Model fluoview 1000

A confocal laser scanning microscope (CLSM) was used to analyse the thickness of biofilms developed for 24 h, as previously described, but using 24-well polystyrene microtiter plates (Orange Scientific, Braine-l Alleud, Belgium) (1 ml per well). The biofilms were stained with 1% (v v À1 ) Calcofluor white (Sigma-Aldrich, St Louis, Missouri) for 10 min at room temperature in the dark and then observed with a CLSM (Olympus BX61, Model FluoView 1000, Portugal). Excitation line 405 and emission filters BA 430-470 (blue channel) were used and images were acquired with the program FV10-ASW 4.2 (Olympus). The biofilm thickness was analysed in three areas of each image and the median thickness value was calculated for each replicate.

In order to study the oligomers' effect in the bio lm thickness the confocal laser scanning microscopy (CLSM) was used. For that, the 24 h bio lms were stained with 1% (v/v) of Calco uor white (Sigma-Aldrich, St Louis, MO, USA) for 10 min at room temperature in the dark and then observed with a CLSM (Olympus BX61, Model FluoView 1000, Portugal). The excitation line 405 and the emission lters BA 430-470 (blue channel) were used, and images were acquired with the program FV10-ASW 4.2 (Olympus).

For the study of chitosan adhesion during the gastrointestinal simulation tests, confocal images were taken in the beginning and at the end of the gastric and intestinal digestion steps of the harmonized in vitro digestion protocol. All samples were analyzed by confocal microscopy (Olympus BX61, Model FluoView 1000, Munich, Germany).

M. avium strain 104 expressing recombinant GFP [37] was used to visualize intracellular mycobacteria, similarly to a previously described method [38] . BMMΦ were seeded at 5 × 10 5 cells/well in 24-well plates with 12 mm diameter coverslips. Mycobacteria were added at a multiplicity of infection of 10 and, after 4 h, each well was extensively washed 4 times with pre-warmed DMEM. The macrophages were then incubated for different time points with LLKKK18 and HA nanogels fluorescently labeled with TAMRA (Schafer-N, Denmark) or Alexa Fluor 488, respectively. Free LLKKK18 was added at a concentration below the toxicity threshold (10 μM), whereas encapsulated LLKKK18 was added at 30 μM per well in 0.5 mg/ml HA nanogels. At the defined endpoints, cells were fixed with 2% paraformaldehyde (in PBS) for 20 min and the coverslip placed on top of a microscopy slide for observation under a Confocal Scanning Laser Microscope (CLSM, Olympus BX61, Model FluoView 1000), comprising a Laser DM 405/488/515/559/635 and the following emission filters: BA 505-540 (green channel), BA 575-620 (orange channel) and BA 655-755 (red channel). Images were analyzed on the software FV10-ASW 4.2.

FISH results were analyzed using an epifluorescence microscope (Olympus BX51) coupled with a DP71 digital camera and three sets of filters (DAPI -365-370/421; FITC -470-490/516; and TRITC -530-550/591) (OlympusPortugal SA, Porto, Portugal). All images were acquired using the Olympus Cell-B software. Since the BX51 microscope used does not include adequate filters to allow the discrimination of the Cy3/Cy5 combination (PGAciLO/ACA), samples with this particular combination were analyzed using a CLSM (Olympus BX61, Model FluoView 1000). Phage-infected P. aeruginosa cells were also visualized by CLSM to observe infection details within the cells. The laser DM 405/488/559/635 and the emission filters BA 505-540 (green channel), BA 575-620 (orange channel) and BA 655-755 (red channel) were used, and images were acquired with the program FV10-ASW 4.2 (Olympus). The settings for samples hybridized with the PGAciLO probe (attached to Cy3) corresponded to the properties of Cy3, that has a maximum emission lying in the orange region, but images were colored as green to facilitate the discrimination of phage infection from Acinetobacter cells that were red.