Confocal images of the newly developed functional food products based on carrots and LAB were acquired with a Zeiss confocal laser scanning system (LSM 710), which was equipped with a diode laser (405 nm), Ar laser (458, 488, 514 nm), DPSS laser (diode-pumped solid-state—561 nm), and HeNe laser (633 nm). To observe the vegetal microstructures and the L. plantarum BL4 cells in detail, the Live/Dead backlight bacterial viability stain kit (Molecular Probes, Eugene, OR, USA) was used according to the manufacturer’s instructions so that only one drop was applied directly to the surface of each sample. The stain kit consisted of a mixture of two nucleic acid-binding stains: SYTO9, which stained all the viable bacteria (shown in green), while the propidium iodide stained the non-viable bacteria (shown in red), after 15 min of dark incubation [25 (link)]. The excitation and emission wavelengths were 480 and 500 nm for SYTO9 and 490 and 635 nm for propidium iodide, respectively. The samples were observed with a Zeiss Axio Observer Z1 inverted microscope equipped with a 40× apochromatic objective (numerical aperture 1.4). The 3D images were rendered and analyzed with the ZEN 2012 SP1 Black edition software. For each sample, twenty fields were evaluated, the viability counts were determined in two independent experiments, and each assay was performed in triplicate.
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