Microscopy and image processing
This protocol is extracted from research article:
Swimming bacteria power microspin cycles
Sci Adv, Dec 19, 2018; DOI: 10.1126/sciadv.aau0125

All movies were acquired at 32 frames per second (fps) on a Zeiss Axio Observer inverted microscope equipped with a Hamamatsu ORCA-Flash4.0 CMOS camera. Movies were captured with a 63× oil immersion objective (1.4 NA), focused equidistant from coverslips. All movies were either DIC or wide-field fluorescence using Zeiss Filter Set 43 (HE DsRed). Slides were discarded after 3 min on the microscope (5.5 min after centrifugation ends), before oxygen consumption led to oxygen gradient formation and altered cell motility.

In general, video microscopy images were acquired for 30 s. These images were used to determine the probability that the bacteria would undergo periodic reversals and to determine the characteristics of the velocity fields, such as maximum velocity, average velocity, and the velocity as a function of radial position. For conditions where periodic reversals were not observed (i.e., cephalexin-treated cells that were longer than 6 μm), some 10-s videos were used to quantify the flow characteristics.

Droplet velocity fields were calculated by defining the region of interest about a droplet using a custom MATLAB algorithm to threshold a given droplet region in each frame of a time-lapse movie. This sequence of binary masks was then used along with a gradient-based optical flow method (22) to determine the velocity fields. The velocity fields were validated by simulating the motion of virtual tracer particles in the computed flow, as described in (22).

Flow patterns in the droplets were classified using the dynamics of the order parameter ψ as a metric (fig. S1). Plots of ψ versus time that showed periodicity (which was almost always a square wave shape) were classified as periodic reversals. When the magnitude of ψ was >0.5 for the length of the movie and did not change sign, the droplet was classified as a stable vortex. These droplets were further classified as either a single stable vortex or a counterrotating vortex based on whether or not the optical flow–extracted velocity fields had counter flow at the edge of the droplet. All droplets that were not classified as any of these patterns were categorized as showing random motion.

The half-period (T1/2) was measured by hand using the order parameter versus time plots. These plots were opened in ImageJ (29), and the time it took to go from one handedness of flow (e.g., counterclockwise) to the other (e.g., clockwise) was measured for each transition. The half-period was defined as the average value of these measurements.

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