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

This work was funded by the Danish Council for Independent Research [grant number 5054-00165B].

1. Liposome Preparation
NOTE: Briefly, preparation of liposomes usually includes three crucial steps: 1) preparation of dry lipid films of the desired lipid composition; 2) rehydration of the lipids for formation of liposomes; and 3) controlling the size and lamellarity of the liposome population.
<ol>
	<li>Weigh out the lipids and dissolve them in tert-butanol:water (9:1) in glass vials.
	<ol>
		<li>Dissolve POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine; MW = 760 g/mol) to 50 mM.</li>...

<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. 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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. 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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. 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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. 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It is important to note that while we describe in detail how the single liposomes assay can be used to study the compositional inhomogeneity between individual liposomes, the platform is very versatile. As previously shown and discussed in the introduction, the protocol can easily be adapted to study aspects of membrane-membrane fusion, protein-membrane interactions, or liposomal drug carrier characterization. For any scientific questions being addressed, the power of the single liposome assay lies in the ability to dete...

Liposomes are spherical phospholipid-based vesicles that are heavily used both in basic and applied research. They function as excellent membrane model systems, because their physiochemical properties can be easily manipulated by varying the lipid components making up the liposome1,2. Also, liposomes constitute the most used drug delivery nanocarrier system, offering improved pharmacokinetics and pharmacodynamics as well as high biocompatibility3.
For many years, liposomes have primarily been studied using bulk techniques, giving only access to ensemble average read-out values. This has led the majority of these studies to assume that all liposomes in the ensemble are identical. However, such ensemble-averaged values are only correct if the underlying dataset is uniformly distributed around the mean value, but can represent a false and biased conclusion if the dataset includes multiple independent populations, for example. Additionally, assuming the ensemble mean to represent the whole population can overlook the information harbored within the inhomogeneity between liposomes. Only recently have quantitative assays emerged that are able to probe single liposomes, revealing large inhomogeneities between individual liposomes with respect to important physicochemical properties including liposome size4, lipid composition5,6, and encapsulation efficiency7, highlighting the importance of studying liposomes at the single liposome level.
A research area where ensemble averaging of liposome properties has been shown to bias results is studying liposome size-dependent protein-membrane interactions8,9. Traditionally, researchers studying such processes have been restricted to preparing liposomes with different ensemble average diameters by extrusion through filters with different pore sizes9. However, extracting the diameter of individual liposomes using single liposome assays has revealed large population overlaps, with liposomes extruded using 100 nm and 200 nm filters displaying up to 70% overlap in their size distribution4. This could severely bias bulk measurements of liposome size-dependent protein-membrane interactions10. Performing the membrane-protein interaction studies using the single liposome assay, researchers instead took advantage of the size-polydispersity within the sample, allowing them to study a wide range of liposome diameters within each single experiment, facilitating new discoveries of how membrane curvature and composition can affect protein recruitment to membranes4,11,12. Another field where the application of single liposome assays has proven instrumental is in mechanistic studies of protein-mediated membrane fusion13,14. For such kinetic measurements, the ability to study individual fusion events alleviated the need for the experimental synchronization of the fusion process, allowing new mechanistic insights that would otherwise have been lost in the spatiotemporal averaging done in bulk ensemble measurements. Additionally, single liposomes have been used as a membrane scaffold, allowing the measurement of individual proteins and offering new knowledge on transmembrane protein structural dynamics15,16. Furthermore, such proteoliposome-based setups made it possible to study the function of individual transmembrane transporters17 and pore-forming protein complexes18 as well as the mechanism of bioactive membrane-permeabilizing peptides19. Single liposomes have also been used as soft matter nanofluidics with surface-immobilized single liposomes serving as chambers for enzymatic reactions in volumes of 10-19 L, increasing the throughput and complexity of the screening assays with minimal product consumption20.
Recently, single liposome assays have been used for characterizing drug delivery liposomes at a previously unprecendented level of detail. Researchers were able to quantify significant inhomogeneities in the amount of polymer attached to the surface of individual liposomes21. The single liposome assays also allowed measurements of drug delivery liposomes in complex media, such as blood plasma, revealing how elements anchored to the liposome surface through lipid anchors can be susceptible to dissociation when liposomes are exposed to conditions mimicking those experienced during in vivo circulation22. Overall, the versatility and usefulness of the single liposome assays are substantiated by the great variety of problems these setups have been employed to address, and we envision that the methodology will continue to be developed and find use in new scientific fields.
Here we describe a fluorescence microscopy-based single liposome assay that allows individual liposomes to be studied in a high-throughput manner (Figure 1). To illustrate the method, we use it to quantify the size and compositional inhomogeneity between individual liposomes within an ensemble. The assay employs fluorescence microscope imaging of single liposomes immobilized on a passivated glass surface. We first describe the critical steps in the liposome fabrication process that ensures proper fluorescent liposome labeling and immobilization. Then, we describe the surface preparation needed to facilitate liposome immobilization before outlining the procedure for ensuring appropriate liposome surface densities. We discuss the microscopy parameters important for acquiring high-quality images and delineate how to perform simple data analysis, allowing the extraction of liposome size and compositional inhomogeneity. This generic protocol should provide a good basis for the interested researcher to develop the assay further for his or her specific research interest.

<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

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

Following the protocol described makes it possible to image single liposomes in a massive parallel manner (Figure 1). The successful surface immobilization of liposomes should be immediately apparent upon the addition of the liposome solution to the chamber (step 3.6 in the protocol) as diffraction limited intensity spots should appear in the image (Figure 1B and Figure 1C</s...

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

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

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

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