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The light source was a laser beam combiner (Omicron, LightHUB), including 488 and 642 nm laser lines, and a 470 nm collimated LED (Thorlabs, M470L3-C5). For the single colour imaging of McjD-EGFP and PH1735-EGFP E. coli cells, the filter sets consisted of a 484 nm beam splitter (Semrock, FF484-FDi0) as dichroic filter, and a 512/25 nm bandpass filter (Semrock FF01-512/25-25) as emission filter. For the two-colour imaging of McjD-EGFP and membrane- DiSC₃(5) E. coli cells, the filter sets consisted of quad-edge dichroic beam splitter (Semrock, Di01-R405/488/543/635-25×36) as dichroic filter and quad-band bandpass filter (Semrock, FF01-446/523/600/677-25) as emission filter, also used in the DNA origami nanoruler measurement.

Cells were imaged using a Zeiss 200M inverted microscope, equipped with an alpha plan-apochromat 100X, 1.46 NA oil-immersion objective (Zeiss). A 405 nm laser (100 mW, CUBE, Coherent, Santa Clara, CA) was used to activate the fluorescent protein mEos3 (30–35 W/cm²). The green fluorescence of mEos3 was obtained with an ArKr mixed-gas ion laser (2.5 W all lines, Stabilite 2018-RM, Spectra-Physics, Mountain View, CA) at 488 nm and the orange fluorescence of activated mEos3 was obtained with a solid-state laser (150 mW, Excelsior One, Newport, Santa Clara, CA) at 561 nm with an excitation density of 2 kW/cm². Both activation and excitation beams passed through a λ/4 wave plate (ThorLabs, Newton, NJ), generating circularly polarized light. A λ/2 wave plate was used to rotate the angle of linear polarization of the excitation lasers. The sample was illuminated via narrow-field epifluorescence (Yang et al., 2004 (link)), that is, a 300 µm pinhole was placed within a specimen-conjugate plane in the activation/excitation beam path, thereby restricting the specimen illumination area to ~7 μm diameter. After passing through a quad-bandpass filter (FF01-446/523/600/677-25, Semrock, Rochester, NY), the fluorescence emission was collected with an EMCCD camera (Evolve 128, Photometrics, Tucson, AZ). Image acquisition was controlled by MetaMorph (Molecular Devices, Sunnyvale, CA).