Nis elements 5.0 imaging software

Overnight cultures of S. parasanguinis (mCherry), S. mutans (GFP), or C. albicans were subcultured and grown to an optical density of 0.5 at an absorbance of 600nm. All biofilms were grown in TSBYE + 1% sucrose with or without 2mM nitrite in µ-Slide 8 well slides (Ibidi, Gräfelfing, Germany, Cat #: 80826) and all inocula were seeded at 1x10⁴ CFU/mL. Biofilms were allowed to grow for 16 hours at 37°C with 5% CO₂. All biofilm wells were washed with PBS and wells with C. albicans were stained with calcofluor white for 15 minutes before imaging. A Nikon A1 + confocal laser scanning microscope (CLSM) (Nikon Instruments Inc., Melville, NY, USA) was used to image biofilms at 60x magnification and 3D images were acquired using the Nis Elements 5.0 Imaging Software (Nikon Instruments Inc., Melville, NY, USA). To enumerate colony forming units, all biofilms were gently washed with sterile PBS twice before adding 200 µL of sterile PBS for plating. The biofilms were scraped up with a 200 µL tip, vortexed for 10 s, and serially diluted. All dilutions were plated on Todd-Hewitt Broth or blood agar plates and incubated at 37 °C with 5% CO₂ for a minimum of 16 h before counting.

MDA MB231 cells were grown overnight on poly-D lysine coated 20 mm, no. 1 cover slips (Neuvitro, Vancouver, WA) in a 24-well plate (Corning, Manassas, VA) at a density of 2 x10⁵ cells/coverslip. Cells were treated with a colloidal dispersion of 10 to 20 nm cerium dioxide (30% colloidal suspension in water produced by Alfa Aesar, Ward Hill, MA) particles at a concentration of 50 μg/mL in complete L-15 media for 72 h. Cells were washed three times with 1X PBST, fixed with 4% paraformaldehyde for 10 min at room temperature, and then permeabilized with 0.2% triton X-100 for 10 min at room temperature. Cells were washed and then stained with Hoechst 33342 (Abcam, Cambridge, MA), mounted and sealed with nail polish. Slides were imaged with Reflectance Structured Illumination Microscopy (R-SIM) (Nikon Corp., Japan) with Orca Flash 4 Camera (Hamamatsu, Japan). To image with R-SIM, a half-mirror was placed in the light path instead of the dichroic and the configuration was set up in the fourth channel using 488 nm laser. The dichroic was set to BS20/80 with all light paths set to “through”. Images were processed and analyzed using Nikon Nis Elements 5.0 Imaging Software. Nanoparticles were displayed in red and cell nucleus in blue for reflectance images. Cells were shown in black and white for differential interference contrast images.

The IVIS Lumina imaging system (Perkin Elmer, Waltham, MA) and Nikon A1R Confocal microscope were used to non-destructively image global and local fluorescence within 3D tumor models incubated with Hypoxisense 680 (Perkin Elmer) to monitor hypoxia. One day prior to imaging, 100 pM Hypoxisense 680 was injected into each 3D tumor model and incubated statically for 2 hours (37°C, 5% CO₂). After dye incubation, media circulation was resumed. The following day 3D tumor models were sterilely disconnected from the flow loop and closed to surroundings in a laminar flow hood. 3D tumor models were imaged with the IVIS Lumina (Ex: 680/Em: 710, 1 second exposure, bin 8, f/stop 2.), and Nikon A1R (Objective: Plan Apo λ 10x, na 0.5, wd 4000; laser lines (nm): 405, 488, 567, 637) using Nis Elements 5.0 Imaging Software on days 5 and on day 10 before fixation, with Hoechst incubation prior to day 10 imaging and fixation. Identical square regions of interest (ROI) were drawn around tumor models to evaluate global fluorescent signal with the IVIS Lumina using Living Image Software (Perkin Elmer). Regional fluorescent signal was measured in confocal Z-stacks using the ImageJ open source processing package FIJI (24 (link)).