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. 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. 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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. 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>

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 ...

Most research with liposomes rely on bold techniques intrinsically assuming all liposomes to be identical. By studying single liposomes, significant inhomogeneities can be observed. The single liposome technique presented here allow us to study hundreds of liposomes simultaneously and detect inhomogeneities in their size and composition.The method can easily be adjusted to study other systems on a single liposome level such as protein membrane interactions. All you need is the compound you study to be fluorescently labeled. With this video, we provide researchers with the tool on how they can perform single liposome studies.We hope that it is clear that the assay can easily be modified to study a range of other scientific topics. To begin, mix the prepared lipid stocks 138 microliters of POPC and 120 microliters of cholesterol in a fresh glass vial. Then add 500 microliters each of two fluorescently labeled lipids and 50 microliters of DSPE-PEG-biotin.Loosen the lid of the glass vial and snap freeze the vial in liquid nitrogen for one to two minutes. Lyophilize the frozen lipid mixture overnight in a freeze dryer. In the morning, add one milliliter of 200 millimolar D-sorbitol buffer to the dry lipids.Heat the mixture to 45 degrees Celsius and expose it to magnetic stirring for at least one hour. Then freeze the lipid suspension by dipping the vial in liquid nitrogen and wait until the suspension is completely frozen. Next, dip the frozen suspension in a heating bath at 55 degrees Celsius until the mixture is completely thawed.Expose the mixture to a total of 11 freeze-thaw cycles. After that, using a mini extrusion kit, extrude the liposome suspension once through an 800 nanometer polycarbonate filter. Mix 1, 200 microliters of BSA and 120 microliters of BSA biotin.Add 300 microliters of the mixture to each well in an eight-well slide and incubate the slide for 20 minutes at room temperature. Slightly tilt the slide and from the edge of each well aspirate the medium. Immediately add 300 microliters of HEPES buffer into each well to wash.Wash each well eight times in total. Add 250 microliters of streptavidin and incubate for 10 minutes at room temperature. Repeat the eight washes as previously described.Place the slide on the microscope and adjust the microscope joystick to focus on the surface of the chamber. This can easily be done using the increased laser reflection signal from the glass buffer interface as a guide. In a microcentrifuge tube, add 10 microliters of the diluted liposome stock containing 20 micromolar total lipid.Transfer 100 microliters of buffer from the chamber to the microcentrifuge tube, mix properly, and transfer the 110 microliters back into the chamber. Put the chamber under the microscope and use a rudimental microscope setting capable of detecting signal from the liposome fluorophores to observe the liposomes being immobilized on the surface. Set up the microscope as described in the manuscript.To image both the DOPE ATTO488 and DOPE ATTO655 fluorophores in the liposomes, image multiple channels. Make sure each channel is imaged sequentially to avoid cross-excitation. For example, first take one image by exciting at 488 nanometers and reading emission at 495 to 590 nanometers.Thereafter, change the settings and take another image by exciting at 633 nanometers but reading emission only at 660 to 710 nanometers.Analyze. In the image menu, choose color and use the merge channel function to create a composite of the two channels. Observe if the liposomes imaged in two different channels display good co-localization or whether visible drift occurred.To detect particles, open the plugins menu, choose ComDet and click detect particles. In ComDet, check that the settings are correct and press OK.After running the analysis, two popup windows with results and summary show. The results table contains the intensity ratio between the two channels.Export the data table results containing the co-localization data. Fit the intensity ratio histogram with a Gaussian function and extract the mean and standard deviation. The mean and standard deviation can be used to calculate the degree of inhomogeneity.Prepare a liposome formulation that has been extruded several times through a 50 nanometer filter. Calibrate the size of the liposomes by comparing the fluorescence intensity to size data from DLS. With the same experimental microscope settings as previously, image the calibration liposomes.Plot a square root intensity histogram of the fluorescence intensity of the calibration liposomes. Fit the histogram with a log normal distribution and extract the average fluorescence intensity of the calibration liposomes as the geometric mean. To determine the relation between the square root intensity and liposome size, calculate the correction factor by dividing the average liposome diameter obtained from the dynamic light scattering measurements with the geometric mean of the square root intensity.Calculate square root intensity values for the liposomes in the compositional inhomogeneity experiment and convert these two diameters by multiplying with the correction factor. Plot the intensity ratio value as a function of diameter for the compositional inhomogeneity liposomes, thus achieving the inhomogeneity as a function of liposome size for a population of liposomes spanning from approximately 50 nanometers to 800 nanometers. In this protocol, the successful surface immobilization of liposomes was immediately apparent upon the addition of the liposome solution to the chamber indicated by the diffraction limited intensity spots.It is recommended to immobilize enough liposomes to achieve a density of 300 to 400 liposomes per frame. A lower density makes the data analysis more time consuming while a higher density makes it challenging to distinguish single liposomes. If the whole field of view is not in proper focus, the sample plate might be tilting.Also, if the liposomes seem large and blurry, this indicates the buffer in the chamber might have evaporated and the surface dried out. The intensity ratio histograms typically reveal a Gaussian distribution around a mean ratio value. If strong deviations from a Gaussian distribution are observed, it indicates a detection sensitivity issue suggesting that a subset of liposomes showing a weaker signal have been excluded.After fitting the intensity ratio histogram with a Gaussian function, a degree of inhomogeneity value of 0.23 was translated to 32%of the population differing by more than 23%from the mean molar ratio of the ensemble. The quantification of the experimental uncertainty was found to be 0.1. The assay will never get better than the materials you use so high quality unilaminar liposomes are essential.So when you prepare these, do not cut any corners such as leaving out freeze-thaw cycles. Poor images will lead to high experimental uncertainty and make you overestimate the inhomogeneity so take the time to acquire good in-focus images. What the assay basically do is to study the surface density of the fluorescent label.If this fluorescent label is on a percolated lipid, then this assay can be used to quality test liposomes for drug delivery. The setup has been applied to study how peptides bind to membranes and sense the curvature. This has given researchers unique insights to the molecular cues that govern how peptides are sorted inside cells.

a-quantitative-fluorescence-microscopy-based-single-liposome-assay

Research

JoVE Journal

Bioengineering

7.7K vistas.  Technical University of Denmark. La mayoría de las investigaciones con liposomas se basan en técnicas audaces que intrínsecamente asumen que todos los liposomas son idénticos. Mediante el estudio de liposomas individuales, se pueden observar inhomogeneidades significativas. La única técnica de liposomas presentada aquí nos permite estudiar cientos de liposomas simultáneamente y detectar inhomogeneidades en su tamaño y composición.El método se puede ajustar fácilmente para estudiar otros sistemas en un solo...