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>

<ol>
	<li>Richter, C. P., Matic, A. I., Wells, J. D., Jansen, E. D., Walsh, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stimulation+with+optical+radiation.">Neural stimulation with optical radiation.</a> Laser. Photonics Rev. 5 (1), 68-80 (2011).</li><li>Dittami, G. M., Rajguru, S. M., Lasher, R. A., Hitchcock, R. W., Rabbitt, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+calcium+transients+evoked+by+pulsed+infrared+radiation+in+neonatal+cardiomyocytes.">Intracellular calcium transients evoked by pulsed infrared radiation in neonatal cardiomyocytes.</a> J. Physiol. 589 (6), 1295-1306 (2011).</li><li>Thompson, A. C., Wade, S. A., Brown, W. G. A., Stoddart, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+of+light+absorption+in+tissue+during+infrared+neural+stimulation.">Modeling of light absorption in tissue during infrared neural stimulation.</a> J. Biomed. Opt. 17 (7), 075002-075002 (2012).</li><li>Thompson, A. C., Wade, S. A., Cadusch, P. J., Brown, W. G., Stoddart, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+of+the+temporal+effects+of+heating+during+infrared+neural+stimulation.">Modeling of the temporal effects of heating during infrared neural stimulation.</a> J. Biomed. Opt. 18 (3), 035004 (2013).</li><li>Huang, H., Delikanli, S., Zeng, H., Ferkey, D. M., Pralle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+control+of+ion+channels+and+neurons+through+magnetic-field+heating+of+nanoparticles.">Remote control of ion channels and neurons through magnetic-field heating of nanoparticles.</a> Nat. Nanotechnol. 5 (8), 602-606 (2010).</li><li>Farah, N., 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=Holographically+patterned+activation+using+photo-absorber+induced+neural-thermal+stimulation.">Holographically patterned activation using photo-absorber induced neural-thermal stimulation.</a> J. Neural. Eng. 10 (5), (2013).</li><li>Ciofani, G., 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=Enhancement+of+neurite+outgrowth+in+neuronal-like+cells+following+boron+nitride+nanotube-mediated+stimulation.">Enhancement of neurite outgrowth in neuronal-like cells following boron nitride nanotube-mediated stimulation.</a> ACS Nano. 4 (10), 6267-6277 (2010).</li><li>Kim, J. 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=Enhancement+of+neurite+outgrowth+in+PC12+cells+by+iron+oxide+nanoparticles.">Enhancement of neurite outgrowth in PC12 cells by iron oxide nanoparticles.</a> Biomaterials. 32 (11), 2871-2877 (2011).</li><li>Myroshnychenko, V., 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=Modelling+the+optical+response+of+gold+nanoparticles.">Modelling the optical response of gold nanoparticles.</a> Chem. Soc. Rev. 37 (9), 1792-1805 (2008).</li><li>Choi, W. I., Sahu, A., Kim, Y. H., Tae, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photothermal+cancer+therapy+and+imaging+based+on+gold+nanorods.">Photothermal cancer therapy and imaging based on gold nanorods.</a> Ann. Biomed. Eng. 40 (2), 534-546 (2011).</li><li>Zhan, Q., Qian, J., Li, X., He, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+mesoporous+silica-encapsulated+gold+nanorods+as+enhanced+light+scattering+probes+for+cancer+cell+imaging.">A study of mesoporous silica-encapsulated gold nanorods as enhanced light scattering probes for cancer cell imaging.</a> Nanotechnology. 21 (5), 055704 (2010).</li><li>Paviolo, 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=Laser+exposure+of+gold+nanorods+can+increase+neuronal+cell+outgrowth.">Laser exposure of gold nanorods can increase neuronal cell outgrowth.</a> Biotechnol. Bioeng. 110 (8), 2277-2291 (2013).</li><li>Paviolo, C., Haycock, J. W., Cadusch, P. J., McArthur, S. L., Stoddart, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+exposure+of+gold+nanorods+can+induce+intracellular+calcium+transients.">Laser exposure of gold nanorods can induce intracellular calcium transients.</a> J. Biophotonics. 7 (10), 761-765 (2014).</li><li>Yong, J., 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=Gold-nanorod-assisted+near-infrared+stimulation+of+primary+auditory+neurons.">Gold-nanorod-assisted near-infrared stimulation of primary auditory neurons.</a> Adv. Healthcare Mater. , (2014).</li><li>Eom, K., 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=Enhanced+infrared+neural+stimulation+using+localized+surface+plasmon+resonance+of+gold+nanorods.">Enhanced infrared neural stimulation using localized surface plasmon resonance of gold nanorods.</a> Small. , (2014).</li><li>Juste, J., Pastoriza-Santos, I., Liz-Marz&#225;n, L. M., 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> Coordination Chemistry Reviews. (17-18), 1870-1901 (2005).</li><li>Shang, J., Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+counting:+towards+accurate+determination+of+the+molar+concentration.">Nanoparticle counting: towards accurate determination of the molar concentration.</a> Chem. Soc. Rev. 43 (21), 7267-7278 (2014).</li><li>Sharma, V., Park, K., Srinivasarao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape+separation+of+gold+nanorods+using+centrifugation.">Shape separation of gold nanorods using centrifugation.</a> Proc. Natl. Acad. Sci. 106 (13), 4981-4985 (2009).</li><li>Kaewkhaw, R., Scutt, A. M., Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+site+influences+the+differentiation+of+adipose-derived+stem+cells+for+schwann-cell+phenotype+and+function.">Anatomical site influences the differentiation of adipose-derived stem cells for schwann-cell phenotype and function.</a> Glia. 59 (5), 734-749 (2011).</li><li>Brown, W. G. A., Needham, K., Nayagam, B. A., Stoddart, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+cell+patch+clamp+for+investigating+the+mechanisms+of+infrared+neural+stimulation.">Whole cell patch clamp for investigating the mechanisms of infrared neural stimulation.</a> JoVE. (77), (2013).</li><li>Cadusch, P. J., Hlaing, M. M., Wade, S. A., McArthur, S. L., Stoddart, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+for+fluorescence+background+subtraction+from+Raman+spectra.">Improved methods for fluorescence background subtraction from Raman spectra.</a> J. Raman Spectrosc. 44 (11), 1587-1595 (2013).</li><li>Daud, M. F. B., Pawar, K. C., Claeyssens, F., Ryan, A. J., Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+aligned+3D+neuronal-glial+co-culture+model+for+peripheral+nerve+studies.">An aligned 3D neuronal-glial co-culture model for peripheral nerve studies.</a> Biomaterials. 33 (25), 5901-5913 (2012).</li><li>Jung, 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=Intracellular+gold+nanoparticles+increase+neuronal+excitability+and+aggravate+seizure+activity+in+the+mouse+brain.">Intracellular gold nanoparticles increase neuronal excitability and aggravate seizure activity in the mouse brain.</a> PLoS ONE. 9 (3), e91360 (2014).</li><li>Salinas, K., Kereselidze, Z., DeLuna, F., Peralta, X., Santamaria, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+extracellular+application+of+gold+nanostars+increases+hippocampal+neuronal+activity.">Transient extracellular application of gold nanostars increases hippocampal neuronal activity.</a> J. 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. Chem. 280 (6), 4761-4771 (2005).</li><li>Beauvoit, B., Kitai, T., Chance, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+mitochondrial+compartment+to+the+optical+properties+pf+the+rat+liver:+a+theoretical+and+practical+approach.">Contribution of the mitochondrial compartment to the optical properties pf the rat liver: a theoretical and practical approach.</a> Biophys. J. 67 (6), 2501-2510 (1994).</li></ol>

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 ，并且可以扩展至原代细胞，组织样本和体内研究。 该凹卢比需要满足以用于生物应用的主要要求是稳定性（包括化学和物理）和生物相容性。后者是特别关键的，由于阳离子表面活性剂的存在下（常见LY CTAB）在金表面上。 CTAB是已知诱导的细胞毒性在体外28和体内29和它在合成过程中是常用的，以驱动NR-形状形成30。稳定性和生物相容性通常通过沉积在Au NR表面附加涂层（ 例如聚乙二醇，二氧化硅）31改善。为了评估生物相容性，不同测定可在体外使用（ 例如活/死，MTS，MTT等），而在组织实验7,8,12,15经常进行组织学分析。 另一个挑战面对当与纳米材料的工作是正确地确定它们的摩尔浓度的难度。在形状，大小和化学性质方面的巨大的各种纳米颗粒的使得技术当前可用的适合只有某些种类的颗粒。例如，在标准动态光scatt化工e圈方法假定纳米粒具有球形形状和散射光各向同性17。因此，应用此方法坳自然保护区结果测得的浓度与真钞存在差异的。 NP浓度的问题是特别成问题的，如果相关的纳米域，其中需要施用的浓度，以便最大限度地处理（ 例如，用于药物递送的应用程序）的功效和减少的纳米材料17的毒性被精确地控制。在此处报道的研究中，所使用的光学密度，得到良好的结果中的细胞生存力和粒子数计。 由于它们的固有吸收性能，金纳米粒子经常结合使用的激光源。在曝光，吸收和散射是可能发生在纳米颗粒的表面上的两个主要过程。如果激光波长的等离子体共振波长相匹配时，吸收常战胜散射，令人兴奋的传导电子在NP表面。这些形成远离其平衡位置，并创建一个谐振相干振荡称为局域型表面等离子体共振（LSPR）的电子气。能量随后被转移到对NP晶格热，这是其后迅速消退到周围环境32。因为热是NR LSPR的激发后所观察到的主要效果，最好是监视激光照射后的细胞活力。 据推测，对神经突增生和细胞内Ca 2+途径所观察到的影响是由于从LSPR的激励而产生的瞬时加热。这种假设是在与温度敏感的TRPV1通道的铁素体纳米粒子5的磁曝光后的激活线。这个过程也与观测的热敏感TRPV4渠道解放军一致的y红外线神经刺激33中起重要作用。在分子水平上，它最近已表明，TRPV4是热敏唯一的磷酸肌醇-4,5- -biphosphate与通道34的相互作用（PIP 2）之后。因此，它是可能的NR激发后所产生的瞬态加热可有助于加速和/或诱导的TRPV4通道的开口。此假设可以通过将来的Ca 2+的实验来确认通过使用钙离子-在画中画2耗尽的培养基和/或分子的控制。 不同的基团还表明，小的温度梯度超过生理温度范围内，可用于诱导其它反应，如神经元生长锥指导25或去极化在人胚肾细胞35的电流。勇和同事记录在初级听觉神经元邪教电信号活性显著增加为带有网络实名制在LSPR的激光照射后的硅涂层金卢比。由激光照射的粒子所产生的加热，使用膜片钳技术测定14。最近，严等人记录的3.4×10 9欧NRs的激光曝光后的增加CNAPS的振幅时在大鼠坐骨神经15的神经束的护套的附近连续灌注。 在实验中观察到的780nm的激光二极管的刺激作用是未链接的热量被水吸收而产生，因为这是公知的可以忽略不计在780nm处36。以前发表的结果也报道了780纳米的光在体内对37,38神经组织的刺激作用， 在体外研究已证明的活性氧，在过程39中的参与。然而，详细的机制，其中近红外刺激影响基因transcription和其他细胞活动目前尚未解决。当前数据表明，在不同的刺激效果，包括在线粒体内发色团吸收光子的相互作用（能量在780nm处的近50％的可能是由细胞色素C氧化酶被吸收）37,39-41，对钙改变膜的渗透性37和抑制炎症活性的细胞中的37。 在这个手稿中提出的研究结果表明，纳米颗粒吸收按住神经细胞的光刺激未来应用前景广阔。的主要优势在于NIR光的深入渗透组织（治疗窗）的能力。这些结果还表明，纳米颗粒吸收剂可以增强和/或替换的基于水的吸收红外线神经刺激的过程。用于神经假体未来的应用，这将是感兴趣的，研究了不同的表面functionalizat离子与神经元轴突，这对于许多光学刺激应用的主要目标化学亲和力。

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><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-&amp;beta;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>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>&amp;mu;-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神经细胞的分化诱导后检测到的主要形态变化是扩散的阻滞，βIII微管蛋白的蛋白质的表达和神经突的生长，这是在最大长度和数目22来分析。 样品自然保护区培养呈突起长度增加在激光irradi在这里测量ANCE水平（1.25和7.5 W之间·厘米-2）。对照样品（未经细胞培养的纳米粒和辐照以相同的激光功率）显示在长度没有显著变化。使用的7.5瓦·厘米-2，对NG108-15用Au卢比培养最终神经突长度的照射剂量为更高大致36％（P &lt;0.01），比非激光照射样品。这种行为不是专门联系到自然保护区的表面化学。这些值比由NG108开发-15单独并暴露于激光曝光（P &lt;0.05）12的值相同的突起较大，几乎20％的。这些结果与对PC12神经元细胞与压电碳纳米管培养并照射超声波辐射7先前公布的研究一致。 无金自然保护区控制实验还表明在神经元的比例有突起NE和数量方面的780纳米的光的某些刺激​​作用每个神经元urites。这种刺激是在较低的激光功率（为1.25W∙厘米-2），随后跌幅最高激光能量（7.5瓦∙厘米-2）12更有效。没有任何激光照射引起的纳米粒适度刺激（聚（苯乙烯磺酸盐）包被的和二氧化硅被覆只）中的神经元与突起12的比例进行检测。这些结果与最近发表的观察结果金纳米颗粒可以增加神经元的活动的体外 23,24线; 图2示出了分化NG108-15神经细胞单独培养（ 图2A）或用Au卢比的落射荧光图像（一个例子图2B ），并照射具有不同的激光功率（在图中表示）。 按照协议1，2使用脉冲近红外光的光生细胞内钙释放的潜力进行了评估，并4.钙离子起到不同的细胞活动，如有丝分裂，肌肉收缩和神经突伸展25,26中起重要作用。在响应于刺激， 钙离子增加时，振荡与降低，导致活化，调制特定细胞功能或终止。近来， 钙瞬变也已观察到心肌细胞红外激光曝光的结果。在这项工作中， 钙离子的反应诱发后IR暴露表现出较低的振幅和更快的恢复时间比自发钙瞬变2， 图3示出了装载的Fluo-4 AM和成象用NG108-15神经元细胞的一个例子共聚焦显微镜。的Fluo-4AM，观察到在非破坏性的方式进入细胞膜，从而导致整个细胞质指示器的大致均匀分布。检测只有轻微的核或细胞质细胞器染色。如前面所指出的，预计卢比将位于细胞内12。 NG108-15单独的神经元细胞或用Au卢比和聚（苯乙烯磺酸盐）培养的包被的金卢比被随后暴露于激光辐照0.07之间Ĵ∙cm -2并且370Ĵ∙cm -2时 ，用激光频率调制的范围0.5-2赫兹。 图4示出的响应的振幅如何被映射为时间的函数的代表性例子。的钙离子的反应而变化的辐射暴露（ 图4A - C）的振幅，并观察到不被一致地通过激光脉冲（ 图4C）13触发。最有可能的解释是细胞内Ca 2+存储归因于不完全的Ca 2+负荷2或在神经元细胞的不同效率NR内化的瞬态损耗。当文化性都没有使用卢比E（对照实验中， 图4D），也观察到了780nm的光的刺激作用。然而，这产生较低的荧光振幅的峰和激活的概率较低（中只有16％的分析样品的检测）。总体而言，NR激光诱导的细胞活化的48％的概率达到尽管背景事件由于780纳米的光，NR处理的细胞的曝光证明具有较低的激光能量和响应13的更高的峰值更高的刺激效率。事实上，钙的反应，发现峰在0.33Ĵ∙cm -2的在NR治疗的细胞。这是由于热抑制13。在实验过程中，没有证据起泡或任何其它形式的细胞膜破坏的检测，这与Huang 等人的结果所报告的蜂窝光破坏与为19W∙cm的相对较高的功率密度一致-2施加给4 2分钟7。被记录在NR处理的细胞无自发活动，而激光曝光。 <img alt="图1" src="/files/ftp_upload/52566/52566fig1.jpg" /> 图1:光学纤维实验装置（A）和平均激光辐照的激光功率区的激光束的函数等于至0.4mm 2（B）中 。束参数是:（A）光出射光纤（θ），光束半径（r）和所述光纤和所述样品（D）之间的距离的最大锥的半角。 <img alt="图2" src="/files/ftp_upload/52566/52566fig2.jpg" /> 图2:单独（A）中培养分化NG108-15神经元细胞或用Au卢比（B）的落射荧光图像的实施例和照射不同的激光功率（在图中表示）。样品我ncubated一天激光照射之前。细胞固定并标记为抗β-III微管蛋白（红色）和DAPI（蓝色）激光照射后三天。比例尺是100微米。 <img alt="图3" src="/files/ftp_upload/52566/52566fig3.jpg" /> 图3:装入Fluo4-AM的Ca 2+指示剂分化NG108-15神经细胞的实施例使用一个倒置的共焦显微镜用40X油浸物镜的图像拍摄。 <img alt="图4" src="/files/ftp_upload/52566/52566fig4.jpg" /> 图4:激光诱导的Ca 2+变化作为时间的NG108-15神经细胞三天用在无血清条件下培养的函数的代表性实例（A） 的聚（苯乙烯磺酸盐）-Au卢比，（B，C）的金自然保护区，和（D）无卢比（对照样品）。这些结果为100毫秒（A，C，D）和50毫秒（B）的激光脉冲获得。用于实验（垂直虚线）的频率分别为1赫兹（A - C）和0.5 Hz的频率（D）中 。计算出的辐射暴露为69.4Ĵ∙厘米-2（A），34.7Ĵ∙厘米-2（B），0.37Ĵ∙厘米-2（C）和138.87Ĵ∙厘米-2（D）。 F 最大 / f 0的表示NG108 -15神经细胞检测与离子霉素的最大荧光增加，从校准（转载许可13）。

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

Universidad San Sebastian

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

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.

Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits.

使用光刺激神经元的负担得起的LED阵列

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

科研

神经科学

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

光纤植入慢性Optogenetic刺激脑组织

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

单个锥体神经元的高保真光遗传学控制方法<em&gt;在体内</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into glass capillaries, pulled to a fine taper, and then sealed using an epoxy to create electrode materials that are used for fast scan cyclic voltammetry testing. The use of bare CFMEs has several limitations, though. First and foremost, the carbon fiber contains mostly basal plane carbon, which has a relatively low surface area and yields lower sensitivities than other nanomaterials. Furthermore, the graphitic carbon is limited by its temporal resolution, and its relatively low conductivity. Lastly, neurochemicals and macromolecules have been known to foul at the surface of carbon electrodes where they form non-conductive polymers that block further neurotransmitter adsorption. For this study, we modify CFMEs with gold nanoparticles to enhance neurochemical testing with fast scan cyclic voltammetry. Au3+ was electrodeposited or dipcoated from a colloidal solution onto the surface of CFMEs. Since gold is a stable and relatively inert metal, it is an ideal electrode material for analytical measurements of neurochemicals. Gold nanoparticle modified (AuNP-CFMEs) had a stability to dopamine response for over 4 h. Moreover, AuNP-CFMEs exhibit an increased sensitivity (higher peak oxidative current of the cyclic voltammograms) and faster electron transfer kinetics (lower &#916;EP or peak separation) than bare unmodified CFMEs. The development of AuNP-CFMEs provides the creation of novel electrochemical sensors for detecting fast changes in dopamine concentration and other neurochemicals at lower limits of detection. This work has vast applications for the enhancement of neurochemical measurements. The generation of gold nanoparticle modified CFMEs will be vitally important for the development of novel electrode sensors to detect neurotransmitters in vivo in rodent and other models to study neurochemical effects of drug abuse, depression, stroke, ischemia, and other behavioral and disease states.

In this study, we modify carbon-fiber microelectrodes with gold nanoparticles to enhance the sensitivity of neurotransmitter detection.

用于增强神经化学检测的金纳米粒子改性碳纤维微电极

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into ...

化学

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

含有金纳米棒蜂窝车辆的制备及光声分析

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

生物工程

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

由共轭聚合物活细胞电活动的光控制

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

听觉神经的光遗传学刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.