Analysis of Polyhydroxyalkanoates Granules in Haloferax mediterranei by Double-Fluorescence Staining with Nile Red and SYBR Green by Confocal Fluorescence Microscopy

Haloferaxmediterranei is a haloarchaeon of high interest in biotechnology because it produces and mobilizes intracellular polyhydroxyalkanoate (PHA) granules during growth under stress conditions (limitation of phosphorous in the culture media), among other interesting metabolites (enzymes, carotenoids, etc.). The capability of PHA production by microbes can be monitored with the use of staining-based methods. However, the staining of haloarchaea cells is a challenging task; firstly, due to the high ionic strength of the medium, which is inappropriate for most of dyes, and secondly, due to the low permeability of the haloarchaea S-layer to macromolecules. In this work, Haloferax mediterranei is used as a halophilic archaeon model to describe an optimized protocol for the visualization and analysis of intracellular PHA granules in living cells. The method is based on double-fluorescence staining using Nile red and SYBR Green by confocal fluorescence microscopy. Thanks to this method, the capability of PHA production by new haloarchaea isolates could be easily monitored.

Biomimetic Curvature and Tension-Driven Membrane Fusion Induced by Silica Nanoparticles

Membrane fusion is a key process to develop new technologies in synthetic biology, where artificial cells function as biomimetic chemical microreactors. Fusion events in living cells are intricate phenomena that require the coordinate action of multicomponent protein complexes. However, simpler synthetic tools to control membrane fusion in artificial cells are highly desirable. Native membrane fusion machinery mediates fusion driving a delicate balance of membrane curvature and tension between two closely apposed membranes. Here we show that silica nanoparticles (SiO₂ NPs) at a size close to the cross-over between tension-driven and curvature-driven interaction regimes initiate efficient fusion of biomimetic model membranes. Fusion efficiency and mechanisms are studied by Förster Resonance Energy Transfer (FRET) and confocal fluorescence microscopy. SiO₂ NPs induce a slight increase in lipid packing likely to increase the lateral tension of the membrane. We observe a connection between membrane tension and fusion efficiency. Finally, real-time confocal fluorescence microscopy reveals three distinct mechanistic pathways for membrane fusion. SiO₂ NPs show significant potential for inclusion in the synthetic biology toolkit for membrane remodelling and fusion in artificial cells.

Biomimetic Curvature and Tension-Driven Membrane Fusion Induced by Silica Nanoparticles

Membrane fusion is a key process to develop new technologies in synthetic biology, where artificial cells function as biomimetic chemical microreactors. Fusion events in living cells are intricate phenomena that require the coordinate action of multicomponent protein complexes. However, simpler synthetic tools to control membrane fusion in artificial cells are highly desirable. Native membrane fusion machinery mediates fusion driving a delicate balance of membrane curvature and tension between two closely apposed membranes. Here we show that silica nanoparticles (SiO₂ NPs) at a size close to the cross-over between tension-driven and curvature-driven interaction regimes initiate efficient fusion of biomimetic model membranes. Fusion efficiency and mechanisms are studied by Förster Resonance Energy Transfer (FRET) and confocal fluorescence microscopy. SiO₂ NPs induce a slight increase in lipid packing likely to increase the lateral tension of the membrane. We observe a connection between membrane tension and fusion efficiency. Finally, real-time confocal fluorescence microscopy reveals three distinct mechanistic pathways for membrane fusion. SiO₂ NPs show significant potential for inclusion in the synthetic biology toolkit for membrane remodelling and fusion in artificial cells.

Confocal Fluorescence Microscopy as a Tool for Assessment of Photoluminescent Pigments Print on Polyester Fabric

The size and distribution of the photoluminescent pigment particles within the selected binder may affect the quality and appearance of the final print significantly. Yet, the techniques for precise evaluation of size distri¬bution of the pigment particles within a 3D fabric space are rather limited, based on their intrinsic fluorescent properties. The presented work demonstrates a simple screen-printing process for the sustainable application of three different types of commercial fluorescent pigments on polyester (PES) fabric, using polydimethylsiloxane (PDMS) as a binder. A comprehensive toolbox was used to compare and study different commercial photo¬luminescent pigments and their corresponding prints, by means of size distribution and concentration effect of emission intensity, including Confocal Fluorescence Microscopy (CFM) and Scanning Electron Microscopy (SEM) in combination with complementary spectroscopic techniques, i.e. Energy Dispersive X-ray Spectroscopy (EDX) and Ultraviolet-visible (UV-vis) spectroscopy. The focus is on CFM utilised as a non-destructive tool, used for the evaluation of photoluminescent pigments´ spatial distribution within printing pastes, as well as on/within the PES fabrics.