How To Characterize Individual Nanosize Liposomes with Simple Self-Calibrating Fluorescence Microscopy

Abstract

Nanosize lipid vesicles are used extensively at the interface between nanotechnology and biology, e.g., as containers for chemical reactions at minute concentrations and vehicles for targeted delivery of pharmaceuticals. Typically, vesicle samples are heterogeneous as regards vesicle size and structural properties. Consequently, vesicles must be characterized individually to ensure correct interpretation of experimental results. Here we do that using dual-color fluorescence labeling of vesicles-of their lipid bilayers and lumens, separately. A vesicle then images as two spots, one in each color channel. A simple image analysis determines the total intensity and width of each spot. These four data all depend on the vesicle radius in a simple manner for vesicles that are spherical, unilamellar, and optimal encapsulators of molecular cargo. This permits identification of such ideal vesicles. They in turn enable calibration of the dual-color fluorescence microscopy images they appear in. Since this calibration is not a separate experiment but an analysis of images of vesicles to be characterized, it eliminates the potential source of error that a separate calibration experiment would have been. Nonideal vesicles in the same images were characterized by how their four data violate the calibrated relationship established for ideal vesicles. In this way, our method yields size, shape, lamellarity, and encapsulation efficiency of each imaged vesicle. Applying this procedure to extruded samples of vesicles, we found that, contrary to common assumptions, only a fraction of vesicles are ideal.

A large size-selective DNA nanopore with sensing applications

“…24). ATTO 655 was encapsulated following recently published methodology 40,48 . Glass slides with attached sticky-Slide VI 0.4 from Ibidi were functionalized with PLL-g-PEG and PLL-g-PEG–biotin in a 100:1 ratio and consequently covered by a neutravidin layer 39 .…”

Controlled Peptide-Mediated Vesicle Fusion Assessed by Simultaneous Dual-Colour Time-Lapsed Fluorescence Microscopy

“…A 10-fold increase in GUV diameter corresponds to a 1000-fold increase in the volume of a GUV 50 , so many more fusion events between LUVs and GUVs would need to occur in the 15 µm GUV, which is 27 times larger than a 5 µm GUV, in order to detect inner content mixing. In addition not all the vesicles are identical, these differences are averaged out in bulk measurements but in single-vesicle experiments such as this, differences in lipid composition 51,52 , and encapsulation efficiency 53,54 , for example can lead to large differences in the observed rate of fusion and even in the ability of a vesicle to fuse at all. The exclusion of lipopeptide CP n K 4 from the membrane of the GUVs in control experiments did not lead to content-mixing, (Fig.…”

Single particle combinatorial multiplexed liposome fusion mediated by DNA

“…Target liposomes were prepared as described with addition of 0.1% biotinylated lipids and no membrane fluorescent markers were added and functionalized using 1:500 LiNA to lipid ratios. 500 µM fluorescein was encapsulated during rehydration following recently published methodology 28,51 . Cargo liposomes were prepared with addition of 0.5% ATTO 655 DOPE and functionalized using 1:2000 LiNA to lipid ratios, complimentary to the targets using LiNA D for targets and D' for cargo vesicles.…”