Using Imaging Flow Cytometry to Quantify and Optimize Giant Vesicle Production by Water-in-oil Emulsion Transfer Methods

Abstract

Many biologists, biochemists, and biophysicists study giant vesicles, which have a diameter of >1 μm, owing to their ease of characterization using standard optical methods. More recently, there has been interest in using giant vesicles as model systems for living cells and for the construction of artificial cells. In fact, there have been a number of reports about functionalizing giant vesicles using membrane-bound pore proteins and encapsulating biochemical reactions. Among the various methods for preparing giant vesicles, the water-in-oil emulsion transfer method is particularly well established. However, the giant vesicles prepared by this method have complex and heterogeneous properties, such as particle size and membrane structure. Here, we demonstrate the characterization of giant vesicles by imaging flow cytometry to provide quantitative and qualitative information about the vesicle products prepared by the water-in-oil emulsion transfer method. Through image-based analyses, several kinds of protocol byproducts, such as oil droplets and vesicles encapsulating no target molecules, were identified and successfully quantified. Further, the optimal agitation conditions for the water-in-oil emulsion transfer method were found from detailed analysis of imaging flow cytometry data. Our results indicate that a sonication-based water-in-oil emulsion transfer method exhibited a higher efficiency in producing giant vesicles, about 10 times or higher than that of vortex and rumble strip-based methods. It is anticipated that these approaches will be useful for fine-tuning giant vesicle production and subsequent applications.

Current problems and future avenues in proteoliposome research

“…In contrast to "classical" liposomes, GUVs are much larger in size (1 -100 µm) and can directly be observed by light microscopic techniques. 168 On the downside, GUVs are less robust and their preparation is less straightforward (see [169][170][171][172][173][174][175][176][177][178] for literature on GUV formation). Compared to SUVs, GUVs have a vastly increased surface and inner volume that allows for the encapsulation of entire protein machineries, small vesicles or even whole bacteria, in order to mimic increasingly complex functions of living cells.…”

Primordial mimicry induces morphological change in Escherichia coli

“…The cells (data points) used for the morphology analysis were further gated (Supplementary Fig. 1B ) according to the sharpness quality of the cell images, i.e., the fluorescence gradient RMS (root mean square for image sharpness), as described previously 79 . Only the cell images in focus were used for the analysis.…”

Giant Unilamellar Vesicle Electroformation: What to Use, What to Avoid, and How to Quantify the Results

“…For particles up to a diameter of around 1 µm, methods such as dynamic light scattering can be used to easily obtain the average diameter value [99]. Since GUVs fall out of this size category, the most commonly used light scattering method is flow cytometry [38,85,[100][101][102][103][104]. Studies have been performed to confirm the correlation between the forward scatter (FSC) and side scatter (SSC) signal intensity and particle size.…”