Raw holograms frames of Bacillus Subtilis at different temperatures

We describe a system for high-temperature investigations of bacterial motility using a digital holographic microscope completely submerged in heated water. Temperatures above 90 ºC could be achieved, with a constant 5 ºC offset between the sample temperature and the surrounding water bath. Using this system, we observed active motility in Bacillus subtilis up to 66 ºC. As temperatures rose, most cells became immobilized on the surface, but a fraction of cells remained highly motile at distances of > 100 µm above the surface. Suspended non-motile cells showed Brownian motion that scaled consistently with temperature and viscosity. A novel open-source automated tracking package was used to obtain 2D tracks of motile cells and quantify motility parameters, showing that swimming speed increased with temperature until ~40 ºC, then plateaued. These findings are consistent with the observed heterogeneity of B. subtilis populations, and represent the highest reported temperature for swimming in this species. This technique is a simple, low-cost method for quantifying motility at high temperatures and could be useful for investigation of many different cell types, including thermophilic archaea. Methods Data were acquired using a custom, open-source software package, DHMx (https://github.com/dhm-org/dhm_suite). The holograms for each recording were either median subtracted (as we described previously (Bedrossian et al., 2020)) or frame-to-frame subtracted then reconstructed in amplitude using Fiji (ImageJ)(RRID:SCR_002285) (Schindelin et al., 2012). Reconstructions were performed using the angular spectrum method (Mann et al., 2005) implemented in a custom plug-in described in detail elsewhere (Cohoe et al., 2019) and available from our update site (https://github.com/sudgy/). Bacteria were tracked using a custom software package, Holographic Examination for Life-like Motility (HELM) (https://github.com/JPLMLIA/OWLS-Autonomy), which was developed to autonomously detect, track, and characterize motile cells