for in vivo, single-cell imaging bacterial cells are commonly immobilised via physical confinement or surface attachment. Different surface attachment methods have been used both for atomic force and optical microscopy (including super resolution), and some have been reported to affect bacterial physiology. However, a systematic comparison of the effects these attachment methods have on the bacterial physiology is lacking. Here we present such a comparison for bacterium Escherichia coli, and assess the growth rate, size and intracellular pH of cells growing attached to different, commonly used, surfaces. We demonstrate that E. coli grow at the same rate, length and internal pH on all the tested surfaces when in the same growth medium. The result suggests that tested attachment methods can be used interchangeably when studying E. coli physiology. Microscopy has been a powerful tool for studying biological processes on the cellular level, ever since the first discovery of microorganisms by Antonie van Leeuwenhoek back in 17th century 1. Recently employed single-cell imaging allowed scientists to study population diversity 2 , physiology 3 , sub-cellular features 4 , and protein dynamics 5 in real-time. Single cell imaging of bacteria is particularly dependent on immobilisation, as majority of bacteria are small in size and capable of swimming. Immobilisation methods vary depending on the application, but typically fall into one of the two categories: use of physical confinement or attachment to the surface via specific molecules. The former group includes microfluidic platforms capable of mechanical trapping 6,7 , where some popular examples include the "mother machine" 8 , CellASIC 9 or MACS 10 devices, and porous membranes such as agarose gel pads 2,11-13. Physical confinement methods, while higher in throughput, can have drawbacks. For example, agarose gel pads do not allow fast medium exchange, and when mechanically confining bacteria the choice of enclosure dimensions should be done carefully in order to avoid influencing the growth and morphology with mechanical forces 14. Furthermore, mechanically confined bacteria cannot be used for studies of bacterial motility or energetics via detection of bacteria flagellar motor rotation 15-17. Chemical attachment methods rely on the interaction of various adhesive molecules, deposited on the cover glass surface, with the cell itself. Adhesion can be a result of electrostatic (polyethylenimine (PEI) 18,19 , poly-L-lysine (PLL) 15,17,19) or covalent interactions (3-aminopropyltriethoxysilane (APTES) 19), or a combination, such as with polyphenolic proteins (Cell-Tak) 19. Time scales on which researchers perform single-cell experiments vary. For example, scanning methods, like atomic force microscopy (AFM) or confocal laser scanning microscopy (CLSM) 19,20 , require enough time to probe each point of the sample, and stochastic approaches of super resolution microscopy (e.g. PALM and STORM) use low activation rate of fluorophores to achieve a single fluorophore localisati...
