Synthesis of IMM layer. We synthesized a layer of each IMM onto glass substrates, including a glass slip (Matsunami Glass Ind., Ltd., Japan, RI=1.5255) and a glass bottom dish (AGC Techno Glass CO., LTD., Japan). An MY-133-EA (MY Polymers Ltd., Israel) layer was prepared by photocuring, wherein we spin-coated the MY-133-EA solution on the glass substrates at 4000 rpm for 30 s, followed by photo-initiated curing with a 365-nm lamp under an atmosphere of nitrogen to generate a layer of ~20-mm thick. Poly(N-hydroxymethyl acrylamide) hydrogel was prepared by chemical gelation16. A pre-gel aqueous solution consisting of 8 wt% N-hydroxymethyl acrylamide (Tokyo Chemical Industry Co., Ltd., Japan), N, N’-Methylenebisacrylamide (Tokyo Chemical Industry Co., Ltd., Japan), N, N, N’, N’- tetramethylethylenediamine (Tokyo Chemical Industry Co., Ltd., Japan), ammonium peroxodisulfate (Tokyo Chemical Industry Co., Ltd., Japan), and distilled water, was poured into a silicon frame (SYLGARDTM 184 silicone elastomer; Dow Corning Tray Co., Ltd., Japan) adhered to a glass bottom dish. The frame was sealed with a plate and incubated at room temperature (25°C) for gelation. Agar gel was prepared by physical gelation16. The agar (0.8 wv%, Nacalai Tesque, Inc., Japan) was dissolved in PBS buffer (Fujifilm Wako Pure Chemical Co., Japan) while heating. A droplet of agar solution (150 mL) was placed on a coverslip and covered with another coverslip, followed by cooling at room temperature for gelation to make a layer of even ~0.5-mm thick. One side of coverslip was then slide off gently.
Strains and culture conditions. The strains used in this study were Schizosaccharomyces pombe JY1 and Pseudomonas aeruginosa PAO117,18. S. pombe JY1 was cultured in yeast extract-peptone-dextrose (YPD) medium (BD Bioscience, USA) while shaking (190 rpm) overnight at 30°C. P. aeruginosa PAO1 was cultured in LB medium (Nacalai tesque, Inc., Japan) while shaking (190 rpm) overnight at 37°C. For biofilm formation, the culture of P. aeruginosa PAO1 was inoculated into fresh LB medium supplemented with 100 mM KNO3 (Fujifilm Wako Pure Chemical Co., Japan) to adjust the optical density of the medium at 600 nm of 0.01 and was placed in a 25-μL frame-sealTM incubation chamber (Bio-Rad Laboratories, Inc., USA) to adhere to the MY-133-EA layer. The chamber was sealed with silicon resin and incubated at 37 °C under aerobic conditions.
Experimental set up. Figure 1 illustrates the experimental setup. The sample was illuminated with a 561-nm continuous wave laser. The reflected light passed through a half mirror and 1.2 Airy-unit (AU) pinhole and was detected with a photomultiplier tube in the inverted confocal system (NIKON A1; NIKON Solutions Co., Ltd., Japan). The average signal intensities in a field of view, except for the region of paint mark, were calculated by processing the image using a custom MATLAB (MathWorks, Inc., USA) routine. We reconstructed 3D projections from Z-stack images using NIS elements software (NIKON Solutions Co., Ltd., Japan).