Name: a quantitative fluorescence microscopy-based single liposome assay for detecting the compositional inhomogeneity between individual liposomes
Uploaded: 2019-12-13T16:00:00
Description: Watch this Scientific Journal Video about A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes at JoVE.com

这项工作由丹麦独立研究理事会资助[赠款号5054-00165B]。

1. 脂体制备
注：简要地说，脂质体制备通常包括三个关键步骤：1）制备所需脂质成分的干脂膜;2） 脂质的补液形成脂质体;3） 控制脂质体种群的大小和粒度。
<ol><li>称量脂质并将其溶解在玻璃瓶中的三丁醇：水（9：1）。<ol>
<li>溶解POPC（1-棕榈-2-醇-甘油-3-磷胆碱）;MW = 760 g/mol） 至 50 mM。</li>
		<li>将胆固醇 （MW = 387 g/mol） 溶解到 25 mM。</li>
		<li>溶解 DOPE-Atto488 （1，2-二氧化?...

<ol>
	<li>Chan, Y. H. M., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+membrane+systems+and+their+applications.">Model membrane systems and their applications.</a> Current Opinion in Chemical Biology. 11 (6), 581-587 (2007).</li><li>Veatch, S. L., Keller, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+spots:+Complex+phase+behavior+in+simple+membranes.">Seeing spots: Complex phase behavior in simple membranes.</a> Biochimica Et Biophysica Acta-Molecular Cell Research. 1746 (3), 172-185 (2005).</li><li>Sercombe, 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=Advances+and+Challenges+of+Liposome+Assisted+Drug+Delivery.">Advances and Challenges of Liposome Assisted Drug Delivery.</a> Frontiers in Pharmacology. 6, 13 (2015).</li><li>Hatzakis, N. 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=How+curved+membranes+recruit+amphipathic+helices+and+protein+anchoring+motifs.">How curved membranes recruit amphipathic helices and protein anchoring motifs.</a> Nature Chemical Biology. 5 (11), 835-841 (2009).</li><li>Elizondo, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+the+Preparation+Route+on+the+Supramolecular+Organization+of+Lipids+in+a+Vesicular+System.">Influence of the Preparation Route on the Supramolecular Organization of Lipids in a Vesicular System.</a> Journal of the American Chemical Society. 134 (4), 1918-1921 (2012).</li><li>Larsen, J., Hatzakis, N. S., Stamou, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+Inhomogeneity+in+the+Lipid+Composition+of+Individual+Nanoscale+Liposomes.">Observation of Inhomogeneity in the Lipid Composition of Individual Nanoscale Liposomes.</a> Journal of the American Chemical Society. 133 (28), 10685-10687 (2011).</li><li>Lohse, B., Bolinger, P. Y., Stamou, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+Efficiency+Measured+on+Single+Small+Unilamellar+Vesicles.">Encapsulation Efficiency Measured on Single Small Unilamellar Vesicles.</a> Journal of the American Chemical Society. 130 (44), 14372 (2008).</li><li>Iversen, L., Mathiasen, S., Larsen, J. B., Stamou, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+curvature+bends+the+laws+of+physics+and+chemistry.">Membrane curvature bends the laws of physics and chemistry.</a> Nature Chemical Biology. 11 (11), 822-825 (2015).</li><li>Bhatia, V. K., Hatzakis, N. S., Stamou, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unifying+mechanism+accounts+for+sensing+of+membrane+curvature+by+BAR+domains,+amphipathic+helices+and+membrane-anchored+proteins.">A unifying mechanism accounts for sensing of membrane curvature by BAR domains, amphipathic helices and membrane-anchored proteins.</a> Seminars in Cell &amp; Developmental Biology. 21 (4), 381-390 (2010).</li><li>Bhatia, V. 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=Amphipathic+motifs+in+BAR+domains+are+essential+for+membrane+curvature+sensing.">Amphipathic motifs in BAR domains are essential for membrane curvature sensing.</a> EMBO Journal. 28 (21), 3303-3314 (2009).</li><li>Larsen, J. B., 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=Membrane+curvature+enables+N-Ras+lipid+anchor+sorting+to+liquid-ordered+membrane+phases.">Membrane curvature enables N-Ras lipid anchor sorting to liquid-ordered membrane phases.</a> Nature Chemical Biology. 11 (3), 192 (2015).</li><li>Larsen, J. B., 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=Membrane+Curvature+and+Lipid+Composition+Synergize+To+Regulate+N-Ras+Anchor+Recruitment.">Membrane Curvature and Lipid Composition Synergize To Regulate N-Ras Anchor Recruitment.</a> Biophysical Journal. 113 (6), 1269-1279 (2017).</li><li>Yoon, T. Y., Okumus, B., Zhang, F., Shin, Y. K., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+intermediates+in+SNARE-induced+membrane+fusion.">Multiple intermediates in SNARE-induced membrane fusion.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (52), 19731-19736 (2006).</li><li>Fix, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+single+membrane+fusion+events+mediated+by+SNARE+proteins.">Imaging single membrane fusion events mediated by SNARE proteins.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (19), 7311-7316 (2004).</li><li>Hatzakis, N. 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=Single+Enzyme+Studies+Reveal+the+Existence+of+Discrete+Functional+States+for+Monomeric+Enzymes+and+How+They+Are+"Selected"+upon+Allosteric+Regulation.">Single Enzyme Studies Reveal the Existence of Discrete Functional States for Monomeric Enzymes and How They Are "Selected" upon Allosteric Regulation.</a> Journal of the American Chemical Society. 134 (22), 9296-9302 (2012).</li><li>Mathiasen, 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=Nanoscale+high-content+analysis+using+compositional+heterogeneities+of+single+proteoliposomes.">Nanoscale high-content analysis using compositional heterogeneities of single proteoliposomes.</a> Nature Methods. 11 (9), 931-934 (2014).</li><li>Veshaguri, 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=Direct+observation+of+proton+pumping+by+a+eukaryotic+P-type+ATPase.">Direct observation of proton pumping by a eukaryotic P-type ATPase.</a> Science. 351 (6280), 1469-1473 (2016).</li><li>Tonnesen, A., Christensen, S. M., Tkach, V., Stamou, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometrical+Membrane+Curvature+as+an+Allosteric+Regulator+of+Membrane+Protein+Structure+and+Function.">Geometrical Membrane Curvature as an Allosteric Regulator of Membrane Protein Structure and Function.</a> Biophysical Journal. 106 (1), 201-209 (2014).</li><li>Kristensen, K., Ehrlich, N., Henriksen, J. R., Andresen, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Vesicle+Detection+and+Analysis+of+Peptide-Induced+Membrane+Permeabilization.">Single-Vesicle Detection and Analysis of Peptide-Induced Membrane Permeabilization.</a> Langmuir. 31 (8), 2472-2483 (2015).</li><li>Christensen, S. M., Bolinger, P. Y., Hatzakis, N. S., Mortensen, M. W., Stamou, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixing+subattolitre+volumes+in+a+quantitative+and+highly+parallel+manner+with+soft+matter+nanofluidics.">Mixing subattolitre volumes in a quantitative and highly parallel manner with soft matter nanofluidics.</a> Nature Nanotechnology. 7 (1), 51-55 (2012).</li><li>Eliasen, R., Andresen, T. L., Larsen, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEG-Lipid+Post+Insertion+into+Drug+Delivery+Liposomes+Quantified+at+the+Single+Liposome+Level.">PEG-Lipid Post Insertion into Drug Delivery Liposomes Quantified at the Single Liposome Level.</a> Advanced Materials Interfaces. 6 (9), 1801807 (2019).</li><li>M&#252;nter, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociation+of+fluorescently+labeled+lipids+from+liposomes+in+biological+environments+challenges+the+interpretation+of+uptake+studies.">Dissociation of fluorescently labeled lipids from liposomes in biological environments challenges the interpretation of uptake studies.</a> Nanoscale. 10 (48), 22720-22724 (2018).</li><li>Traikia, M., Warschawski, D. E., Recouvreur, M., Cartaud, J., Devaux, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+unilamellar+vesicles+by+repetitive+freeze-thaw+cycles:+characterization+by+electron+microscope+and+P-31-nuclear+magnetic+resonance.">Formation of unilamellar vesicles by repetitive freeze-thaw cycles: characterization by electron microscope and P-31-nuclear magnetic resonance.</a> European Biophysics Journal with Biophysics Letters. 29 (3), 184-195 (2000).</li><li>Nele, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Formulation+Method,+Lipid+Composition,+and+PEGylation+on+Vesicle+Lamellarity:+A+Small-Angle+Neutron+Scattering+Study.">Effect of Formulation Method, Lipid Composition, and PEGylation on Vesicle Lamellarity: A Small-Angle Neutron Scattering Study.</a> Langmuir. 35 (18), 6064-6074 (2019).</li><li>Kunding, A. H., Mortensen, M. W., Christensen, S. M., Stamou, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence-based+technique+to+construct+size+distributions+from+single-object+measurements:+Application+to+the+extrusion+of+lipid+vesicles.">A fluorescence-based technique to construct size distributions from single-object measurements: Application to the extrusion of lipid vesicles.</a> Biophysical Journal. 95 (3), 1176-1188 (2008).</li><li>Hughes, L. D., Rawle, R. J., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choose+Your+Label+Wisely:+Water-Soluble+Fluorophores+Often+Interact+with+Lipid+Bilayers.">Choose Your Label Wisely: Water-Soluble Fluorophores Often Interact with Lipid Bilayers.</a> PLoS One. 9 (2), e87649 (2014).</li></ol>

需要注意的是，虽然我们详细描述了如何使用单脂体测定来研究单个脂质体之间的组成不均匀性，但该平台非常通用。如前面所示和介绍中讨论，该协议可以很容易地适应研究膜膜融合、蛋白膜相互作用或脂质体药物载体表征的各个方面。对于任何正在解决的科学问题，单脂质测定的力量在于能够检测合奏的各个成分，从而具有定量读出，而不是通过集合平均效应进行偏颇。
<p class="jove_content"...

脂质体是以球面磷脂为基础的囊泡，在基础和应用研究中被大量使用。它们作为优秀的膜模型系统，因为它们的物理化学性质可以很容易地通过改变组成脂质体1，2的脂质成分来操纵。此外，脂体构成最常用的药物输送纳米载体系统，提供改进的药代动力学和药效学以及高生物相容性3。
多年来，脂体主要使用散装技术进行研究，仅允许访问整体平均读出值。这导致大多数这些研究假设，在集合中的所有脂体是相同的。但是，仅当基础数据集在平均值周围均匀分布时，此类集合平均值才正确，但如果数据集包含多个独立总体，则可以表示错误和偏置的结论。此外，假设表示整个人群的合奏意味着可以忽视脂质体之间不均匀性中蕴藏的信息。直到最近才出现能够探测单脂质体的定量测定，揭示了单个脂质体之间相对于重要的物理化学特性（包括脂质体大小4、脂质组合物5、6和封装效率7）之间的大不均匀性，突出了在单脂质体水平上研究脂质体的重要性。
一个研究领域，其中脂质体特性的一组平均已被证明有偏差的结果是研究脂质体大小依赖蛋白膜相互作用8，9。传统上，研究此类过程的研究人员仅限于通过不同孔径的过滤器，以制备不同集合平均直径的脂质体。然而，使用单脂体测定提取单个脂体直径后发现，脂体种群重叠较大，使用100nm和200nm滤波器拉伸的脂体在其大小分布4中显示高达70%的重叠。这可能严重偏置脂体大小依赖蛋白膜相互作用的批量测量10。研究人员使用单脂体测定法进行膜蛋白相互作用研究，而是利用样品中的大小-多分散性，允许他们在每个实验中研究广泛的脂质体直径，从而促进新发现膜曲率和组成如何影响膜4、11、12的蛋白质招募。单脂体测定的应用证明有用的另一个领域是蛋白质介导膜融合的力学研究13，14。对于这种动力学测量，研究单个融合事件的能力减轻了对融合过程的实验同步的需要，使得新的机械洞察力，否则在散装集合测量中完成的时空平均中会丢失。此外，单脂体已被用作膜支架，允许测量单个蛋白质，并提供跨膜蛋白结构动力学的新知识15，16。此外，这种基于蛋白酶体的设置使得研究单个跨膜输送机17和孔隙形成蛋白复合物18的功能以及生物活性膜渗透肽19的机理成为可能。单脂质体也被用作软物质纳米流体，表面固定单脂质体作为10-19 L体积的酶反应室，以最小的产品消耗20增加筛选测定的通量和复杂性。
最近，单脂体测定已用于在以前未预先拟定的细节水平上对药物输送脂量体进行特征化。研究人员能够量化附着在单个脂质体表面的聚合物数量中显著的不均匀性。单脂体测定还允许在复杂的介质（如血浆）中测量药物输送脂质体，揭示当脂质体暴露于模仿体内循环过程中经历的条件时，通过脂质锚锚锚定位到脂质体表面的元素如何容易分离。总体而言，单脂体测定的多功能性和实用性得到了这些装置用于解决的各种问题的证实，我们设想，该方法将继续开发，并在新的科学领域得到应用。
在这里，我们描述了一种基于荧光显微镜的单脂体测定，它允许以高通量方式研究单个脂体（图1）。为了说明该方法，我们用它来量化集合中单个脂质体之间的大小和组成不均匀性。该测定采用荧光显微镜成像单脂质体固定在钝化玻璃表面。我们首先描述了脂体制造过程中的关键步骤，以确保适当的荧光脂体标记和固定。然后，在概述确保适当脂体表面密度的程序之前，我们描述了促进脂体固定所需的表面制备。我们讨论了对获取高质量图像非常重要的显微镜参数，并描述如何执行简单的数据分析，允许提取脂质体大小和成分不均匀性。此通用协议应为感兴趣的研究人员为进一步开发其特定研究兴趣的测定提供良好基础。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>8-well microscopy slides (&#181; slides)</td>
			<td>Ibidi</td>
			<td>80827</td>
			<td>Microscopy slides with glass bottom</td>
		</tr>
		<tr>
			<td>Avanti Mini Extrusion kit</td>
			<td>Avanti Polar Lipids</td>
			<td>610000</td>
			<td>Consumables (Whatman filters) can be aquired from GE Healthcare</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA-Biotin</td>
			<td>Sigma</td>
			<td>A8549</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Avanti Polar Lipids</td>
			<td>700000</td>
			<td>Traded trough Sigma</td>
		</tr>
		<tr>
			<td>Computer with FIJI (Fiji Is Just ImageJ)</td>
			<td></td>
			<td></td>
			<td>ComDet plugin must be installed. Also, a data handling software (Excel, MatLab, OpenOffice, GraphPad Prism etc.) able to load .txt files will be needed to plot the data</td>
		</tr>
		<tr>
			<td>DOPE-Atto488</td>
			<td>Atto-Tech</td>
			<td>AD488-165</td>
			<td></td>
		</tr>
		<tr>
			<td>DOPE-Atto655</td>
			<td>Atto-Tech</td>
			<td>AD655-165</td>
			<td></td>
		</tr>
		<tr>
			<td>DOPE-PEG-Biotin</td>
			<td>Avanti Polar Lipids</td>
			<td>880129</td>
			<td>Traded trough Sigma</td>
		</tr>
		<tr>
			<td>D-Sorbitol</td>
			<td>Sigma</td>
			<td>S-6021</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze-dryer</td>
			<td></td>
			<td></td>
			<td>e.g. ScanVac Coolsafe from Labogene</td>
		</tr>
		<tr>
			<td>Glass vials</td>
			<td>Brown Chromatography</td>
			<td>150903</td>
			<td>Glass vials that can resist snap-freezing in liquid nitrogen. The 8 mL version of the vials has a size that also fits with the syringes of the extrusion kit</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Honeywell Fluka</td>
			<td>258148</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating bath</td>
			<td></td>
			<td></td>
			<td>Capable of heating to minimum 65C</td>
		</tr>
		<tr>
			<td>Heating plate w. Magnet stirring</td>
			<td></td>
			<td></td>
			<td>Capable of heating to minimum 65C</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td>Including container for storage, e.g. Rubber-bath</td>
		</tr>
		<tr>
			<td>Magnetic stirring bars</td>
			<td>VWR</td>
			<td>442-4520 (EU)</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 1.5 mL</td>
			<td>Eppendorf</td>
			<td>0030 120.086 (EU)</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td></td>
			<td></td>
			<td>For the images in this protocol a Leica SP5 confocal microscope has been used</td>
		</tr>
		<tr>
			<td>Na HEPES</td>
			<td>Sigma</td>
			<td>H7006</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Honeywell Fluka</td>
			<td>71686</td>
			<td></td>
		</tr>
		<tr>
			<td>POPC</td>
			<td>Avanti Polar Lipids</td>
			<td>850457</td>
			<td>Traded trough Sigma</td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Sigma</td>
			<td>S4762</td>
			<td></td>
		</tr>
		<tr>
			<td>tert-Butanol (2-methyl-2-propanol)</td>
			<td>Honeywell Riedel-de Ha&#235;n</td>
			<td>24127</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td></td>
			<td></td>
			<td>e.g. MilliQ</td>
		</tr>
	</tbody>
</table>

a quantitative fluorescence microscopy-based single liposome assay for detecting the compositional inhomogeneity between individual liposomes

大多数使用脂体作为膜模型系统或药物输送载体的研究都依赖于批量读出技术，因此在本质上假定整体的所有脂体都是相同的。然而，能够在单粒子水平上观察脂质体的新实验平台使得对单个脂质体的蛋白质膜相互作用或药物载体特性进行高度精密的定量研究成为可能，从而避免从集合平均错误。在这里，我们提出了一个协议，用于准备，检测和分析单脂体使用荧光为基础的显微镜测定，促进这种单粒子测量。该设置允许以大规模并行方式成像单个脂质体，并用于揭示样品内大小和成分不均匀性。此外，该协议还描述了在单脂体水平上研究脂体的优点、测定的局限性以及修改其以研究其他研究问题时需要考虑的重要特征。

遵循所述协议，可以大规模并行方式对单个脂体进行成像（图1）。在将脂体溶液添加到腔室（协议步骤 3.6）时，脂体的成功表面固定应立即显现出来，因为衍射有限强度点应出现在图像中（图 1B和图 1C）。
为了实现良好的统计和利?...

Watch this Scientific Journal Video about A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes at JoVE.com

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

该协议描述了脂体的制造，以及如何用荧光显微镜将这些脂体固定在表面上，并单独以大规模平行的方式成像。这允许定量种群单脂质体之间的大小和组成不均匀性。

定量荧光显微镜为基础的单脂体测定，用于检测单个脂质体之间的成分不均匀性

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

大多数对脂质体的研究都依赖于大胆的技术，内在假设所有的脂质体是相同的。通过研究单脂体，可以观察到显著的异质性。这里介绍的单脂体技术使我们能够同时研究数百种脂质体，并检测其大小和成分的不均匀性。该方法可以很容易地调整，以研究其他系统在一个单一的脂体水平，如蛋白质膜相互作用。你所需要的只是你研究的化合物被荧光标记。通过这个视频，我们为研究人员提供了他们如何执行单脂体研究的工具。我们希望，很明显，该分析可以很容易地修改，以研究一系列其他科学课题。首先，将准备好的脂质储存138微升的波普克和120微升胆固醇混合在新鲜的玻璃瓶中。然后添加500微升，每个两个荧光标记脂质和50微升的DSPE-PEG-生物素。松开玻璃瓶盖，将小瓶在液氮中捕捉冻结一到两分钟。在冷冻干燥机中冷冻脂质混合物过夜。早晨，在干脂中加入一毫升200毫摩尔D-索比托缓冲液。将混合物加热至45摄氏度，使其暴露在磁搅拌中至少一小时。然后将小瓶浸入液氮中，然后冷冻脂质悬浮液，然后等待悬浮液完全冷冻。接下来，将冷冻悬浮液浸入55摄氏度的加热浴中，直到混合物完全解冻。将混合物暴露在总共 11 个冷冻解冻循环中。之后，使用迷你挤出套件，通过 800 纳米聚碳酸酯过滤器挤出脂体悬浮液一次。混合 1，200 微升 BSA 和 120 微升 BSA 生物素。在八井滑梯中，将混合物的300微升加入到每井中，并在室温下孵育20分钟。稍微倾斜幻灯片，从每个滑点的边缘向上倾斜，并吸气介质。立即将 300 微升的 HEPES 缓冲液添加到每个井中进行清洗。每井总共洗八次。加入250微升的链球菌素，在室温下孵育10分钟。重复前面描述的八次洗涤。将幻灯片放在显微镜上，并调整显微镜操纵手柄以聚焦在室表面。这可以很容易地使用从玻璃缓冲接口增加的激光反射信号作为参考。在微离心管中，加入含有20微摩尔总脂质的稀释脂质库存10微升。将 100 微升缓冲液从腔室转移到微离心管，正确混合，然后将 110 微升将缓冲器重新传递回腔室。将室置于显微镜下，并使用能够检测来自脂质荧光团的信号的初级显微镜设置来观察脂质体在表面上被固定。设置手稿中描述的显微镜。要在脂质体中同时成像 DOPE ATTO488 和 DOPE ATTO655 荧光团，请成像多个通道。确保每个通道按顺序成像，以避免交叉激发。例如，首先在 488 纳米时以令人兴奋的速度拍摄一张图片，在 495 到 590 纳米时读取发射。此后，更改设置，以 633 纳米的令人兴奋的速度拍摄另一个图像，但读取发射仅为 660 至 710 纳米。分析。在图像菜单中，选择颜色并使用合并通道功能创建两个通道的合成。观察在两个不同的通道中成像的脂质体是否显示良好的共定位，或者是否发生了可见漂移。要检测粒子，请打开插件菜单，选择 ComDet 并单击检测粒子。在 ComDet 中，检查设置是否正确，然后按"确定"。运行分析后，将显示两个弹出窗口，显示结果和摘要。结果表包含两个通道之间的强度比。导出包含共同本地化数据的数据表结果。将强度比直方图与高斯函数拟合，并提取均值和标准差。均值和标准差可用于计算不均匀性的程度。准备通过 50 纳米过滤器挤出多次的脂体配方。通过将荧光强度与DLS的尺寸数据进行比较，校准脂质体的大小。使用与以前相同的实验显微镜设置，对校准脂质体进行成像。绘制校准脂质体荧光强度的平方根强度直方图。将直方图与对数正态分布拟合，并提取校准脂质体的平均荧光强度作为几何平均值。要确定平方根强度和脂体大小之间的关系，通过将从动态光散射测量中获得的平均脂体直径与平方根强度的几何平均值进行划分来计算校正系数。计算组合不均匀性实验中脂质体的强度值，并将这两个直径与校正因子相乘。绘制强度比值作为组成非等性脂质脂质体直径的函数，从而实现脂质大小的不和源性，为从大约 50 纳米到 800 纳米的脂质体种群。在该协议中，在衍射有限强度点指示的腔室中加入脂质体溶液后，脂质体的成功表面固定立即显现出来。建议固定足够的脂质体，以达到每帧 300 到 400 个脂质体的密度。较低的密度使数据分析更加耗时，而较高的密度则难以区分单个脂质体。如果整个视野没有正确聚焦，则样品板可能会倾斜。此外，如果脂质体看起来又大又模糊，则表示腔室中的缓冲液可能已经蒸发，表面干涸。强度比直方图通常显示围绕平均比率值的高斯分布。如果观察到与高斯分布的强烈偏差，则表示检测灵敏度问题，表明已排除了显示较弱信号的脂质体子集。将强度比直方图与高斯函数拟合后，将0.23的不和质值度转换为32%的人口，与合奏的均摩尔比相差23%以上。实验不确定性的定量为0.1。检测永远不会得到更好的材料，你使用，所以高品质的非摩尔脂质体是必不可少的。因此，当您准备这些，不要削减任何角落，如离开冻结解冻周期。糟糕的图像会导致高实验性的不确定性，并让你高估了不均匀性，所以花时间去获取好的对焦图像。测定基本上要研究荧光标签的表面密度。如果此荧光标签在渗透脂质上，则此检测可用于检测药物输送的脂质体。该设置已被应用于研究肽如何与膜结合和感觉曲率。这给了研究人员独特的见解，指导肽在细胞内如何排序的分子线索。

Research

JoVE Journal

Bioengineering

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

氧微环境中的单个胰岛水平的定量和临时控制

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological ...

科研

生物工程

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

腺苷酸环化酶的药物引起的过敏性：含量简化和小型化的小分子和siRNA筛选应用

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

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

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

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 ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

由微型条纹分析技术在微流控设备混合不均匀

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

生物膜荧光探针力：受体 - 配体动力学并行和定量结合诱导细胞内信号作用于单细胞

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

使用多色荧光检测液滴微流控光纤

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

工程分子识别与单壁碳纳米管仿生聚合物

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

单细胞基因组测序的超高通量微流控平台

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...