Bacteria sense chemicals, surfaces, and other cells and move toward some and away from others. Studying how single bacterial cells in a population move requires sophisticated tracking and imaging techniques. We have established quantitative methodology for label-free imaging and tracking of individual bacterial cells simultaneously within the bulk liquid and at solid-liquid interfaces by utilizing the imaging modes of digital holographic microscopy (DHM) in three dimensions (3D), differential interference contrast (DIC), and total internal reflectance microscopy (TIRM) in two dimensions (2D) combined with analysis protocols employing bespoke software. To exemplify and validate this methodology, we investigated the swimming behavior of a Pseudomonas aeruginosa wild-type strain and isogenic flagellar stator mutants (motAB and motCD) within the bulk liquid and at the surface at the single-cell and population levels. Multiple motile behaviors were observed that could be differentiated by speed and directionality. Both stator mutants swam slower and were unable to adjust to the near-surface environment as effectively as the wild type, highlighting differential roles for the stators in adapting to near-surface environments. A significant reduction in run speed was observed for the P. aeruginosa mot mutants, which decreased further on entering the near-surface environment. These results are consistent with the mot stators playing key roles in responding to the near-surface environment. IMPORTANCE We have established a methodology to enable the movement of individual bacterial cells to be followed within a 3D space without requiring any labeling. Such an approach is important to observe and understand how bacteria interact with surfaces and form biofilm. We investigated the swimming behavior of Pseudomonas aeruginosa, which has two flagellar stators that drive its swimming motion. Mutants that had only either one of the two stators swam slower and were unable to adjust to the near-surface environment as effectively as the wild type. These results are consistent with the mot stators playing key roles in responding to the near-surface environment and could be used by bacteria to sense via their flagella when they are near a surface.

We present a transmission-mode digital holographic microscope that can switch easily between three different imaging modes: inline, dark field off-axis, and bright field off-axis. Our instrument can be used: to track through time in three dimensions microscopic dielectric objects, such as motile micro-organisms; localize brightly scattering nanoparticles, which cannot be seen under conventional bright field illumination; and recover topographic information and measure the refractive index and dry mass of samples via quantitative phase recovery. Holograms are captured on a digital camera capable of high-speed video recording of up to 2000 frames per second. The inline mode of operation can be easily configurable to a large range of magnifications. We demonstrate the efficacy of the inline mode in tracking motile bacteria in three dimensions in a 160 µm × 160 µm × 100 µm volume at 45× magnification. Through the use of a novel physical mask in a conjugate Fourier plane in the imaging path, we use our microscope for high magnification, dark field off-axis holography, demonstrated by localizing 100 nm gold nanoparticles at 225× magnification up to at least 16 µm from the imaging plane. Finally, the bright field off-axis mode facilitates quantitative phase microscopy, which we employ to measure the refractive index of a standard resolution test target and to measure the dry mass of human erythrocytes.