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 border="0" cellpadding="0" cellspacing="0" style="width:764px;" width="764">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Name of Material/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Escherichia coli expressing the luxABCDE operon&#160;</td>
			<td style="width:119px;"></td>
			<td>kindly provided by Jos&#233; A. Bengoechea with permission from Herbert P. Schweizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Klebsiella pneumoniae 52145&#160;</td>
			<td style="width:175px;"></td>
			<td style="width:119px;"></td>
			<td>52145 is a serotype K2 reference strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria Bertani (LB)&#160;</td>
			<td style="width:175px;"></td>
			<td style="width:119px;"></td>
			<td>standard growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Q-Tracker 705&#160;</td>
			<td style="width:175px;">Life Sciences</td>
			<td>Q21061MP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Q-Tracker 800</td>
			<td style="width:175px;">Life Sciences</td>
			<td>Q21071MP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Alexa 555</td>
			<td style="width:175px;">Life Sciences</td>
			<td style="width:119px;">S21381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Alexa 568</td>
			<td style="width:175px;">Life Sciences</td>
			<td style="width:119px;">S11226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Alexa 633</td>
			<td style="width:175px;">Life Sciences</td>
			<td style="width:119px;">S21375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Alexa 700</td>
			<td style="width:175px;">Life Sciences</td>
			<td style="width:119px;">S21383</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Non-fluorescent microspheres</td>
			<td style="width:175px;">Polysciences, Inc</td>
			<td align="right" style="width:119px;">15913</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Pink microspheres</td>
			<td style="width:175px;">Life Sciences</td>
			<td>F8887</td>
			<td>40nm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">yellow microspheres</td>
			<td style="width:175px;">Life Sciences</td>
			<td style="width:119px;">F8888</td>
			<td>40nm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Ivis Spectrum</td>
			<td style="width:175px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZ-Link Sulfo-NHS-LC-Biotin</td>
			<td style="width:175px;">Thermo Scientific Pierce</td>
			<td align="right">21425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeba Spin Desalting Columns 7K MWCO</td>
			<td style="width:175px;">Thermo Scientific Pierce</td>
			<td align="right">21425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HABA assay kit&#160;</td>
			<td style="width:175px;">Thermo Scientific Pierce</td>
			<td align="right">28005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Bradford assay</td>
			<td style="width:175px;">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

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

荧光光谱法测定膜蛋白质折叠的热力学

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

科研

生物工程

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

在活细胞中的荧光寿命成像分子转子

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &amp;#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

产生和回收的β-细胞的细胞球体从逐步增长的PEG-肽水凝胶

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

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

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

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

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3d轨道跟踪的改进双光子显微镜之应用：细胞内的囊泡的跟踪

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Engineering &#039;Golden&#039; Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

用质谱和荧光法研究非规范氨基酸和蛋白质分析的选择性压力下工程 "金" 荧光

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

可伸缩、双通道、微流控芯片制造

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

使用一个荧光显微镜进行多种成像模式

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.