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

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.