Optical microscopy images of TW and SC double emulsions were obtained to confirm the correct formation and droplet structure of the different systems. A vertical microscope Axioskop (Carl Zeiss, Oberkochen, Germany) with a Zeiss Plan-Neofluar lens (Carl Zeiss, Göttingen, Germany) coupled to a Leica DMC 6200 pixel-shift camera (Leica Microsystems, Wetzlar, Germany) was used. Samples were prepared by depositing one drop of each emulsion on microscope slides, without using stains. Samples were observed at 40× and 100× using an open condenser and illumination (level four).
Confocal laser scanning microscopy analysis was also performed to evaluate the microstructure of the emulsion particles and their progression during the in vitro gastro-intestinal digestion assay. A droplet of each sample was placed on a microscope slide and stained with 10 µL of Nile red solution (1 mg of Nile red/mL of acetone). Fluorescence excitation and emission were measured at 488 and 523–650 nm, respectively. Then, samples were studied with a confocal multispectral TCS SP5 system (Leica Microsystems, Germany) at 20× and at 40× using a Zeiss Plan-Neofluar lens.
Confocal laser scanning microscopy analysis was also performed to evaluate the microstructure of the emulsion particles and their progression during the in vitro gastro-intestinal digestion assay. A droplet of each sample was placed on a microscope slide and stained with 10 µL of Nile red solution (1 mg of Nile red/mL of acetone). Fluorescence excitation and emission were measured at 488 and 523–650 nm, respectively. Then, samples were studied with a confocal multispectral TCS SP5 system (Leica Microsystems, Germany) at 20× and at 40× using a Zeiss Plan-Neofluar lens.