Pe 4000

All data were acquired with a commercial Nikon Eclipse Ti2 inverted microscope (Nikon instruments, Tokyo, Japan), equipped with A1R confocal scanhead, N-SIM and N-STORM modules (Nikon instruments, Tokyo, Japan). The fully motorized automated microscope was controlled by the NIS Elements software (version 5.42.01, Laboratory Imaging s.r.o, Praha, Czech Republic). The system performs multicolor widefield, confocal, and single-molecule localization imaging thanks to a pE-4000 (CoolLED, Andover, UK) light source with 16 selectable LED wavelengths (Widefield microscopy) and a LU-NV laser unit (Nikon instruments, Tokyo, Japan) equipped with 5 laser lines (405 nm (23.1 mW), 440 nm (25.5 mW) 488 nm (79.1 mW), 561 nm (79 mW), and 647 nm (137 mW)) (Confocal, SIM, TIRF and dSTORM microscopy). The microscope is equipped with fluorescence filter sets for the optimal detection of the employed fluorochromes thanks to a double-layer turret allowing the combination of 5 cubes per layer. The upper turret is devoted to widefield and STORM imaging, with the lower one acting as an additional filter wheel. SIM images were instead collected through the lower layer by spatially calibrated filter cubes. Emitted light was collected by a CMOS camera (Dual ORCA Flash 4.0 Digital CMOS camera C13440, Hamamatsu, Tokio, Japan) set on a 16-bit scale detection modality (Widefield/SIM/TIRF/dSTORM).

For the examination of the cross-sections of the paint samples, a Nikon ECLIPSE LV100ND microscope attached to a CoolLED pE-4000 UV light source and equipped with a Nikon DS-Fi3 camera (Nikon, Leuven, Belgium) was used (Figure 2 and Supplementary Figure S2). An Olympus Digital Microscope DSX510 (3D microscope) allowed the verification of the efficiency of the varnish removal by the selected hydrogel composite (Olympus, Hamburg, Germany) (Figure 10a,b).

Axenic cultures (5 ml) of each strain were grown to OD_600nm = 0.6–0.7. Cells were harvested and resuspended in 7H9 (100 μl) and labelled with alk-DADA (2.5 μl) at 37°C for 60 min. Cells were centrifuged, washed in 1X PBS (Lonza) and fixed overnight in 250 μl of 2.5% glutaraldehyde (Sigma) at room temperature. Fixed cells were centrifuged at 13 000 rpm for 2 min and resuspended in PBS-Tween-BSA (PBSTB) buffer (250 μl) and centrifuged at 13 000 rpm for 2 min. Pellets were resuspended in PBSTB (230 μl), to which CuACC reaction components were added in the following order: 10 mM CuSO₄ (5 μl), 6.4 mM TBTA (5 μl), 60 mM Sodium Ascorbate (5 μl), 1 mM azido-probe (5 μl). The CuACC reaction was incubated at room temperature for 30 min and harvested by centrifuging the cells at 13 000 rpm for 2 min. The cells were washed three times in 1X PBS and added to an agarose pad as described previously. Images were captured using the Nikon Eclipse T12 and PE4000 LED light source, in the TL DIC channel (200 msec exposure) and pre-configured azo488 channel (excitation 490 nm, 50% power; emission 512 nm; 3 sec exposure).