Eight chambered glass bottom microscopy slide

Fluorescence confocal laser scanning microscopy was performed on an inverted fluorescence microscope LSM780 (Zeiss), using oil immersion 100× objective (NA = 1.46, Zeiss) for E. coli cells and oil immersion 63× objective (NA = 1.4, Zeiss) for mammalian cells. Solutions of E. coli cells were diluted to the A₆₀₀ of ∼0.1 and placed into the eight chambered glass bottom microscopy slide (ibidi). Live HEK293T cells were maintained at 37 °C and 5% CO₂ in INUBG2H-ELY incubator (TOKAI HIT) during the measurements.
Laser excitation of the fluorescence was done using Ar-Ion laser (Lasos, Germany) at 458 nm wavelength. Fluorescence images were obtained by a 34-channel QUASAR detector (Zeiss) set to the 460 to 690 nm wavelength range. Fluorescence spectra for E. coli cultures expressing different mutants were used to provide reference spectra for the spectral unmixing of the mixture of cells expressing different mutants. The same approach was used for HEK293T. Spectral unmixing was performed using the fnnls Python library by Joshua Vendrow and Jamie Haddock (https://pypi.org/project/fnnls/) (51 ).

Fluorescence lifetime measurements and FLIM were performed on an inverted fluorescence microscope LSM780 (Zeiss), using oil immersion 63× objective (NA = 1.4, Zeiss). Solutions of E. coli cells were diluted to the A₆₀₀ of ∼0.1 and placed into the eight chambered glass bottom microscopy slide (ibidi). Two photon excitation was achieved using Insight DeepSee laser (λ_exc = 920 nm; 80 MHz, 150 fs pulse; Newport Spectra Physics). Fluorescence intensity decays were generated using time-correlated single photon counting with SPC-150 modules (Becker & Hickl, Germany). Average lifetimes were calculated using monoexponential fitting with a binning parameter of three in SPCImage software (Becker & Hickl) and averaged to obtain τ_av. CagFbFP solutions with concentration of ∼0.1 mg/ml were measured using the same experimental setup.