Data are from Bacillus subtilis cells expressing SecA-mNeonGreen or SecDF-mNeonGreen fusions from the original gene locus. They are related to the submitted manuscript "Strach et al., 2022). "TIF" images and "stk" stacks can be opened using software "ImageJ" (or "Fiji") (https://imagej.nih.gov/ij/). For the individual molecules were tracked using custom-made slim-field setup on an inverted fluorescence microscope (Nikon Eclipse Ti-E, Nikon Instruments Inc.). An EMCCD camera (ImagEM X2 EM-CCD, Hamamatsu Photonics KK) was used to ensure high-resolution detection of the emission signal, resulting in a calculated resolution of the position of the molecule down to 20 nm. The central part of a 514 nm laser diode (max power 100 mW, TOPTICA Beam Smart) was used with up to 20% of the intensity (about 160 W cm-2) in the image plane) to excite samples fused to mVenus/ mNeonGreen (using a laser filter set with BrightLine 500/24, dichroic mirror 520 and BrightLine 542/27), by focusing the beam onto the back focal plane of the objective. A CFI Apochromat objective (TIRF 100 x Oil, NA 1.49) was used in the setup.