作者想（扩大他们的感激之情从纽约巴斯德基金会的资金支持（以法学博士，CS，CS），欧盟第七框架计划"自动化"（以SLS），研究所卡诺计划11至JD，AH ，AR，RT，SLS）和项目IMNOS（至RT，SLS），该的Conny-梅芙慈善基金会（SLS），欧洲大师赛在分子成像（它），该地区法兰西岛计划MODEXA（SLS），芝麻（SLS）和DimMalInf（SLS，RT），该ANR程序GRANDES投资公司DE L&#39;AVENIR基础设施NATIONALES恩Biologie -桑特: 法国LifebioImaging（FLI）法国寿命成像（RT，SLS）， 法国生物成像 （法学博士，SLS）和巴斯德研究所，巴黎。此外，作者要感谢，何塞Bengoechea和赫伯特·施韦策的试剂。此外，作者要感谢辛迪热夜谁产生的抗体。

1，试剂<ol><li>购买或开发发光细菌和合适的培养基，如表达勒克斯 ABCDE 21改性大肠杆菌 ， 弧菌鲵，发光菌菌肺炎克雷伯氏菌22。 </li><li>根据标准配方制备的生理盐水（0.9％NaCl）中和培养基的解决方案。E.大肠杆菌和K。肺炎链球菌在37℃，V.生长的Luria BERTANI（LB） 鲵和发光菌菌在LB中添加0.5 M氯化钠在22°C。 </li><li>购买或获取的荧光探针，例如Q-跟踪器705（40纳米的直径，被称为QD705），Q-跟踪器800（40纳米的直径，被称为QD800），荧光（40纳米的直径）和非荧光聚苯乙烯微球（ 48纳米的直径），和传统的荧光染料。 </li><li>准备细菌标记所需的试剂。 </li><li>确保获得整体动物的生物发光成像仪，如IVIS光谱，其能够在宽范围的发射波长和曝光时间的检测信号。酶标仪能生物发光测量将足以满足大多数这里所描述的实验。 </li></ol>燃料2。基本重演<ol><li>从标准的培养皿中单个菌落起，开始发光细菌的过夜培养。在这里， 五鲵被使用。 </li><li>实验当天，发起新的亚文化，让他们进步，直到外径600 1-1.5的实现。为了产生类似的结果，最好是利用细菌在其产生强烈的信号相似的生长状态。这可以通过保持OD 600恒定得到保证。 </li><li>结合100微升等分从每任5微升的QD705或生理盐水（PS），然后加入895微升的PS成STANDA第三届分光比色皿。将装满试管放入IVIS频谱，并采取在适当的过滤器设置的测量。在这里，710-730 nm发射滤波器使用。 </li></ol> 3，燃油在不同的距离<ol><li>填补2减小的体积（1ml）的塑料比色皿光度与任何50μl的QD705或97.2μl，以1毫升的PS的总体积非荧光48纳米的聚苯乙烯微球。这确保每两个实体之间的总体积类似的固体的表面积。 </li><li>通过装箱第三试管与标准黑胶带或能够阻挡光线的一些其它不透明材料准备的光源比色皿。仔细的比色皿的两侧准备两个相同的光学窗口。 </li><li>将预先填充的比色皿，直接到光源比色皿的任一侧。添加1毫升等分五的鲵或其他文化（ 即发光大肠杆菌或发光菌 sP）从一个新鲜传代培养到光源比​​色杯中，用不​​透明的材料，如黑色的纸，以减少任何光污染覆盖它。 </li><li>可视化三个试管在适当的发射器，如总轻和710-730 nm发射器，分别为10曝光时间，30和30秒。 </li><li>在从中央实验皿等效更远的距离定位两个外部比色皿，然后再次显现。重复，直到最后面到面（中央对减小体积比色皿）的距离为3cm的实现。在每个步骤中，获取荧光图像（450-480 nm激发，710-730 nm发射）来验证QD705位置。 </li></ol> 4，调查潜在的燃料双<ol><li>填补2减小的体积比色皿用含有两种荧光团或感兴趣的荧光纳米粒子在1和PS或PS与非荧光纳米粒子作为阴性对照在其他1毫升的解决方案。在日此处，工作证明，各种潜在的市售燃料的荧光基团和荧光纳米粒子进行了调查，如表1所示 。 </li><li>添加1毫升等分的新鲜五鲵或其他发光细菌含有位于两侧两个物理光学等于一个窗户涂黑的第三个试管。盖在诸如一张黑纸的不透明物质的反应杯。 </li><li>在一个很短但距离相等的地方上的涂黑的比色皿两侧的两个体积减少比色皿，并根据适当的过滤器可视化。这里，使用了0.7厘米的距离。重复与其他荧光团。 </li></ol> 5，有针对性的燃料<ol><li>准备特定的发光细菌的生物素化的抗体。例如，这里第一抗体特异性，以K.肺炎链球菌 （α-KP）使用含有EZ-Link的磺基-NHS-LC-生物素的溶液中的生物素化。 </l我&gt; <li>使用2分钟下离心（1,500 XG）脱盐柱的抗体去除过量的试剂。 </li><li>使用一个标准的Bradford法测定蛋白浓度，并确定由HABA试验抗体生物素化的程度。 </li><li>通过将100微升（10 mM时，溶解在1×PBS中）与40μl的α-KP抗体，从而导致每16抗体生物素残基和一个最终替代的最终程度得到α-KP-比奥生物素化抗体（抗体）批10毫克毫升-1蛋白浓度。 </li><li>使用多个复制过夜培养，目标与上述抗体下面相应的协议洗过的细菌。 <ol><li>具体来说，洗K。肺炎在1x PBS，重悬在1x PBS中至4的外径（ 约 4×10 8 CFU毫升-1 OD -1）。 </li><li>对于每个复制，孵化100微升PBS，α -KP抗体（10微升α-KP-biotB或10微升PBS的对照）和100μl的细胞90分钟，在30℃下在1x PBS在196微升PBS洗细胞三次，重悬。 </li></ol></li><li>标记的细胞与链霉亲和QD705共轭，并将其分成两个相等的体积。 </li><li>洗1溶液3次，然后重悬到适当的体积。 </li><li>分发100微升的洗涤和未洗涤溶液加入到黑色96孔板的各孔中。根据测量的总光产生的发光，490-510纳米，和710-730纳米的过滤器。荧光团浓度可以用450-480 nm激发和710-730 nm发射来确定。 </li></ol>

<ol>
	<li>Hu, 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=A+human+immunodeficiency+virus+type+1+protease+biosensor+assay+using+bioluminescence+resonance+energy+transfer.">A human immunodeficiency virus type 1 protease biosensor assay using bioluminescence resonance energy transfer.</a> J Virol Methods. 128, 93-103 (2005).</li><li>Contag, C. 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=Visualizing+gene+expression+in+living+mammals+using+a+bioluminescent+reporter.">Visualizing gene expression in living mammals using a bioluminescent reporter.</a> Photochem Photobiol. 66, 523-531 (1997).</li><li>Badr, C. E., Tannous, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging:+progress+and+applications.">Bioluminescence imaging: progress and applications.</a> Trends in Biotechnology. 29, 624-633 (2011).</li><li>Maywood, 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=Analysis+of+core+circadian+feedback+loop+in+suprachiasmatic+nucleus+of+mCry1-luc+transgenic+reporter+mouse.">Analysis of core circadian feedback loop in suprachiasmatic nucleus of mCry1-luc transgenic reporter mouse.</a> Proc Natl Acad Sci USA. 110, 9547-9552 (2013).</li><li>Condeelis, J., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Imaging+in+Cancer.">In Vivo Imaging in Cancer.</a> Cold Spring Harbor Perspectives in Biology. , (2010).</li><li>Madero-Visbal, R. 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=Bioluminescence+imaging+correlates+with+tumor+progression+in+an+orthotopic+mouse+model+of+lung+cancer.">Bioluminescence imaging correlates with tumor progression in an orthotopic mouse model of lung cancer.</a> Surgical Oncology. 21, 23-29 (2012).</li><li>Thalhofer, C. 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=In+vivo+imaging+of+transgenic+Leishmania+parasites+in+a+live+host.">In vivo imaging of transgenic Leishmania parasites in a live host.</a> J Vis Exp. 10, (2010).</li><li>Troy, T., Jekic-McMullen, D., Sambucetti, L., Rice, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+comparison+of+the+sensitivity+of+detection+of+fluorescent+and+bioluminescent+reporters+in+animal+models.">Quantitative comparison of the sensitivity of detection of fluorescent and bioluminescent reporters in animal models.</a> Molecular Imaging: Official Journal of the Society for Molecular Imaging. 3, 9-23 (2004).</li><li>Michelini, E., Cevenini, L., Mezzanotte, L., Roda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminescent+probes+and+visualization+of+bioluminescence.">Luminescent probes and visualization of bioluminescence.</a> Methods Mol Biol. 574, 1-13 (2009).</li><li>Branchini, B. R., Ablamsky, D. M., Rosenberg, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+Modified+Firefly+Luciferase+Is+an+Efficient+Source+of+Near-Infrared+Light.">Chemically Modified Firefly Luciferase Is an Efficient Source of Near-Infrared Light.</a> Bioconjugate Chemistry. 21, 2023-2030 (2010).</li><li>Saito, 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=Luminescent+proteins+for+high-speed+single-cell+and+whole-body+imaging.">Luminescent proteins for high-speed single-cell and whole-body imaging.</a> Nat Commun. 3, 1262 (2012).</li><li>Dragulescu-Andrasi, A., Chan, C. T., De, A., Massoud, T. F., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+resonance+energy+transfer+(BRET)+imaging+of+protein-protein+interactions+within+deep+tissues+of+living+subjects.">Bioluminescence resonance energy transfer (BRET) imaging of protein-protein interactions within deep tissues of living subjects.</a> Proc Natl Acad Sci USA. 108, 12060-12065 (2011).</li><li>Bacart, J., Corbel, C., Jockers, R., Bach, S., Couturier, 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+BRET+technology+and+its+application+to+screening+assays.">The BRET technology and its application to screening assays.</a> Biotechnology Journal. 3, 311-324 (2008).</li><li>So, M. -. K., Xu, C., Loening, A. M., Gambhir, S. S., Rao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-illuminating+quantum+dot+conjugates+for+in+vivo+imaging.">Self-illuminating quantum dot conjugates for in vivo imaging.</a> Nat Biotechnol. 24, 339-343 (2006).</li><li>Wu, Q., Chu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-illuminating+quantum+dots+for+highly+sensitive+in+vivo+real-time+luminescent+mapping+of+sentinel+lymph+nodes.">Self-illuminating quantum dots for highly sensitive in vivo real-time luminescent mapping of sentinel lymph nodes.</a> Int J Nanomedicine. 7, 3433-3443 (2012).</li><li>Wu, 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=In+vivo+far-red+luminescence+imaging+of+a+biomarker+based+on+BRET+from+Cypridina+bioluminescence+to+an+organic+dye.">In vivo far-red luminescence imaging of a biomarker based on BRET from Cypridina bioluminescence to an organic dye.</a> Proc Natl Acad Sci USA. 106, 15599-15603 (2009).</li><li>Piston, D. W., Kremers, G. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein+FRET:+the+good,+the+bad+and+the+ugly.">Fluorescent protein FRET: the good, the bad and the ugly.</a> Trends Biochem Sci. 32, 407-414 (2007).</li><li>Dragavon, 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=in+Imaging,+Manipulation,+and+Analysis+of+Biomolecules,+Cells,+and+Tissues.">in Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues.</a> , 790210-790219 (2011).</li><li>Dragavon, 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=In+vivo+excitation+of+nanoparticles+using+luminescent+bacteria.">In vivo excitation of nanoparticles using luminescent bacteria.</a> Proceedings of the National Academy of Sciences. 109, 8890-8895 (2012).</li><li>Holland, A. D., 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=In+vitro+characterization+of+fluorescence+by+unbound+excitation+from+luminescence:+Broadening+the+scope+of+energy+transfer.">In vitro characterization of fluorescence by unbound excitation from luminescence: Broadening the scope of energy transfer.</a> Methods. , (2013).</li><li>Choi, K. -. H., Schweizer, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mini-Tn7+insertion+in+bacteria+with+single+attTn7+sites:+example+Pseudomonas+aeruginosa.">mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa.</a> Nat. Prot. 1, 153-161 (2006).</li><li>Riottot, M. M., Fournier, J. M., Jouin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+for+the+involvement+of+capsular+polysaccharide+in+the+immunoprotective+activity+of+Klebsiella+pneumoniae+ribosomal+preparations.">Direct evidence for the involvement of capsular polysaccharide in the immunoprotective activity of Klebsiella pneumoniae ribosomal preparations.</a> Infect Immun. 31, 71-77 (1981).</li><li>Becker, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+lifetime+imaging--techniques+and+applications.">Fluorescence lifetime imaging--techniques and applications.</a> J Microsc. 247, 119-136 (2012).</li><li>Rogers, K. L., 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=Visualization+of+local+Ca2++dynamics+with+genetically+encoded+bioluminescent+reporters.">Visualization of local Ca2+ dynamics with genetically encoded bioluminescent reporters.</a> Eur J Neurosci. 21, 597-610 (2005).</li><li>Dubochet, J., McDowall, A. W., Menge, B., Schmid, E. N., Lickfeld, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy+of+frozen-hydrated+bacteria.">Electron microscopy of frozen-hydrated bacteria.</a> Journal of Bacteriology. 155, 381-390 (1983).</li><li>Graham, L. L., Harris, R., Villiger, W., Beveridge, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze-substitution+of+gram-negative+eubacteria:+general+cell+morphology+and+envelope+profiles.">Freeze-substitution of gram-negative eubacteria: general cell morphology and envelope profiles.</a> Journal of Bacteriology. 173, 1623-1633 (1991).</li><li>Hobot, J. A., Carlemalm, E., Villiger, W., Kellenberger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periplasmic+gel:+new+concept+resulting+from+the+reinvestigation+of+bacterial+cell+envelope+ultrastructure+by+new+methods.">Periplasmic gel: new concept resulting from the reinvestigation of bacterial cell envelope ultrastructure by new methods.</a> Journal of Bacteriology. 160, 143-152 (1984).</li></ol>

作者宣称竞争经济利益。法学博士，SB，凯龙瑞和SLS促成了欧洲专利EP10290158.4和世界知识产权组织专利申请WO/2011/117847。

燃料的基本演示可以简单地通过混合发光细菌荧光纳米颗粒或量子点来实现。这两个实体将被物理上分开，并保持超出了任何有效的RET距离。更困难的是燃油信号优化在体外和体内 。下在体外条件下，具有和不具有光吸收体存在，通常是加入过量的荧光团将足以最大化燃料反应。然而，在高浓度的现象，如静态或碰撞猝灭可导致荧光信号的损失。由独立改变发光光源和荧光团的浓度进行稀释系列，将有助于优化所需的浓度。在体内的浓度建立和燃料的优化要困难得多，需要在逐案基础上加以解决。它可能很难建立一个共同ndition其中所述荧光实体可以被光学地由发光源访问。因此，与直接合作，注射两个部分的开头可以提供关于燃料的最佳条件下的成功信息。 标准协议标签细菌和真核细胞存在荧光实体，如Alexa的系列和量子点。通常这需要表面功能化或活化的抗体，从而导致像减少细胞存活力的或改变代谢活性的不想要的效果。为了克服这一点，以确定所需要的抗体或活化剂的最佳量是同时最大化的荧光标记细胞的最小扰动是很重要的。利用量子点是因为他们的典型宽的激发光谱，窄，可调发射光谱，和一个大的斯托​​克斯位移的可能性是有利的。然而，量子点可以是细胞毒性的，可能不希望在某些情况下。 <p级="jove_content"&gt;燃油是一种现象，它存在于许多BRET实验13，并适用于各种发光和荧光源。到现在为止，从燃料产生的光子被视为非特异性相互作用或设计不当的BRET实验中产生的一个不幸的背景信号的产品。只有用实验证明在这里，我们能够确定这种无用信号的工具类型。在所示的例子中，发光细菌作为漫射声源能够引起从各种各样的荧光实体的标准荧光反应。此外，由于大量的工作距离，它是安全的结论是，虽然燃料可以在不BRET的发生来构成，在一般的BRET不能在没有从燃料中贡献观察。重要的是，由于缺少靶向要求，燃料可以被用于覆盖的空间间隙是BRET和c之间存在onventional全动物成像​​技术。

生物，如病毒1，2，3的细菌，或小哺乳动物的遗传修饰4要么诱导或组成型表达的生物发光，已非常成功并广泛证明5-7。生物发光， 在体内的化学发光反应涉及天然存在的试剂，有而不需要外部光源产生的光的优点。因此，生物发光成像不会从自动和非特异性信号的发现从荧光成像8的共同缺点的。因此，生物发光具有显著信号 - 噪声比，因为任何检测到的信号来源于完全从预期的来源。而许多模型已经利用从发光光的勒克斯操纵子（480和490毫微米之间的中心发射最大值）为在体外和体内应用9，其在小MAMM使用ALS一直是个问题，由于成像条件的性质;光吸收剂，如血红蛋白，和散射剂，如组织和骨的弥漫存在，强烈地影响蓝色到黄色的波长3。一个精心设计的萤火虫荧光素酶（最大发射位于617nm）的表达，最近已经开发并结合，提供了一个工具，极大地克服了光吸收10，但仍受到散射的影响。 作为响应，已经有多种尝试红移发射的信号转换为650-900纳米，最小的吸收和散射的区域所需的光学窗口，用生物发光共振能量转移（BRET）11-13。作为一种工具，以提高信号的检测，BRET，其采用生物发光信号源作为供体和一个附加的荧光团作为受体，目前已发现了有限的成功。作为这种现象的精液例如，"自发光的量子点4; （SIQDs）14包括修改海肾reniformis荧光素酶绑定到外部聚合物-赖氨酸层 市售的量子点（QDs）。后基板此外，所得到的生物发光反应诱导的量子点的荧光发射，产生显著生产的红色光子。然而，这些SIQDs已经在生理有关事件体内可视化有限适用性。这个有限的适用性很可能是由于在双探头连接到器官，细胞或感兴趣的基因，由于SIQDs不能被遗传编码的，因此，就需要在聚合物壳的二次改性的难度。以改善它们的适用性，可替代SIQDs，其中所述荧光素酶直接结合到所述发光芯，最近已采用15。建设关SIQD理念，更适用BRET系统是由连接海萤荧光素酶的实现中docyanine染料16，这是能特异性靶向肿瘤的小鼠同时产生大量的红色转变，从460纳米到675纳米。接受非辐射能量转移，BRET遵循相同的主约束其荧光对应:必须有供体发射和受体的激发光谱和两个基团之间的工作距离之间有很强的光谱重叠必须是顺序福斯特半径（5-14纳米取决于供体-受体对，与福斯特半径17倍的有效的最大距离）。这个距离的依赖性大大限制了可以使用的BRET以提高检测的装置被观察的事件的类型。 最近一种新的方法被确定，并根据表明在体外和体内条件。关栋BRET，通过荧光激发无限制自发光（燃料）18，19的基础</suP&gt;还要求发光和荧光成分之间有很强的光谱重叠。然而，不同于BRET，燃料是一种完全的辐射过程，由此从所述发光光源所发射的光子是由一个光学访问的荧光团，随后根据所述荧光团的量子产率发出红移光子吸收。类似于BRET，这种方法也可以用来克服成像的约束在光吸收剂的存在。所得到的红移提供了在检测到的信号，由于在衰减的降低和减少了光的散射效应的整体增加和特异性。燃料已报道的生物发光大肠杆菌表达勒克斯操纵和量子点18,19之间发生。而实验类似于SIQDs，从根本上存在差异:在燃料，这是没有必要的发光源是物理结合的荧光团，其允许FOr为荧光探针的基因编码。由于成功地检测发光细菌和量子点之间的燃料，它是可能的，这种技术可以同时适用于浅表（皮肤）和深部组织（肺，肝）感染，如金黄色葡萄球菌和肺炎Klebsiellia。 由于它的实验意义的报告，燃料已发展到包括可用于预测可接受的发光和荧光对健壮的数学模型20，其应用领域已扩大到包括用于识别的光物理特性，例如量子效率。我们在下面说明一些燃料的基本技巧。首先，我们展示的证据，这种现象在短期（微米）和长（厘米）的工作距离，这从根本上区别于燃料BRET。第二，我们在可能的燃油对通过检查各种荧光基团和荧光纳米颗粒的扩大。锡尔D，燃料应用通过比较目标和非目标燃料对考察。

<table><tbody><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; expressing the &lt;em&gt;lux&lt;/em&gt;ABCDE operon&amp;nbsp;</td><td></td><td></td><td>kindly provided by Jos&amp;eacute; A. Bengoechea with permission from Herbert P. Schweizer</td></tr><tr><td>&lt;em&gt;Klebsiella pneumoniae&lt;/em&gt; 52145&amp;nbsp;</td><td></td><td></td><td>52145 is a serotype K2 reference strain</td></tr><tr><td>Luria Bertani (LB)&amp;nbsp;</td><td></td><td></td><td>standard growth media</td></tr><tr><td>Q-Tracker 705&amp;nbsp;</td><td>Life Sciences</td><td>Q21061MP</td><td></td></tr><tr><td>Q-Tracker 800</td><td>Life Sciences</td><td>Q21071MP</td><td></td></tr><tr><td>Alexa 555</td><td>Life Sciences</td><td>S21381</td><td></td></tr><tr><td>Alexa 568</td><td>Life Sciences</td><td>S11226</td><td></td></tr><tr><td>Alexa 633</td><td>Life Sciences</td><td>S21375</td><td></td></tr><tr><td>Alexa 700</td><td>Life Sciences</td><td>S21383</td><td></td></tr><tr><td>Non-fluorescent microspheres</td><td>Polysciences, Inc</td><td>15913</td><td></td></tr><tr><td>Pink microspheres</td><td>Life Sciences</td><td>F8887</td><td>40 nm diameter</td></tr><tr><td>Yellow microspheres</td><td>Life Sciences</td><td>F8888</td><td>40 nm diameter</td></tr><tr><td>Ivis Spectrum</td><td>PerkinElmer</td><td></td><td></td></tr><tr><td>EZ-Link Sulfo-NHS-LC-Biotin</td><td>Thermo Scientific Pierce</td><td>21425</td><td></td></tr><tr><td>Zeba Spin Desalting Columns 7K MWCO</td><td>Thermo Scientific Pierce</td><td>21425</td><td></td></tr><tr><td>HABA assay kit&amp;nbsp;</td><td>Thermo Scientific Pierce</td><td>28005</td><td></td></tr><tr><td>Bradford assay</td><td>Bio-Rad</td><td>500-0201</td><td></td></tr></tbody></table>

a step beyond bret: fluorescence by unbound excitation from luminescence (fuel)

荧光由未结合的激励从发光（燃料）是产生的增加信号和对比度增强的体外和体内的辐射激发发光的过程。燃料共享许多相同的基本原则，生物发光共振能量转移（BRET），还大大不同于在发光光源和荧光实体之间的可接受的工作距离。而BRET被有效地限制在一个最大的福斯特半径的2倍，通常小于14纳米，燃料可以在没有光吸收发生在距离可达微米或什至厘米。在这里，我们根据燃料的基础和应用拓展通过审查现象背后的有关原则，并展示其与多种荧光基团和荧光纳米颗粒的兼容性。此外，抗体靶向燃料的效用进行了探讨。这里所示的例子中提供的证据表明，燃料可以用于APPLications其中BRET是不可能的，填充空隙空间的那BRET和传统的整体动物成像之间存在。

共振能量转移（RET）是发光体和荧光受体，由此从退出供体的能量能够通过一个强的偶极-偶极相互作用13诱导的受体的荧光响应之间的非辐射相互作用。再生能源，已使用23荧光，化学发光24和 ​​生物发光13捐助者，主要描述的要求:供体发射和受主激发光谱之间1强光谱重叠;。 2这两个实体之间的适当的旋转对准;和3，一种工作距离不大于0.5至2倍的福斯特半径R 0时 ，供体和受体17之间。用RET对比，就会发生燃料时的发光光源，诸如发光细菌，发射被吸收和再发射由第二实体的光子，如荧光团或荧光纳米粒子，红移的发射断层朗姆酒原发光源的。因此，燃料遵循标准激发发光过程类似于标准啶的条件下，还没有利用聚焦的激发。这方面的证据可以通过发光细菌和高荧光量子点的简单混合可以容易地观察到。在弧菌的存在下，在红色信号显著增加时观察QD705也是在溶液相比，非荧光聚苯乙烯微球的对照（ 图1A）。必要创建生物发光，基本上与醛底物，荧光素酶和ATP的组件，都产生并包含在细胞质内。此外，酶的发光体的生产发生在细菌细胞质中（ 图1B）内。如已在文献，细菌的内和外膜间的距离通常大于30纳米25-27，一个距离，该距离不容许显著RET发生。以及，所述荧光纳米粒子不交叉进入细菌细胞质中，因为没有或内吞作用摄取的其他手段。一起，这些约束表示燃料是当发光细菌与荧光纳米粒子混合时发生的显性激发发射现象。 如先前所示，燃料之外不存在RET的范围内。来调查关于距离的燃料的依赖，减少标准体积分光比色皿，可以使用与含有新鲜发光溶液的等分试样1含荧光团溶液，含有适当的控制，可以控制对散射的第二和第三。至关重要的是，中央比色皿用黑胶带或其它不透明材料，除位于相对的面的至少两个相同的光学窗口，以减少任何可能的光污染来自发光源笼罩。此外，剩下的两个试管需要被等距离地放置在中央比色皿的任一侧。最后，一​​个新鲜的发光液，用细菌或化学发光，应该用每个实验使用，以确保最大光生产。经适当的位置，获得下所需的过滤器的发光信号。的总光量和710-730 nm的滤膜在这种情况下使用，虽然只从后者中的数据如下所示。在每次采集后，逐渐增加的面到面距离为1.0厘米到三厘米（ 图2）。最后，规范所有的数据到最亮的点。在这里所使用的例子中，这发生在发光源和含有QD705反应杯之间的最短距离（D = 1厘米）。使用这种方法，可行的燃料信号可以观察到最后的采集表明，在没有任何光学吸收，燃料可以发生在任何可能产生的共振能量转移距离，只能解释是一个纯粹的辐射效果。拟合数据揭示了作为距离D的下面一个D -1到D -2依赖的函数信号下降。根据不同的几何构型，前者对应于电容器的距离依赖性，后者的平方反比定律为点源。因此，该结果与我们的整体获取的设置相一​​致，考虑到反应杯的孔的大小和发光光源和荧光团之间的距离。 燃料不仅适用于量子点，而且还可以使用范围广泛的荧光团包括从Alexa的系列，以荧光微球（ 表2）中被观察到。为了可比较的对照，各种荧光团的浓度相等，应使用与纳米颗粒的总表面积保持不变（ 表1）。由荧光纳米粒子和比较它们的适当的控制（PS或PS与非荧光聚苯乙烯微球），在信号显著增加的最大发射荧光的每个实体的观察。在信号的最大相对增加，发现发生，其中所述荧光发射最大值是最远离发光发射最大值。这是由于在荧光信号的增加的特异性相比，发光光源的宽的发射光谱的可能性最大。 反之，最不可靠燃料的信号时，发现两个发射最大值没有得到很好的分离。有趣的是，一个可辨别的信号FUEL找到具有黄色的微球，即使频谱差是最小的，由于最可能每圈（350荧光素当量）存在的荧光团的数量可观。这里示出的结果表明，在适当条件下，燃料可以与各种荧光团，从而使两个I下选择了多个相关的应用探针的剪裁可以实现Ñ ​​体外和体内条件。所观察到的燃料的信号的显着性用标准的双尾Student t-检验确定的。因此，只有Alexa555的发现是与所使用的发光源不相容。 为了探索燃料目标的影响，生物素化抗体用于针对发光细菌和链霉联量子点。它是细菌或发光光源提供足够厚，以减少RET的发光体和相应的荧光团之间的可能的层是很重要的。孵育并洗涤去除未结合的抗体后，将生物素标记的细菌，然后暴露于链霉亲和共轭QD705或者非官能化QD705作为对照，分解和一组暴露在进一步洗涤以除去任何非粘附QD705。在这里，所有的四个条件，所产生的发光是根据总的光，490-510纳米，观测到ð710-730 nm的滤光片，虽然只有后两种过滤器被用于数据分析。使用450-480 nm激发和710-730 nm发射滤波器的QD705的存在进行了比较。经过调查我们发现几乎在红移的信号没有什么区别，当溶液处于一种未洗的状态（ 图3）。在此条件下所产生的荧光信号也被发现是相当类似，这表明相同数目的QD705的是在这两个目标和非目标状态存在。然而，洗涤样品以除去任何未结合的QD705提供了近2倍的增加相对于红色信号相比，其非靶向对照的靶向细菌。调查由荧光可以用来验证QD705的存在或不存在。相比之下，有针对性的清洗条件导致了明显少于未洗的状态几乎三倍，但由此产生的红移仅下降了一个荧光强度30％，这表明单纯的约束条件下，细菌的定位将导致增加红移发射。这强烈暗示有针对性燃料的未来应用的实用程序。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51549/51549fig1highres.jpg" src="/files/ftp_upload/51549/51549fig1.jpg" /> 图1。发光细菌和市售的量子点之间的燃料相互作用导致增加红色光子产生两个分光光度比色皿中填充有含有新鲜V. 100微升等份溶液鲵文化1-1.5外径600，895微升的PS，要么5微升的QD705或生理盐水（PS），被放置到一个IVIS频谱和后天的发射光谱之前。红色信号A显著增加，是由于存在实现该QD705，如可以注意到的增加所探测到的光子•秒-1•cm -2的（P•秒-1•厘米-2）相比，710-730 nm发射滤波器（A）根据控制。这里，该种细菌的双膜不包括在发光部分（蓝色圆圈）之间的相互作用和QD705（黑色圆圈）。前者被发现仅在细菌细胞质中，而后者可自由地分布在本体溶液（B）。 N = 3，微生物的解决方案。 <a href="https://www.jove.com/files/ftp_upload/51549/51549fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51549/51549fig2highres.jpg" src="/files/ftp_upload/51549/51549fig2.jpg" /> 发生在距离并发症如图2所示。证据燃料的etely不含共振能量转移。分光比色皿中填充有含有为50微升QD705和950微升生理盐水（PS）或97.2微升的非荧光48纳米的聚苯乙烯微球和902.8微升PS的解决方案，并等距离地放置在相对 ​​的两侧的中央黑色皿含1毫升新鲜的发光发光细菌藻在外径600 1-1.5。中央试管有两个相同的光学窗口，允许发射的光子自由散开。在距离（D）从1.0厘米到3厘米采集图像，观察到的生产红光下降的距离显示的证据表明，燃料可发生于距离不是由共振能量转移实现的功能。强度似乎有丁-2依赖性，类似于平方反比定律（左）。相同的协议，其次为E。大肠杆菌 （右）。由此产生的趋势线保持形式的概念THA吨的细菌被用作点光源。一共有三个独立的测量距离被收购与各使用一个独特的亚文化。误差棒是存在于每一个点。在某些情况下，误差线比所用的符号，表明归一化强度较小。 <a href="https://www.jove.com/files/ftp_upload/51549/51549fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51549/51549fig3highres.jpg" src="/files/ftp_upload/51549/51549fig3.jpg" /> 图3。针对性的（特定的）和非靶向（本体溶液）的燃料。K之间的比较 肺炎被官能化的生物素化的抗体，然后暴露于链霉亲和标记（目标）或非标记（非定向）QD705。由此产生的解决方案也同样迪维DED和一组洗涤三次，用PBS重悬所成的PBS其初始体积之前。然后将溶液分配到黑色96孔板中，根据490-510纳米（500 nm）和710-730纳米（720 nm）的发射滤光片（表示为蓝色条）中观察到的生物发光的各个孔中。在p•秒-1•cm -2的发现从720纳米过滤器每口井进行归一化的各p•秒-1•cm -2的生物发光从500纳米的同一井强度。从表面荧光激发产生的相对荧光强度是用450-480 nm激发和710-730 nm发射器（显示为红色条）而厘定。几乎在生物发光或荧光信号无明显差异，洗过的条件（非目标未洗和未洗的目标）下观察。这表明，结合的和游离漂浮QD705是由生物发光的光子同样兴奋。洗涤后，正早在一个相对的红色信号2倍差异观察到相对于对照（非靶向洗水）的QD705标记的细菌（靶向洗水）。在非针对性的水洗缺乏QD705是由缺乏荧光信号的确认和验证QD705标记的目标水洗状态。这些数据是从K的四个独立的文化肺炎。为清楚起见，传说指示QD705激励源。 <a href="https://www.jove.com/files/ftp_upload/51549/51549fig3highres.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody align="center"><tr height="26"><td height="26" style="height:26px;"> 荧光 </td><td> 浓度 </td><td> 微升 </td><td> PS（微升） </td><td> λ 最大 EX / EM（NM） </td><td> 发射滤波器（N<html> M） </td></tr><tr height="21"><td height="21" style="height:21px;width:108px;"> Alexa的555 </td><td style="width:116px;"> 37.2微米</td><td style="width:88px;"> 4.77 </td><td style="width:88px;"> 995.23 </td><td style="width:119px;">五百六十五分之五百五十五</td><td style="width:152px;"> 570 - 590 </td></tr><tr height="21"><td height="21" style="height:21px;width:108px;"> Alexa的568 </td><td style="width:116px;"> 37.2微米</td><td style="width:88px;"> 4.77 </td><td style="width:88px;"> 995.23 </td><td style="width:119px;">六百○三分之五百七十八</td><td style="width:152px;"> 590 - 610 </td></tr><tr height="21"><td height="21" style="height:21px;width:108px;"> Alexa的633 </td><td style="width:116px;"> 37.2微米</td><td style="width:88px;"> 4.77 </td><td style="width:88px;"> 995.23 </td><td style="width:119px;"> 637分之632 </td><td style="width:152px;"> 650 - 670 </td></tr><tr height="21">宽度:108px;"&gt; Alexa的700 </td><td style="width:116px;"> 37.2微米</td><td style="width:88px;"> 4.77 </td><td style="width:88px;"> 995.23 </td><td style="width:119px;"> 723分之702 </td><td style="width:152px;"> 710 - 730 </td></tr><tr height="21"><td height="21" style="height:21px;width:108px;"></td><td style="width:116px;"></td><td style="width:88px;"></td><td style="width:88px;"></td><td style="width:119px;"></td><td style="width:152px;"></td></tr><tr height="21"><td height="21" style="height:21px;width:108px;">无荧光</td><td style="width:116px;"> 2.62％的固体</td><td style="width:88px;"> 9.72 </td><td style="width:88px;"> 990.28 </td><td style="width:119px;"></td><td style="width:152px;"></td></tr><tr height="21"><td height="21" style="height:21px;width:108px;"> μSph粉红</td><td style="width:116px;"> 5％的固体</td><td style="width:88px;"> 4.77 </td><td style="width:88px;"> 995.23 </td><html><td style="width:119px;">六百零五分之五百八十〇 </td><td style="width:152px;"> 610 - 630 </td></tr><tr height="21"><td height="21" style="height:21px;width:108px;"> μSph黄色</td><td style="width:116px;"> 5％的固体</td><td style="width:88px;"> 4.77 </td><td style="width:88px;"> 995.23 </td><td style="width:119px;">五百一十五分之五百零五</td><td style="width:152px;"> 510 - 530 </td></tr><tr height="21"><td height="21" style="height:21px;width:108px;"></td><td style="width:116px;"></td><td style="width:88px;"></td><td style="width:88px;"></td><td style="width:119px;"></td><td style="width:152px;"></td></tr><tr height="21"><td height="21" style="height:21px;width:108px;"> QD705 </td><td style="width:116px;"> 2微米</td><td style="width:88px;"> 5 </td><td style="width:88px;"> 995 </td><td style="width:119px;"> 705分之465 </td><td style="width:152px;"> 710 - 730 </td></tr><tr height="22">高度:22px;宽度:108px;"&gt; QD800 </td><td style="width:116px;"> 2微米</td><td style="width:88px;"> 5 </td><td style="width:88px;"> 995 </td><td style="width:119px;"> 705分之465 </td><td style="width:152px;"> 790 - 810 </td></tr></tbody></table> 表1。整个燃料示范使用荧光基团的性质。 <table border="0" cellpadding="0" cellspacing="0"><tbody align="center"><tr><td rowspan="2"></td><td rowspan="2"> 控制 </td><td rowspan="2"> 过滤器 </td><td colspan="2"> 荧光 </td><td colspan="2"> 控制 </td><td rowspan="2"> P值 </td></tr><tr height="21"><td> ·P·秒-1·厘米-2 </td><td> SD </td><td> ·P·秒-1·厘米-2 </td><td> SD </td></tr><tr height="21"><td height="21" style="height:21px;"> A555 </td><td> PS </td><td> 580 </td><td> 1.08×10 7 </td><td> 3.31×10 6个</td><td> 9.09×10 6个</td><td> 1.75×10 6个</td><td> 0.233 </td></tr><tr height="21"><td height="21" style="height:21px;"> A568 </td><td> PS </td><td> 600 </td><td> 8.47×10 6个</td><td> 4.23×10 6个</td><td> 4.49×10 6个</td><td> 9.71×10 5个</td><td> 0.094 </td></tr><tr height="21"><td height="21" style="height:21px;"> A633 </td><td> PS </td><td> 660 </td><td> 2.40×10 6个</td><td> 1.25×10 6个</td><td> 7.85×10 5个</td><td> 2.39×10 5个</td><td> 0.046 </td></tr><tr height="21"><td height="21" style="height:21px;"> A700 </td><td> PS </td><td> 720 </td><td> 5.53×10 5个</td><td> 2.46×10 5个</td><td> 1.54×10 5个</td> &lt;TD&gt; 6.05×10 4 </td><td> 0.026 </td></tr><tr height="21"><td height="21" style="height:21px;"> MSPH黄色</td><td>非荧光μspheres </td><td> 520 </td><td> 1.19×10 8个</td><td> 4.85×10 7 </td><td> 5.79×10 7 </td><td> 1.99×10 7 </td><td> 0.057 </td></tr><tr height="21"><td height="21" style="height:21px;"> MSPH粉红</td><td>非荧光μspheres </td><td> 620 </td><td> 2.37×10 7 </td><td> 1.36×10 7个</td><td> 2.16×10 6个</td><td> 8.00×10 5 </td><td> 0.026 </td></tr><tr height="21"><td height="21" style="height:21px;"> QD705 </td><td>非荧光μspheres </td><td> 720 </td><td> 1.76×10 7个</td><td> 7.33×10 6个</td><td> 2.08×10 5个</td><td> 7.16×10 4个</td><td> 0.007 </td></tr><tr height="21"><td height="21" style="height:21px;"> QD800</td><td>非荧光μspheres </td><td> 800 </td><td> 7.79×10 6个</td><td> 4.72×10 6个</td><td> 3.60×10 4个</td><td> 1.52×10 4个</td><td> 0.023 </td></tr></tbody></table> 表2。鉴定荧光和荧光纳米颗粒燃料兼容。

Watch this Scientific Journal Video about A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence FUEL at JoVE.com

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence FUEL

扩大荧光通过调查的有关原则和展示与众多的荧光基团和抗体有针对性的条件，其兼容性的基础和适用性由非绑定从激发发光（燃料）。

一种超越BRET步骤:通过荧光激发无限制自发光（FUEL）

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast ...

a-step-beyond-bret-fluorescence-unbound-excitation-from-luminescence

Research

JoVE Journal

Bioengineering

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

11.5K 次观看.  Institut Pasteur. 扩大荧光通过调查的有关原则和展示与众多的荧光基团和抗体有针对性的条件，其兼容性的基础和适用性由非绑定从激发发光（燃料）。 

视频: A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence FUEL

Synthesis of Luminol

Chemical reactions can be classified as endothermic or exothermic depending on whether the reactions absorb or release heat when they change to products. The difference in energy between reactants and products is known as the enthalpy of the reaction. This is found by calculating the difference in energies between the products and the reactants, or &#916;H. If &#916;H is positive, the reaction is endothermic; if it is negative, the reaction is exothermic. While chemical reactions are traditionally thought of as a conversion of reactants into products, many reactions take place in multiple steps, forming intermediates. In chemiluminescent reactions, these intermediates transition from a high energy state to the ground state, releasing a photon of light that may be visible as the reaction progresses.
<h4>Atomic Energy Levels</h4>
Niels Bohr, a Danish physicist, proposed the theory that an electron orbiting the nucleus of the atom can occupy only certain orbits or energy levels. An atom with all of its electrons in the lowest possible energy level is said to be at its ground state. When an electron occupies an energy level higher than the ground state, the atom is in its excited state.
A key assumption in Bohr&#8217;s theory is that an electron remains in the ground state until it is disturbed. Thus, an electron is only elevated to an excited state by absorbing energy. When the electron relaxes back down to the ground state, it releases that energy. Often, this is in the form of a photon of light, whose wavelength is directly related to the energy difference between the excited and ground states.
When an electron is excited to a higher energy level by absorbing light of a certain energy or wavelength, the phenomenon is called fluorescence. This effect can be seen if you expose a laundered white T-shirt to a black light or UV-light. The characteristic glow is caused by the fluorescence of the detergent and bleaching compounds in the shirt, which make it look white under regular light.
<h4>Chemiluminescence</h4>
In a chemiluminescent reaction, an electron is excited by absorbing heat that is released during a reaction. Then, light is released when the electron relaxes back down to the ground state. The major difference between chemiluminescence and fluorescence is that the energy that excites the electrons in chemiluminescence comes directly from the reaction.
One practical application of chemiluminescence is in commercial glow sticks. A glow stick contains two separate solutions: one containing peroxide and the other containing diphenyl oxalate and a color dye. When the two solutions are mixed, which occurs upon activating the glow stick, the resulting reaction between the peroxide and diphenyl oxalate produces energy that excites the dye to a higher energy state. As the reactants are exhausted, the dye returns to the ground state and releases light. This is what gives glow sticks their characteristic color.
<h4>Luminol</h4>
Luminol is a chemical that exhibits chemiluminescent properties and is utilized in a wide range of applications, most notably in forensics. Luminol (C8H7N3O2) emits blue when it is mixed with an oxidizing agent. In the case of forensics, luminol reacts with the iron in hemoglobin, enabling forensic scientists to identify very small traces of blood.
Luminol is synthesized by the dehydration reaction of 3-nitrophthalic acid with hydrazine. The reaction is heated to remove water, and triethylene glycol is added to further increase the temperature. The nitro group of the 3-nitrophthalhydrazide is then reduced using sodium dithionite to form an amino group at high pH. In basic conditions, the 3-nitrophthalhydrazide is soluble. The addition of glacial acetic acid precipitates the luminol.
With potassium hydroxide, luminol forms a dianion. The hydroxide anions deprotonate the two hydrogens that are attached to the nitrogens in the luminol. Oxygen gas oxidizes luminol to its excited state. As it relaxes back to the ground state, it releases a blue-white light.
<h4>References</h4>
<ol>
	<li style="font-size:17px">Kotz, J.C., Treichel Jr, P.M., Townsend, J.R. (2015). Chemistry and Chemical Reactivity. Belmont, CA: Brooks/Cole, Cengage Learning.</li>
</ol>

Synthesis of Luminol and Chemiluminescence - Concept

鲁米诺的合成

Chemical reactions can be classified as endothermic or exothermic depending on whether the reactions absorb or release heat when they change to products. ...

教育

JoVE Lab Manual

化学

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

科研

生物工程

Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely manipulate neural firing, hormonal secretion and genetically-reprogrammed cells. However, the translation of this technology for medical applications is currently hampered by a lack of biophysical mechanisms by which targeted tissues sense and respond to LIPUS. A suitable approach to identify these mechanisms would be to use optical biosensors in combination with LIPUS to determine underlying signaling pathways. However, implementing LIPUS to a fluorescence microscope may introduce undesired mechanical artefacts due to the presence of physical interfaces that reflect, absorb and refract acoustic waves. This article presents a step-by-step procedure to incorporate LIPUS to commercially-available upright epi-fluorescence microscopes while minimizing the influence of physical interfaces along the acoustic path. A simple procedure is described to operate a single-element ultrasound transducer and to bring the focal zone of the transducer into the objective focal point. The use of LIPUS is illustrated with an example of LIPUS-induced calcium transients in cultured human glioblastoma cells measured using calcium imaging.

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) is a modality for non-invasive mechanical stimulation of endogenous or engineered cells with high spatial and temporal resolution. This article describes how to implement LIPUS to an epi-fluorescence microscope and how to minimize acoustic impedance mismatch along the ultrasound path to prevent unwanted mechanical artefacts.

荧光显微镜对聚焦超声的刺激

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely ...

工程学

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of light-mediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities.
Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence-light emitted by a luciferase enzyme oxidizing a small-molecule substrate, a luciferin-can be harnessed to activate photosensory proteins, thereby adding another dimension to light stimulation and enabling the manipulation of a multitude of light-mediated functions in cells across temporal and spatial scales.

生物发光光遗传学2.0:利用生物发光在体外和体内激活光敏蛋白

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate ...

生物化学

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

在生物环境的新技术的产生和观测化学发光

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and arrhythmias can only be fully understood at the whole organ level. Here, we present the spectroscopic technique of local field fluorescence microscopy (LFFM) that allows the measurement of cellular signals in the intact heart. The technique is based on a combination of a Langendorff perfused heart and optical fibers to record fluorescent signals. LFFM has various applications in the field of cardiovascular physiology to study the heart under normal and pathological conditions. Multiple cardiac variables can be monitored using different fluorescent indicators. These include cytosolic [Ca2+], intra-sarcoplasmic reticulum [Ca2+] and membrane potentials. The exogenous fluorescent probes are excited and the emitted fluorescence detected with three different arrangements of LFFM epifluorescence techniques presented in this paper. The central differences among these techniques are the type of light source used for excitation and on the way the excitation light is modulated. The pulsed LFFM (PLFFM) uses laser light pulses while continuous wave LFFM (CLFFM) uses continuous laser light for excitation. Finally, light-emitting diodes (LEDs) were used as a third light source. This non-coherent arrangement is called pulsed LED fluorescence microscopy (PLEDFM).

In the heart, molecular events coordinate the electrical and contractile function of the organ. A set of local field fluorescence microscopy techniques presented here enables the recording of cellular variables in intact hearts. Identifying mechanisms defining the cardiac function is critical in understanding how the heart works under pathological situations.

局部场荧光显微镜:成像在完整的心蜂窝信号

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and ...

生物学

Introduction to Fluorescence Microscopy

Fluorescence microscopy is a very powerful analytical tool that combines the magnifying properties of light microscopy with visualization of fluorescence. Fluorescence is a phenomenon that involves absorbance and emission of a small range of light wavelengths by a fluorescent molecule known as a fluorophore. Fluorescence microscopy is accomplished in conjunction with the basic light microscope by the addition of a powerful light source, specialized filters, and a means of fluorescently labeling a sample. This video describes the basic principles behind fluorescence microscopy including the mechanism of fluorescence, the Stoke&#x2019;s shift, and photobleaching. It also gives examples of the numerous ways to fluorescently label a sample including the use of fluorescently tagged antibodies and proteins, nucleic acid fluorescent dyes with, and the addition of naturally fluorescent proteins to a specimen. The major components of the fluorescence microscope including a xenon or mercury light source, light filters, the dichroic mirror, and use of the shutter to illuminate the sample are all described. Finally, examples of some of the many applications for fluorescence microscopy are shown.

Fluorescence Microscopy: Concept, Instrumentation, and Applications

荧光显微镜技术的介绍

Fluorescence microscopy is a very powerful analytical tool that combines the magnifying properties of light microscopy with visualization of fluorescence. ...

JoVE Science Education

基础生物学

常用的实验室技术

Photoluminescence: Fluorescence and Phosphorescence

Photoluminescence is a process where a molecule absorbs light energy and re-emits it in the form of light. This phenomenon occurs when a substance absorbs photons, promoting its electrons to higher energy level excited states, followed by a relaxation process in which the electrons return to their original ground state energy levels and emit light. Photoluminescence is widely observed in various materials, including semiconductors, and organic and inorganic compounds.
A pair of electrons in a singlet spin state occupy the same electronic ground state with opposite spins, while a triplet excited state occurs when an electron&#39;s spin is no longer paired with that of the ground state. There are two main types of photoluminescence based on the electron spin states involved: fluorescence and phosphorescence.
Fluorescence is a type of photoluminescence characterized by rapid decay time, typically ranging from nanoseconds to microseconds. In fluorescence, the excited and ground states have the same electron spin multiplicity, meaning that the electron spin remains unchanged during the transition. The process involves singlet-singlet transitions, where both the excited and ground states are singlet states where all electrons are paired.
Phosphorescence is another type of photoluminescence characterized by significantly longer decay times, extending from milliseconds to minutes. In phosphorescence, the excited and ground states have different electron spin multiplicities. The process involves triplet-singlet transitions, where the excited state is a triplet state (two unpaired electrons with parallel spins), and the ground state is a singlet state. These transitions are &#39;spin-forbidden,&#39; meaning the electron spin must change during the transition.
Both fluorescence and phosphorescence can be used in various applications, such as optical sensors, bioimaging, and organic light-emitting diodes. Photoluminescence spectra are recorded by measuring the intensity of emitted radiation in relation to either the excitation wavelength or the emission wavelength. Excitation spectra are obtained by monitoring emission at a fixed wavelength while varying the excitation wavelengths. Emission spectra are obtained by using a fixed wavelength to excite molecules.

光致发光:荧光和磷光

Photoluminescence is a process where a molecule absorbs light energy and re-emits it in the form of light. This phenomenon occurs when a substance absorbs ...

JoVE Core

Analytical Chemistry

Unit 1

Introduction to Molecular Spectroscopy

KEY TERMS AND CONCEPTS

光致发光：荧光和磷光

Photoluminescence: Applications

Photoluminescence offers a wide range of applications due to its inherent sensitivity and selectivity. This technique allows for both direct and indirect analyses of the analyte. Direct quantitative analysis is possible when the analyte exhibits a favorable quantum yield for fluorescence or phosphorescence. However, an indirect analysis may be feasible if the analyte is not fluorescent or phosphorescent, or if the quantum yield is unfavorable. Indirect methods include reacting the analyte with a reagent to form a fluorescent product or measuring the decrease in fluorescence upon adding the analyte to a solution containing a fluorescent molecule.
Inorganic ions, with a few exceptions like UO2+, are generally not sufficiently fluorescent for direct analysis. These ions can be analyzed with an organic ligand to form a fluorescent or phosphorescent metal-ligand complex. Organic compounds containing aromatic rings are typically fluorescent, while aromatic heterocycles tend to be phosphorescent. If the organic analyte is not naturally fluorescent or phosphorescent, it can sometimes be incorporated into a chemical reaction to produce a fluorescent or phosphorescent product. For instance, the enzyme creatine phosphokinase can be determined by catalyzing the formation of creatine from phosphocreatine, which then reacts with ninhydrin to yield a fluorescent product of unknown structure.
Phosphorescence and fluorescence methods are complementary as strongly fluorescing compounds exhibit weak phosphorescence and vice versa. Phosphorimetry has been used to determine a variety of organic and biochemical species. However, it is not as widespread as fluorometry, possibly due to the need for low temperatures and the generally poorer precision of phosphorescence measurements. In recent years, considerable effort has been put into developing phosphorimetric methods that can be carried out at room temperature. These include methods where the analyte is bound to a solid support, such as filter paper or silica gel, or incorporated into the core of micelles or cyclodextrin molecules. In most room-temperature experiments, heavy atoms, such as Tl(I), Pb(II), Ag(I), and halide ions, are used to promote intersystem crossing.

光致发光：应用

Photoluminescence offers a wide range of applications due to its inherent sensitivity and selectivity. This technique allows for both direct and indirect ...

一种超越BRET步骤:通过荧光激发无限制自发光（FUEL）

摘要

探索更多视频

此视频中的章节

鲁米诺的合成

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

荧光显微镜对聚焦超声的刺激

生物发光光遗传学2.0:利用生物发光在体外和体内激活光敏蛋白

在生物环境的新技术的产生和观测化学发光

局部场荧光显微镜:成像在完整的心蜂窝信号

荧光显微镜技术的介绍

光致发光:荧光和磷光

光致发光:荧光和磷光

光致发光：应用