笔者要感谢NanoVentures澳大利亚旅行的资金支持，并教授约翰·海科克为被部分托管于该研究在谢菲尔德和Jaimee梅恩女士的大学为她拍摄时的帮助。

1.金自然保护区准备注意：金卢比可通过许多配方16来合成，或者从商业供应商购买。 <ol><li>通过UV-Vis光谱，通过与0.5-2纳米的分辨率记录从300纳米到1000纳米的吸收值测量在Au NR溶液的初始光密度（OD）。而变化，以用于与可用比色皿中的溶液的体积。 </li><li>评估的初始的NP摩尔浓度与合适的技术17（ 例如 UV-Vis光谱，单粒电感耦合等离子体质谱法，透射电子显微镜）或使用由供应商提供的浓度值。 </li><li>通过稀释的初始的Au NR样品达到的OD = 1。对于最好的重复性，保持外径恒定对于所有测试的样品制备5 ml储备溶液。按照商业供应商的协议，用于稀释的溶剂的组合物。如果不确定中，使用去离子水。 </li><li>离心机将1ml的Au NR溶液两次15分钟，在7800×g离心以除去任何化学过量的溶液。离心循环可以在力量和时间（ 如 20分钟5600×克）18有所不同。 </li><li>除去上清液并重新悬浮在去离子水中的卢比。作为重悬浮颗粒可能会形成聚集在溶液中，每天准备他们有最好的结果。可替代地，在冰箱商店不超过1周。不要冷冻。 </li><li>细胞培养条件下，使用前，超声处理在Au NR溶液中5分钟，然后用UV光杀菌30分钟（UV辐射强度不小于400毫瓦∙米-2在254nm）。 </li></ol> 2. NG108-15神经细胞培养和分化<ol><li>对于细胞培养基，制成500毫升之无菌的Dulbecco含有10％（重量/体积）胎牛血清（FCS）的改良的Eagle培养基（DMEM），1％（重量/体积）的L-谷氨酰胺，1％的（重量/体积）青霉素/链霉素和0.5％（重量/体积）的两性霉素B. 注意：补充剂可以等分，储存在-20℃，并加入到所需要的天介质。细胞培养基可冷藏在无菌条件下，最多为1个月。 </li><li>对于细胞分化培养基，制备50毫升含有1％无菌DMEM中（重量/体积）的L-谷氨酰胺，1％（重量/体积）青霉素/链霉素和0.5％（重量/体积）的两性霉素B. </li><li>生长在（在37℃，5％CO 2）的聚苯乙烯制在潮湿气氛的培养箱T75烧瓶NG108-15神经细胞在10ml细胞培养基。通常情况下，种子1.5-2×10 5个细胞在每个烧瓶中，以准备在3-4天。改变细胞培养基每两天。 注意：为防止遗传漂移或更改，请不要使用超过21通道的实验细胞。 </li><li>当70-80％融合的文化，用温暖的新鲜细胞培养基改变介质。机械轻轻KNOC分离细胞王汇合烧瓶底部。不要使用胰蛋白酶。 </li><li>离心细胞悬浮液5分钟，于600×g离心并重新悬浮细胞沉淀在2ml温暖细胞分化培养基。 </li><li>种子2×10 4细胞/ cm 2在组织培养聚苯乙烯96孔板用200μl细胞分化培养基。孵育实验1天在5％的CO 2/37℃。 </li><li>添加的金NR溶液之间3.2×10 9 -4.2×1 0 10个粒子/ ml，第2天，并培育它用于另外24小时。没有为对照实验中添加粒子。 注意：作为一个替代的控制，可用于比较目的的14金纳米粒子具有分化良好的峰值吸收波长。对于一个更加一致的细胞行为，保持细胞密度不变，不修改之前细胞种植井表面。 </li></ol> 3.突起生长增强<ol><li>对与单模光纤的激光器（数值孔径= 0.13），并用一光纤连接器终止它（FC连接器是方便和通常可获得的）。测量输出激光功率与标准功率计。以获得最有效的结果，匹配激光到卢比的等离子体共振峰的峰值波长。 </li><li>在第3天以下NR孵化，修复FC连接器的很好。照射样品和对照在室温连续波1分钟，为不同的激光功率。允许培养至在5％的CO 2/37℃进行3天的附加 ​​。重复激光照射了至少3个独立的测量。 注意：不同的照射时间和脉冲频率可被选择，这取决于应用。 </li><li>表征激光光束直径和激光辐照方面（W∙厘米-2）。 <ol><li>对于标准单模光纤，用NA = N∙罪θ，其中n是所使用的介质的折射率，θ是光离开所述光纤的锥体的半角（参照图1A）。从三角，R =黄褐色θ∙d，其中r是光束半径，d是纤维和样品之间的距离。在FC连接器的直径匹配良好，因此照明在距离 d 2.70±0.20毫米样品=。光从水中纤维（N = 1.33），使R =棕褐色（SIN-1（NA / N））∙ð。 </li><li>使用后者方程，计算出的激光束的半径，对应的光束面积和平均激光辐照（激光功率由波束面积除以）在目标（参见图1B的例子中图）。这些值代表平均照度以上的照明面积。 </li><li>评估与错误传播的一般理论的测量误差。 注意：距离between对FC连接器和所述样品可通过实验装置使用适当的软件（ 例如 ImageJ的）图像和后处理的照片来测量。 </li></ol></li><li>在第5天，从实验除去细胞分化培养基和固定样品以3.7％为10分钟（V / V）的甲醛溶液，再透化的细胞含有0.1％（体积/体积）的Triton X-100进行20分钟。 </li><li>加入3％（重量/体积）牛血清白蛋白（BSA）的样品60分钟，以封闭未反应的蛋白质结合位点。标记的样品用于抗βIII微管蛋白过夜（5微克/毫升的PBS补充有1％BSA的）在4℃下。 </li><li>孵育所述细胞90分钟，在黑暗中与合适的二级抗体（ 例如，TRITC标记的抗小鼠IgG抗体），在PBS中的1％BSA中使用0.4-2微克/ ml的浓度。标记细胞核用DAPI（0.1微克/毫升的去离子水）10分钟。 注：抗体偶联和浓度MIGHT根据公司的协议而变化。洗涤样品，用PBS两次，每次染色阶段之后5分钟。 <img alt="图1" src="/files/ftp_upload/52566/52566fig1.jpg" /></li><li>图像通过荧光或共聚焦显微镜使用至少一个20×的目标样本。选择显微镜相应滤波器的二级抗体，选择DAPI过滤器（λ= EX 358纳米;λEM = 488纳米），以可视化的细胞核。 </li><li>通过评估分析图像：ⅰ）的最大神经突长度（记录从突起到细胞体的开头的前端的长度），ⅱ）每神经细胞神经突的数目（总结所有每个细胞的神经突的）和iii）细胞与神经突的百分比（除以细胞表达βIII微管蛋白通过细胞与阳性染色细胞核的总数）19的总数。 </li></ol> 4.激光诱导细胞内Ca 2+成像<ol&gt; <li>制备20％（重量/体积）的库存的尼克F-127的溶液通过在10ml无水二甲基亚砜（DMSO）中溶解2克溶质。热尼克F-127的在40℃的溶液中约20分钟，以增加溶解度。它准备提前如果储存在室温。 </li><li>用于以下NR孵育3天，准备一个平衡盐溶液（BSS; 135 mM氯化钠，4.5 mM的氯化钾，1.5mM的氯化钙 ，0.5mM的氯化镁 ，5.6毫摩尔葡萄糖和10mM HEPES，pH 7.4）中，并用5补充它的尼克F-127溶液的Fluo-4 AM在DMSO中和0.1％μM（重量/体积）。 注意：BSS解决方案可以预先准备并冷藏最长不超过1个月。添加补充剂（的Fluo-4 AM和尼克F-127），在实验的日子。 </li><li>除去细胞的分化培养基，并将其与补充的BSS溶液替代。以获得具有一个倒置的共焦显微镜的最好的结果，孵化的细胞中的微滑动良好。 </li><li>负载NG108-15细胞荧光 - 上午04点对于在5％的CO 2/37℃，在黑暗中30分钟。继孵化时间，洗两次样本BSS，以消除任何外卸载荧光。 </li><li>使用标准技术耦合激光器与单模光纤和劈开的尖端。观察所得的尖端在光学显微镜下，以确保高质量的表面（ 即尖端应垂直于纤维轴和平坦经显微镜检查）。 <ol><li>测量输出激光功率与标准功率计。插入的光传输光纤到光纤保持器，并将其粘贴到一个微定位。参阅Brown 等人的 20对光纤制备和定位的更多细节。 注意：激光功率的测量过程中遵循激光安全的一般规则（ 例如，不要直视激光束，在操作时戴好激光护目镜，防止杂散光接触到其他实验室用户）&lt;。/ li&gt; </ol></li><li>示波器连接到便携式激光监视光调制。使用二进制信号具有可变的频率（0.5-2赫兹）和脉冲长度（20-100毫秒）。连接使用调制信号作为晶体管-晶体管逻辑（TTL）的激光输入的显微镜，按照Paviolo 等人13所示的设置。 <ol><li>放置细胞的倒置共焦显微镜下的光递送纤维250±50μm的位置远离透射照明模式中的目标小区。在这个距离上，使用提出的方程3.3.1来计算光束半径的目标。 </li></ol></li><li>使用氩离子激光器（488纳米）激发内在的Fluo-4AM染料（λEX = 494纳米;λEM = 516 nm），而同步的LD的内吞卢比的激励。通过收集时间序列的扫描执行成像时，使用至少一个40×物镜样品和对照的逆转录具有256×256像素/英寸往返模式帧的分辨率至少为4赫兹的频率。没有任何的LD激励执行记录，以确定任何氩离子激光干涉（基线噪声）。 </li><li>删除数据使用自适应加权惩罚最小二乘算法21的背景。绘制诱导的Ca 2+变化作为时间的函数。在一定的水平分析使用由阈值图像后处理软件中的荧光强度瞬时峰值的基线噪声（3σ）的3倍的标准偏差。 </li></ol>

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Nanobiotechnology. 12 (1), 31 (2014).</li><li>Ebbesen, C. L., Bruus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+laser-induced+heating+in+optical+neuronal+guidance.">Analysis of laser-induced heating in optical neuronal guidance.</a> J. Neurosci. Meth. 209 (1), 168-177 (2012).</li><li>Iwanaga, S., 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=Location-dependent+photogeneration+of+calcium+waves+in+HeLa+cells.">Location-dependent photogeneration of calcium waves in HeLa cells.</a> Cell Biochem. Biophys. 45 (2), 167-176 (2006).</li><li>Huang, X., 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=Determination+of+the+minimum+temperature+required+for+selective+photothermal+destruction+of+cancer+cells+with+the+use+of+immunotargeted+gold+nanoparticles.">Determination of the minimum temperature required for selective photothermal destruction of cancer cells with the use of immunotargeted gold nanoparticles.</a> Photochem. Photobiol. 82 (2), 412-417 (2006).</li><li>Connor, E. E., Mwamuka, J., Gole, A., Murphy, C. J., Wyatt, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanoparticles+are+taken+up+by+human+cells+but+do+not+cause+acute+cytotoxicity.">Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity.</a> Small. 1 (3), 325-327 (2005).</li><li>Isomaa, B., Reuter, J., Djupsund, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+subacute+and+chronic+toxicity+of+cetyltrimethylammonium+bromide+(CTAB),+a+cationic+surfactant,+in+the+rat.">The subacute and chronic toxicity of cetyltrimethylammonium bromide (CTAB), a cationic surfactant, in the rat.</a> Arch. Toxicol. 35 (2), 91-96 (1976).</li><li>Juste, J., Pastoriza-Santos, I., Liz-Marz&#225;n, L., Mulvaney, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanorods:+synthesis,+characterization+and+applications.">Gold nanorods: synthesis, characterization and applications.</a> Coord. Chem. Rev. 249 (17-18), 1870-1901 (2005).</li><li>Dreaden, E. C., Alkilany, A. M., Huang, X., Murphy, C. J., 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=The+golden+age:+gold+nanoparticles+for+biomedicine.">The golden age: gold nanoparticles for biomedicine.</a> Chem. Soc. Rev. 41 (7), 2740-2779 (2012).</li><li>Huang, X., 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=Plasmonic+photothermal+therapy+(PPTT)+using+gold+nanoparticles.">Plasmonic photothermal therapy (PPTT) using gold nanoparticles.</a> Lasers Med. Sci. 23 (3), 217-228 (2008).</li><li>Albert, E. S., 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=TRPV4+channels+mediate+the+infrared+laser-evoked+response+in+sensory+neurons.">TRPV4 channels mediate the infrared laser-evoked response in sensory neurons.</a> J. Neurophysiol. 107 (12), 3227-3234 (2012).</li><li>Garcia-Elias, A., 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=Phosphatidylinositol-4,5-biphosphate-dependent+rearrangement+of+TRPV4+cytosolic+tails+enables+channel+activation+by+physiological+stimuli.">Phosphatidylinositol-4,5-biphosphate-dependent rearrangement of TRPV4 cytosolic tails enables channel activation by physiological stimuli.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (23), 9553-9558 (2013).</li><li>Shapiro, M. G., Homma, K., Villarreal, S., Richter, C. -. P., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+light+excites+cells+by+changing+their+electrical+capacitance.">Infrared light excites cells by changing their electrical capacitance.</a> Nat. Commun. 3 (736), (2012).</li><li>Roggan, A., Friebel, M., D&#246;rschel, K., Hahn, A., M&#252;ller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+properties+of+circulating+human+blood+in+the+wavelength+range+400-2500+nm.">Optical properties of circulating human blood in the wavelength range 400-2500 nm.</a> J. Biomed. Opt. 4 (1), 36-46 (1999).</li><li>Byrnes, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+promotes+regeneration+and+functional+recovery+and+alters+the+immune+response+after+spinal+cord+injury.">Light promotes regeneration and functional recovery and alters the immune response after spinal cord injury.</a> Lasers Surg. Med. 36 (3), 171-185 (2005).</li><li>Wu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=810+nm+wavelength+light:+an+effective+therapy+for+transected+or+contused+rat+spinal+cord.">810 nm wavelength light: an effective therapy for transected or contused rat spinal cord.</a> Lasers Surg. Med. 41 (1), 36-41 (2009).</li><li>Grossman, N., Schneid, N., Reuveni, H., Halevy, S., Lubart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=780+nm+low+power+diode+laser+irradiation+stimulates+proliferation+of+keratinocyte+cultures:+involvement+of+reactive+oxygen+species.">780 nm low power diode laser irradiation stimulates proliferation of keratinocyte cultures: involvement of reactive oxygen species.</a> Lasers Surg. Med. 22 (4), 212-218 (1998).</li><li>Wong-Riley, M. T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobiomodulation+directly+benefits+primary+neurons+functionally+inactivated+by+toxins+-+Role+of+cytochrome+c+oxidase.">Photobiomodulation directly benefits primary neurons functionally inactivated by toxins - Role of cytochrome c oxidase.</a> J. Biol. 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The authors have no competing interests to disclose.

在这个演讲中概述的协议描述了如何培养，区分和使用光学吸收外在刺激神经细胞。了NR特性（ 例如尺寸，形状，等离子体共振波长和表面化学）和激光的刺激参数（如波长，脉冲长度，重复率等 ）可以变化，以满足不同的实验的需要。可以使用标准的生物测定法和材料来监测对细胞行为的影响。总的来说，方法提供了一种简单，但功能强大，这样照射细胞群在体外 ，并且?...

最近的研究表明，用红外光的由水（波长&gt; 1400纳米）的吸收相关的瞬态加热可以用于诱导动作电位的神经组织1，并在心肌2细胞内钙瞬变的。使用红外光的提出用于神经假体的应用极大的兴趣，由于潜在的更精细的空间分辨率，缺乏与组织去除的必要基因的直接接触，最小化刺激伪影，并修改之前刺激细胞（根据需要在光遗传学）1。尽管所有的这些优点，最近开发的热模型表明，目标组织/细胞可能受到的累积热效应，当多个刺激位点和/或高重复率使用3,4。 为了应对这些挑战，研究人员已经认识到使用外在absor电位的BER为神经刺激，以产生在组织中更局部加热效应。 Huang 等人通过使用超顺磁性铁氧体纳米远程激活温度敏感的TRPV1通道的HEK 293细胞与射频磁场表现出5这一原则。尽管这种技术可以允许更深的渗透（磁场相互作用相对较弱与组织），则反应进行仅记录在几秒钟期间，而不是在仿生器件5所需的毫秒的持续时间。大鼠皮质神经元用黑色微粒在体外的类似的，法拉等人证明电刺激。它们表现出刺激细胞级精度使用脉冲持续时间数百微秒和能量在μJ的范围的顺序，潜在地允许更快的重复率6。 使用外在吸收剂也被应用以诱导形态学变化的抑制作用 。 Ciofani 等显示在神经元细胞生长增加了〜40％的使用由超声7激发压电氮化硼纳米管。同样地，内吞氧化铁纳米颗粒在PC12细胞中已经报道了以提高神经突分化以剂量依赖的方式，由于细胞粘附分子的活化与氧化铁8。 近日，在吸收外在的利益，协助神经刺激还注重利用金纳米粒子（金纳米粒子）。金纳米粒子具有有效地吸收激光光线的电浆峰和消散它到周围环境中的热量9的形式的能力。在所有可用的粒子形状，金纳米棒的光吸收（凹NRS）方便地匹配的生物组织的治疗窗（近红外-红外，750-1,400纳米之间的波长）10。此外，在续神经刺激的电话分机，采用的Au卢比的提供了相对良好的生物相容性和广泛的表面功能选项11。最近的研究已经表明，在分化的刺激作用能够的Au卢比的NG108-15神经细胞12连续激光照射后所诱导。同样，细胞内钙瞬变记录后激光照射调制可变频率和脉冲长度13金自然保护区培养神经细胞。金自然保护区的螺旋神经节神经元14的原代培养NIR激光照射后，还记录了细胞膜去极化。首先在与辐照金自然保护区体内应用已被证明只是最近。 EOM和同事露出的Au卢比在其电浆峰和记录在6倍的增加的化合物的神​​经动作电位（CNAPS）的幅度和一个3倍的降低，在刺激阈值中的大鼠坐骨神经。带连接hanced响应归因于从NR电浆峰15的激发导致局部加热效应。 在本论文，调查激光刺激与金自然保护区培养NG108-15神经细胞的影响，协议规定。这些方法提供了一个简单，但强大的方式使用标准的生物技术和材料照射细胞群在体外 。所述协议是基于一个光纤耦合激光二极管（LD），允许安全操作和重复的排列。在Au NR样品制备和激光照射方法可以进一步扩展到不同的颗粒形状和神经元细胞培养物，提供了具体的合成和培养协议是已知的，分别。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Au NR</td>
			<td>Sigma Aldrich</td>
			<td>716812</td>
			<td></td>
		</tr>
		<tr>
			<td>NG108-15</td>
			<td>Sigma Aldrich</td>
			<td>8811230</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma Aldrich</td>
			<td>D6546</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Life Technologies</td>
			<td>10100147</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Life Technologies</td>
			<td>15290018</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>BDH</td>
			<td>T8532</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>A2058</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#946;III-tubulin</td>
			<td>Promega</td>
			<td>G7121</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC-conjugated anti-mouse IgG antibody</td>
			<td>Sigma Aldrich</td>
			<td>T5393</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-4 AM</td>
			<td>Invitrogen</td>
			<td>F14201</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Invitrogen</td>
			<td>P6867</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Vis spectrometer</td>
			<td>Varian Medical Systems Inc.</td>
			<td>Cary 50 Bio</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini centrifuge</td>
			<td>Eppendorf</td>
			<td>Mini Spin</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonic bath</td>
			<td>Unisonics Australia</td>
			<td>FPX 10D</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Kendro</td>
			<td>Hera Cell 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture centrifuge</td>
			<td>Hettich</td>
			<td>Rotofix 32A</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser diode</td>
			<td>Optotech</td>
			<td>780 nm single mode fibre - coupled LD</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical fiber</td>
			<td>Thorlabs</td>
			<td>780 HP</td>
			<td></td>
		</tr>
		<tr>
			<td>Power meter</td>
			<td>Coherent</td>
			<td>Laser Check</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>http://rsb.info.nih.gov/ij/index.html</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescent microscope</td>
			<td>Axon Instruments</td>
			<td>ImageX-press 5000A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#956;-slide well</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted confocal microscope</td>
			<td>Carl Zeiss Microscopy Ltd.</td>
			<td>LSM 510 meta-confocal microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>Tektronix</td>
			<td>TDS210</td>
			<td></td>
		</tr>
	</tbody>
</table>

gold nanorod-assisted optical stimulation of neuronal cells

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.

通过使用协议1，2和3在此描述时，观察到在NG108-15神经元细胞培养用Au纳米颗粒上分化的刺激作用（凹卢比，聚（苯乙烯磺酸盐）包被的金卢比和二氧化硅被覆的Au NRS）激光后1.25和7.5 W之间曝光·厘米-2。 rhodamineB标记的Au卢比的共焦图像证实，该颗粒是从孵育12的第1天内化。本地化是主要在细胞质观察到的，这表明吸收的优选机制是经由细胞体膜12。在NG108-15神经细胞的分化...

Watch this Scientific Journal Video about Gold Nanorod-assisted Optical Stimulation of Neuronal Cells at JoVE.com

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

金纳米棒辅助神经细胞的刺激光

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared ...

gold-nanorod-assisted-optical-stimulation-of-neuronal-cells

Research

JoVE Journal

Neuroscience

8.9K 次观看.  Swinburne University of Technology. This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

视频: Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

结合经颅磁刺激和功能磁共振成像检查默认模式网络

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

科研

神经科学

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS：使用的Theta突发协议，以探索个人与脆性X综合征与孤独症的可塑性Mechasnism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

NeuroStar TMS的设备进行美国FDA批准用于治疗抑郁症的议定书“

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

大鼠慢性经颅电刺激及 Intracortical 记录

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

小鼠胚胎干细胞体外神经元分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

无转基因生成血液衍生神经元细胞

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...

Methods Article

金纳米棒辅助神经细胞的刺激光