Megaview g2

Procyclic-form RNAi-induced cells, and non-induced controls, were harvested, washed with PBS, stained with DAPI and then directly observed using Zeiss LZSM510 META confocal microscope. Parasites submitted to these same experimental conditions were further analysed using transmission electron microscopy. The cells were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, followed by post fixation for 30 min in 1% OsO₄ in 0.1 M cacodylate buffer, dehydration in increasing concentrations of acetone and embedding in Epon (Polybed 812) resin. Ultra-thin sections were harvested on 300 mesh copper grids, stained with 5% uranyl acetate and 1% lead citrate, and observed with a FEI Tecnai Spirit transmission electron microscope. The images were randomly acquired with a CCD camera system (MegaView G2, Olympus, Germany).

Average hydrodynamic diameter (nm), polydispersity index (PdI) and ζ-potential (mV) of the developed nanovesicles were measured by Dynamic and Electrophoretic Light Scattering, DLS/ELS (Zetasizer Nanoseries ZS90) by Malvern instrument (Worcestershire, UK), at 25 °C, with a scattering angle of 90 °C [38 (link),39 (link)]. Glycerosomes and PG-nanovesicles loaded with the essential oils were diluted with ultrapure water before measurements, in order to achieve a suitable scattering intensity. Successively, the two systems loaded with OOEO plus STEO were observed by transmission electron microscope, TEM (CM12 TEM, PHILIPS, Eindhoven, The Netherlands) equipped with an OLYMPUS Megaview G2 camera, with an accelerating voltage of 80 kV. A drop of sample, 5-folds diluted in water, was applied and dried by desiccation on a carbon film copper grid and it was counterstained with 1% w/v of phosphotungstic acid solution for 3 min [40 (link)]. Then, the sample was examined at different amplifications.

The parasites were washed with PBS and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. The cells were then post-fixed for 30 min in 1% OsO₄, dehydrated in acetone and embedded in Epon (Polybed 812). Ultra-thin sections were harvested on 300 mesh copper grids, stained with 5% uranyl acetate and 1% lead citrate, and observed with a JEOL 1210 transmission electron microscope. The images were randomly acquired with a CCD camera system (MegaView G2, Olympus, Germany).

During aa optimization procedures, nanoliposome characterization has been performed by evaluating size, homogeneity, and possible aggregation state, with the aid of dynamic and electrophoretic light scattering (DLS and ELS, Zetasizer Nanoseries ZS90) by Malvern Instruments (Worcestershire, UK), by using a scattering angle of 90 °C at 25 °C [23 (link)]. Autocorrelation functions were analyzed by the cumulant method in order to obtain the average hydrodynamic diameter (AHD, nm), the size distribution expressed as PdI (polydispersity index, dimensionless measurement) and ζ-potential (mV), using the software provided by Malvern. Scattering measurements, in triplicate, were performed on samples, diluted 50/100-fold in ultrapure water. Nanoliposome morphology was investigated for vesicle dispersion, dimension, and deformability by TEM (CM12 TEM, Philips, The Netherlands) equipped with an Olympus Megaview G2 camera, applying an 80 kV accelerating voltage. For this purpose, a drop of the diluted sample, released on a carbon film copper grid, was dried by desiccation, counterstained with 1% (w/v) of phosphotungstic acid solution and examined at different magnifications [24 (link)].

For the analysis of transmission electron microscopy (TEM), samples were properly diluted with water. A drop of diluted sample was placed on 300 mesh carbon coated copper grid (Ted pella). Grid was left for 5 min to settle down the droplets. Excess of liquid was removed using filter paper and grid was left to air dry. Then a drop of 1% phosphotungustic acid in water was added to the grid. Phosphotungustic acid works as negative stain. Again this left for 5 min to settle down and dried as previous. Finally, dried grid was visualized under Jeol TEM, JEM1010 (Japan) at an operating voltage of 80 kV. Images of the vesicles were captured using iTEM software and olympus Megaview G2 top mounted camera.

Morphology of the self-assembled structures was characterized by transmission electron microscopy (TEM). An aqueous droplet of micellar solution (20 uL) was placed on a copper-coated grid (Ted Pella, Inc., Redding, CA, USA). The grid was kept horizontally for 20 s to allow the colloidal aggregates to settle. A drop of 2% solution of phosphotungstic acid (PTA) in PBS (pH = 7.0) was then added to give the negative stain. After 1 min, the excess fluid was removed by using a strip of filter paper. The samples were left to get dry at room temperature and loaded into a JEM-1010 Transmission electron microscope (JEOL, Tokyo, Japan) operating at an acceleration voltage of 80 kV. Images were recorded with a high-speed read-out side-mounted MegaView^G2 (Olympus, Hamburg, Germany) camera and processed with iTEM (Olympus Soft Imaging Solutions GmbH, Münster, Germany) software.