‘Simultaneous tracking of cell motility in liquid and at the solid-liquid interface’

Abstract

25Bacteria sense chemicals, surfaces and other cells and move toward some and away from others. Studying 26 how single bacterial cells in a population move requires sophisticated tracking and imaging techniques. We 27 have established quantitative methodology for label-free imaging and tracking of individual bacterial cells 28 simultaneously within the bulk liquid and at solid-liquid interfaces by utilizing the imaging modes of digital 29 holographic microscopy (DHM) in 3D, differential interference contrast (DIC) and total internal reflectance 30 microscopy (TIRM) in 2D combined with analysis protocols employing bespoke software. To exemplify 31 and validate this methodology, we investigated the swimming behavior of Pseudomonas aeruginosa wild 32 type and isogenic flagellar stator mutants (motAB and motCD) respectively within the bulk liquid and at the 33 surface at the single cell and population levels. Multiple motile behaviours were observed that could be 34Page 2 of 39 differentiated by speed and directionality. Both stator mutants swam slower and were unable to adjust to the 35 near surface environment as effectively as the wildtype highlighting differential roles for the stators in 36 adapting to near surface environments. A significant reduction in run speed was observed for the P. 37 aeruginosa mot mutants, which decreased further on entering the near-surface environment. These results 38 are consistent with the mot stators playing key roles in responding to the near-surface environment. 39 40 Importance 41We have established a methodology to enable the movement of individual bacterial cells to be followed 42 within a 3D space without requiring any labelling. Such an approach is important to observe and understand 43 how bacteria interact with surfaces and form biofilm. We investigated the swimming behavior of 44Pseudomonas aeruginosa, which has two flagellar stators that drive its swimming motion. Mutants that 45 only had either one of the two stators swam slower and were unable to adjust to the near surface 46 environment as effectively as the wildtype. These results are consistent with the mot stators playing key 47 roles in responding to the near-surface environment, and could be used by bacteria to sense when it is near a 48 surface. 49 50 planktonic and surface attached cells will facilitate the acquisition of spatio-temporal dynamics that will aid 59 our understanding of the signal transduction mechanisms that drive bacterial social behaviors. 60 61Pseudomonas aeruginosa is a Gram-negative rod-shaped cell with a single polar flagellum that employs a 62 number of different mechanisms for moving through liquids and across surfaces. These include flagella-63 mediated swimming, spinning, near-surface swimming and swarming, type IV pili-mediated twitching 64 (crawling and walking), gliding (active movement without the use of flagella or pili), and sliding i.e. 65 passive movements over surfaces through the use of surfactants (14, 22). Flagella are also associated with 66 bacterial surface mechanosensin...

Digital holography-based 3D particle localisation for single molecule tweezer techniques

“…HoloMiP does not require generation of look-up tables or any a priori knowledge of the objects being imaged and it can localise them even if their diffraction patterns overlap. In addition, its application is not reliant on the symmetry of the imaged objects, and could be readily applied to non-spherical microscopic objects that scatter light, such as microflakes (18) or rod-shaped bacteria (19). We demonstrate the utility of HoloMiP in fast, multiplexed single molecule force spectroscopy assays using MT to apply force at receptor-ligand bonds attached to a surface by DNA tethers.…”