和吸收（RSA）18。 据了解，由于LSPR，散射特别强烈的电浆结构。根据基本的电磁散射与入射强度的反应应该是线性的。然而，在纳米粒子，散射和吸收紧密通过米氏理论联系在一起，都可以通过电子邮件xpressed中的介电常数的实部和虚部条款。下，一个单一的GNS表现为下光照偶极的假设下，从一个单一的等离子纳米颗粒根据米氏理论的散射系数（Q SCA）和吸收系数（Q 绝对）可以表示为19 <img alt="式（1）" src="/files/ftp_upload/53338/53338eq1.jpg" /> 其中 x是2πa/λ，a是球体的半径，并且m 2 为 ε 米 /εð。这里，ε 米 和 εD分别对应于金属的介电常数和周围的电介质，分别。因为散射系数的形式是类似的第Ë吸收系数，因此它被期望遵守饱和散射在一个电浆纳米粒子20。 最近，在一个孤立的电浆粒子非线性饱和散射证实首次21。值得注意的是，在深度饱和，其实散射强度略有下降，当激发强度增加。更值得注意的是，当激励强度继续后散射趋于饱和增加，散射强度再次上升，呈现出反饱和散射20的效果。 Wavelength-和尺寸相关的研究表明散射21 LSPR和非线性之间有紧密的关系。电浆散射的强度和波长的依赖性非常类似于那些吸收，这表明这些非线性行为基础的共同机制。 在应用方面，它是很好KNOWN该非线性有助于改善光学显微镜的分辨率。在2007年，饱和励磁提出（SAX）显微镜，其可以通过经由激励光束22的时间正弦调制提取饱和信号提高分辨率。 SAX显微镜是基于这样，对于一个激光焦斑，强度在中心处比在外围更强的概念。如果信号（或者荧光或散射）呈现饱和特性，饱和必须从中心开始，而线性响应保持在外围。因此，如果有一种方法，以仅提取饱和部分，它将只离开中心部分，同时抑制周边部，从而有效地增强了空间分辨率。原则上，存在的SAX显微镜没有更低分辨率极限，只要深达到饱和并且有由于强烈照明没有样品的损伤。 已经表明，该resolutioÑ​​荧光成像可以通过利用SAX技术来显著增强。然而，荧光遭受的漂白效果。结合散射非线性的发现和SAX的概念，是根据散射超分辨率显微镜可以实现21。相对于传统的超分辨率显微技术，散射为基础的技术提供了一种新颖的非漂白对比法。在本文中，一步一步的描述给出概述获得和提取等离子散射的非线性要求的程序。识别通过改变入射强度引入散射非线性的方法进行了描述。详情将提供解开这些非线性如何影响单个纳米粒子的图像，以及如何空间分辨率可以相应地由SAX技术来增强。

<table><tbody><tr><td>microscope body</td><td>Olympus, Japan</td><td>BX-51</td><td></td></tr><tr><td>objective lens</td><td>Olympus, Japan</td><td>UPlanSapo, 100X, NA 1.4</td><td></td></tr><tr><td>80-nm gold colloid</td><td>BBI Solutions, UK</td><td>EM.GC80</td><td></td></tr><tr><td>supercontinuum laser</td><td>Fianium, United Kingdom</td><td>SC400-2-PP</td><td></td></tr><tr><td>broadband dielectric mirrors</td><td>Thorlabs, USA</td><td>BB1-E02</td><td></td></tr><tr><td>field emission SEM</td><td>JEOL, Japan</td><td>JSM-6330F</td><td>optional</td></tr><tr><td>spectrometer</td><td>Andor Technology, UK</td><td>Shamrock 163</td><td></td></tr><tr><td>charge-coupled device</td><td>Andor Technology, UK</td><td>iDus DV420A-OE</td><td></td></tr><tr><td>acousto-optic modulators</td><td>IntraAction Corp., USA</td><td>AOM-402AF1</td><td></td></tr><tr><td>lock-in amplifier</td><td>Stanford Research Systems, USA</td><td>SR-830</td><td></td></tr><tr><td>MAS-coated slide glass</td><td>Matsunami Glass, Japan,</td><td>S9215</td><td></td></tr></tbody></table>

measurement of scattering nonlinearities from a single plasmonic nanoparticle

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus have potential applications in nonlinear optics, which requires extraordinary optical intensity. One of the most studied nonlinearities in plasmonics is nonlinear absorption, including saturation and reverse saturation behaviors. Although scattering and absorption in nanoparticles are closely correlated by the Mie theory, there has been no report of nonlinearities in plasmonic scattering until very recently. 
Last year, not only saturation, but also reverse saturation of scattering in an isolated plasmonic particle was demonstrated for the first time. The results showed that saturable scattering exhibits clear wavelength dependence, which seems to be directly linked to the localized surface plasmon resonance (LSPR). Combined with the intensity-dependent measurements, the results suggest the possibility of a common mechanism underlying the nonlinear behaviors of scattering and absorption. These nonlinearities of scattering from a single gold nanosphere (GNS) are widely applicable, including in super-resolution microscopy and optical switches. 
In this paper, it is described in detail how to measure nonlinearity of scattering in a single GNP and how to employ the super-resolution technique to enhance the optical imaging resolution based on saturable scattering. This discovery features the first super-resolution microscopy based on nonlinear scattering, which is a novel non-bleaching contrast method that can achieve a resolution as low as l/8 and will potentially be useful in biomedicine and material studies.

图6显示了从一个80纳米的GNS测量光谱。一组基于米氏理论计算出的曲线给出了相同的情节，表现出良好的协议。该LSPR峰值大约是580纳米。在以下的实验中，激光波长为532纳米，这被选择，因为它位于电浆频带内，以提高光散射与电浆效果和使散射饱和21。 图7呈现在不同的激发强度的散射单个金纳米颗粒的图像，且最底行提供每个粒子以突?...

Watch this Scientific Journal Video about Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle at JoVE.com

Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle

Saturable and reverse saturable scattering were discovered in isolated plasmonic particles and adopted as a novel non-bleaching contrast method in super-resolution microscopy. Here the experimental procedures of detecting and extracting nonlinear scattering are explained in detail, as well as how to enhance resolution with the aid of saturated excitation microscopy.

从单电浆纳米粒子散射非线性测量

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus ...

measurement-scattering-nonlinearities-from-single-plasmonic

Universidad de Cartagena UDEC

Research

JoVE Journal

Engineering

12.8K 次观看.  National Taiwan University. Saturable and reverse saturable scattering were discovered in isolated plasmonic particles and adopted as a novel non-bleaching contrast method in super-resolution microscopy. Here the experimental procedures of detecting and extracting nonlinear scattering are explained in detail, as well as how to enhance resolution with the aid of saturated excitation microscopy. 

视频: Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle

Evaluating Plasmonic Transport in Current-carrying Silver Nanowires

Plasmonics is an emerging technology capable of simultaneously transporting a plasmonic signal and an electronic signal on the same information support1,2,3. In this context, metal nanowires are especially desirable for realizing dense routing networks4. A prerequisite to operate such shared nanowire-based platform relies on our ability to electrically contact individual metal nanowires and efficiently excite surface plasmon polaritons5 in this information support. In this article, we describe a protocol to bring electrical terminals to chemically-synthesized silver nanowires6 randomly distributed on a glass substrate7. The positions of the nanowire ends with respect to predefined landmarks are precisely located using standard optical transmission microscopy before encapsulation in an electron-sensitive resist. Trenches representing the electrode layout are subsequently designed by electron-beam lithography. Metal electrodes are then fabricated by thermally evaporating a Cr/Au layer followed by a chemical lift-off. The contacted silver nanowires are finally transferred to a leakage radiation microscope for surface plasmon excitation and characterization8,9. Surface plasmons are launched in the nanowires by focusing a near infrared laser beam on a diffraction-limited spot overlapping one nanowire extremity5,9. For sufficiently large nanowires, the surface plasmon mode leaks into the glass substrate9,10. This leakage radiation is readily detected, imaged, and analyzed in the different conjugate planes in leakage radiation microscopy9,11. The electrical terminals do not affect the plasmon propagation. However, a current-induced morphological deterioration of the nanowire drastically degrades the flow of surface plasmons. The combination of surface plasmon leakage radiation microscopy with a simultaneous analysis of the nanowire electrical transport characteristics reveals the intrinsic limitations of such plasmonic circuitry.

Silver nanowires can simultaneously transport electrons and optical information in the form of surface plasmons. A procedure is described here to realize such a shared circuitry and the limitations at propagating both information carriers are evaluated.

Plasmonics is an emerging technology capable of simultaneously transporting a plasmonic signal and an electronic signal on the same information ...

科研

工程学

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

为增强微型和纳米操纵利用电浆和光子晶体纳米结构

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

生物工程

Analyzing the Movement of the Nauplius 'Artemia salina' by Optical Tracking of Plasmonic Nanoparticles

We demonstrate how optical tweezers may provide a sensitive tool to analyze the fluidic vibrations generated by the movement of small aquatic organisms. A single gold nanoparticle held by an optical tweezer is used as a sensor to quantify the rhythmic motion of a Nauplius larva (Artemia salina) in a water sample. This is achieved by monitoring the time dependent displacement of the trapped nanoparticle as a consequence of the Nauplius activity. A Fourier analysis of the nanoparticle&#39;s position then yields a frequency spectrum that is characteristic to the motion of the observed species. This experiment demonstrates the capability of this method to measure and characterize the activity of small aquatic larvae without the requirement to observe them directly and to gain information about the position of the larvae with respect to the trapped particle. Overall, this approach could give an insight on the vitality of certain species found in an aquatic ecosystem and could expand the range of conventional methods for analyzing water samples.

Analyzing the Movement of the Nauplius 'Artemia salina' by Optical Tracking of Plasmonic Nanoparticles

We use optical tracking of plasmonic nanoparticles to probe and characterize the frequency movements of aquatic organisms.

分析无节幼体的运动“<em&gt;卤虫</em&gt;&#39;由电浆纳米粒子的光学跟踪

We demonstrate how optical tweezers may provide a sensitive tool to analyze the fluidic vibrations generated by the movement of small aquatic organisms. A ...

Colloidal Synthesis of Nanopatch Antennas for Applications in Plasmonics and Nanophotonics

We present a method for colloidal synthesis of silver nanocubes and the use of these in combination with a smooth gold film, to fabricate plasmonic nanoscale patch antennas. This includes a detailed procedure for the fabrication of thin films with a well-controlled thickness over macroscopic areas using layer-by-layer deposition of polyelectrolyte polymers, namely poly(allylamine) hydrochloride (PAH) and polystyrene sulfonate (PSS). These polyelectrolyte spacer layers serve as a dielectric gap in between silver nanocubes and a gold film. By controlling the size of the nanocubes or the gap thickness, the plasmon resonance can be tuned from about 500 nm to 700 nm. Next, we demonstrate how to incorporate organic sulfo-cyanine5 carboxylic acid (Cy5) dye molecules into the dielectric polymer gap region of the nanopatch antennas. Finally, we show greatly enhanced fluorescence of the Cy5 dyes by spectrally matching the plasmon resonance with the excitation energy and the Cy5 absorption peak. The method presented here enables the fabrication of plasmonic nanopatch antennas with well-controlled dimensions utilizing colloidal synthesis and a layer-by-layer dip-coating process with the potential for low cost and large-scale production. These nanopatch antennas hold great promise for practical applications, for example in sensing, ultrafast optoelectronic devices and for high-efficiency photodetectors.

A protocol for the colloidal synthesis of silver nanocubes and fabrication of plasmonic nanoscale patch antennas with sub-10 nm gaps is presented.

在等离子体激元和纳米光子学应用Nanopatch天线的合成胶体

We present a method for colloidal synthesis of silver nanocubes and the use of these in combination with a smooth gold film, to fabricate plasmonic ...

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this work, we use single particle tracking of fluorescently labeled PNIPAM microgels as a showcase for tuning microgel size by a rapid non-stirred precipitation polymerization procedure. This approach is well suited for prototyping new reaction compositions and conditions or for applications that do not require large amounts of product. Microgel synthesis, particle size and structure determination by dynamic and static light scattering are detailed in the protocol. It is shown that the addition of functional comonomers can have a large influence on the particle nucleation and structure. Single particle tracking by wide-field fluorescence microscopy allows for an investigation of the diffusion of labeled tracer microgels in a concentrated matrix of non-labeled microgels, a system not easily investigated by other methods such as dynamic light scattering.

Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels

Non-stirred precipitation polymerization provides a rapid, reproducible prototyping approach to the synthesis of stimuli-sensitive poly(N-isopropylacrylamide) microgels of narrow size distribution. In this protocol synthesis, light scattering characterization and single particle fluorescence tracking of these microgels in a wide-field microscopy setup are demonstrated.

可控合成和高度统一聚荧光追踪（<em&gt;ñ</em&gt; -isopropylacrylamide）微凝胶

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this ...

化学

Plasmonic Trapping and Release of Nanoparticles in a Monitoring Environment

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few can observe immobilized particles. Moreover, a limited number of studies have experimentally measured the exertable forces on the particles. The designs can be classified as the protruding nanodisk type or the suppressed nanohole type. For the latter, microscopic observation is extremely challenging. In this paper, a new plasmonic tweezer system is introduced to monitor particles, both in directions parallel and orthogonal to the symmetric axis of a plasmonic nanohole structure. This feature enables us to observe the movement of each particle near the rim of the nanohole. Furthermore, we can quantitatively estimate the maximal trapping forces using a new fluidic channel.

A microchip fabrication process that incorporates plasmonic tweezers is presented here. The microchip enables the imaging of a trapped particle to measure maximal trapping forces.

电浆捕获和纳米粒子的释放在监控环境

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few ...

Fabrication of Periodic Gold Nanocup Arrays Using Colloidal Lithography

Within recent years, the field of plasmonics has exploded as researchers have demonstrated exciting applications related to chemical and optical sensing in combination with new nanofabrication techniques. A plasmon is a quantum of charge density oscillation that lends nanoscale metals such as gold and silver unique optical properties. In particular, gold and silver nanoparticles exhibit localized surface plasmon resonances-collective charge density oscillations on the surface of the nanoparticle-in the visible spectrum. Here, we focus on the fabrication of periodic arrays of anisotropic plasmonic nanostructures. These half-shell (or nanocup) structures can exhibit additional unique light-bending and polarization-dependent optical properties that simple isotropic nanostructures cannot. Researchers are interested in the fabrication of periodic arrays of nanocups for a wide variety of applications such as low-cost optical devices, surface-enhanced Raman scattering, and tamper indication. We present a scalable technique based on colloidal lithography in which it is possible to easily fabricate large periodic arrays of nanocups using spin-coating and self-assembled commercially available polymeric nanospheres. Electron microscopy and optical spectroscopy from the visible to near-infrared (near-IR) was performed to confirm successful nanocup fabrication. We conclude with a demonstration of the transfer of nanocups to a flexible, conformal adhesive film.

We demonstrate the fabrication of periodic gold nanocup arrays using colloidal lithographic techniques and discuss the importance of nanoplasmonic films.

用胶体光刻制备周期性金 Nanocup 阵列

Within recent years, the field of plasmonics has exploded as researchers have demonstrated exciting applications related to chemical and optical sensing ...

Determination of the Excitation and Coupling Rates Between Light Emitters and Surface Plasmon Polaritons

We have developed a unique method to measure the excitation and coupling rates between the light emitters and surface plasmon polaritons (SPPs) arising from metallic periodic arrays without involving time-resolved techniques. We have formulated the rates by quantities that can be measured by simple optical measurements. The instrumentation based on angle- and polarization-resolved reflectivity and photoluminescence spectroscopy will be described in detail here. Our approach is intriguing due to its simplicity, which requires routine optics and several mechanical stages, and thus is highly affordable to most of the research laboratories.

This protocol describes the instrumentation for determining the excitation and coupling rates between light emitters and Bloch-like surface plasmon polaritons arising from periodic arrays.

光发射器与表面等离子体极化激元的激发和耦合速率的测定

We have developed a unique method to measure the excitation and coupling rates between the light emitters and surface plasmon polaritons (SPPs) arising ...

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

使用单纳米粒子暗场显微镜可视化细胞膜上金纳米棒的扩散动力学

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

生物化学

Performing Spectroscopy on Plasmonic Nanoparticles with Transmission-Based Nomarski-Type Differential Interference Contrast Microscopy

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using visible-range light. The purpose of this protocol is to detail a proven method for preparing plasmonic nanoparticle samples and performing single particle spectroscopy on them with DIC microscopy. Several important steps must be followed carefully in order to perform repeatable spectroscopy experiments. First, landmarks can be etched into the sample substrate, which aids in locating the sample surface and in tracking the region of interest during experiments. Next, the substrate must be properly cleaned of debris and contaminants that can otherwise hinder or obscure examination of the sample. Once a sample is properly prepared, the optical path of the microscope must be aligned, using Kohler Illumination. With a standard Nomarski style DIC microscope, rotation of the sample may be necessary, particularly when the plasmonic nanoparticles exhibit orientation-dependent optical properties. Because DIC microscopy has two inherent orthogonal polarization fields, the wavelength-dependent DIC contrast pattern reveals the orientation of rod-shaped plasmonic nanoparticles. Finally, data acquisition and data analyses must be carefully performed. It is common to represent DIC-based spectroscopy data as a contrast value, but it is also possible to present it as intensity data. In this demonstration of DIC for single particle spectroscopy, the focus is on spherical and rod-shaped gold nanoparticles.

The goal of this protocol is to detail a proven approach for the preparation of plasmonic nanoparticle samples and for performing single particle spectroscopy on them with differential interference contrast (DIC) microscopy.

从单电浆纳米粒子散射非线性测量

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