A1 confocal laser scanning microscope clsm

The titanium substrates were removed from wells with sterile forceps and gently rinsed three times with Phosphate Buffered Saline (PBS, pH = 7.4) to remove non-adherent or loosely adhered bacteria. The bacteria were then stained with a solution of the FilmTracker^TM Live/Dead^® Biofilm Viability kit (Invitrogen, Life Technologies, Carlsbad, CA, USA). In this study, 1.5 µl of SYTO^®9 and 1 µl of propidium iodide stain were added to 1 ml of PBS. After that, 150 µl of the staining solution was gently added to each substrate and plates were incubated for 15 minutes in the dark. Suspensions were then aspirated and the titanium substrates were transferred into a new well plate with ample PBS to fully immerse the sample. Samples were then visualized with a Nikon A1 confocal laser scanning microscope (CLSM) with a 40x water dipping lens. For the bacterial adhesion assay for 2 hours’ incubation, the surface coverage of bacteria under each field of view was determined by calculating the surface area of live and dead bacteria cells with ImageJ. For the early-stage biofilms formed after 2 days, the biomass under each field of view was determined by COMSTAT2 plugin (Lyngby, Denmark) in ImageJ. For each substrate type, three independent experiments were performed, and on each sampled surface, five equidistant evenly spaced fields (105.96 × 105.96 µm²) were selected.

GFP-labeled UA159 and mCherry-labeled FW213 were used to visualize biofilms. All biofilms for confocal were grown in Ibidi µ-Slide 8 well slides (Cat #: 80826). Wells were gently washed with phosphate-buffered saline (PBS). Wells with C. albicans were stained with calcofluor white in PBS for 15 min before imaging. Biofilms were visualized on a Nikon A1 + confocal laser scanning microscope (CLSM) (Nikon Instruments Inc.) using a 60 × oil immersion lens. Three dimensional biofilm images were acquired using the Nis Elements 5.0 Imaging Software. All images are representative of biofilms from 3 independent experiments.

For cellular uptake experiments, UMUC3 cells were seeded on glass dishes at a density of 5 × 10⁴ cells for 24 h. The medium was then aspirated and replaced with media containing DOX-loaded PCL1-b-PManEA@ConA micelles at a concentration of 2 mg/mL in the absence and presence of 1-methyl α-d-mannopyranoside (MeMan, 20 mM). The cells were cultured for another 2 h, and washed thrice with sterilized doubly distilled water to remove the physically adsorbed DOX-loaded PCL1-b-PManEA@ConA micelles completely. After that, the cells were fixed in formaldehyde aqueous solution (4%, v/v) for 30 min. Following fixation, the cells were observed on a Nikon (A1) Confocal Laser Scanning Microscope (CLSM, Nikon, Tokyo, Japan). For fluorescence imaging, the cells were excited at 488 nm, and the fluorescence in the wavelength region of 570–620 nm was recorded.

Bacterial strains were grown in TSBYE in a sterile 8-well treated μ-Slide (Ibidi) under 5% CO₂ at 37°C for 16 h. The biofilm samples were gently washed with PBS three times to remove unattached cells followed by incubation in 5% bovine serum albumin (BSA) in PBS for 1h. S. parasanguinis was incubated with a polyclonal antibody that recognizes the surface amylase binding protein (AbpA) [17 (link)] or a monoclonal antibody that recognizes the surface fimbriae protein Fap1 [20 (link)]. P. aeruginosa was incubated with a monoclonal antibody that recognizes an outer membrane protein (Omp) (Abcam). Alginate was probed using a polyclonal antibody that was provided as a gift from Dr. Gerald B. Pier at Harvard University [55 (link)]. The biofilms were washed 3 times with PBS to remove the unattached primary antibodies and then incubated with fluorescent-conjugated secondary antibodies (Molecular Probes) for 30 min. Alexa Fluor 594 (red)-conjugated goat anti-mouse IgG and Alexa Fluor 488 (green)-conjugated goat anti-rabbit IgG were used to stain bacterial cells and alginate. The stained samples were then washed 3 times prior to detection using a fluorescence (Nikon X-Cite series 120 PC) or a Nikon A1+ confocal laser scanning microscope (CLSM) (Nikon Instruments Inc.). NIS Elements microscopy imaging software was used to calculate biofilm biomass and co-localization.