Glass slide cover

The protocol for synthesizing the novel hemoglobin microbubbles has been discussed in detail in the supplementary information. After preparing the microbubble solution, the Coulter Counter method was used to measure particle size distribution and concentrations of the microbubbles. A 2 μL sample of microbubbles was diluted in 10 mL of isotone II within a 25 mL cuvette and characterized using a Multisizer 4e Coulter Counter (MS4), Beckman Coulter (Brea, CA). The samples were measured three times, and the size distributions were averaged. A BX50 Upright Microscope (A.C.H. 40X/0.80 ∞/0.17 objective) was used to visualize the microbubbles for brightfield microscopy. A small square-shaped well was created using high vacuum silicone grease, Dow Corning (Midland, MI). 10 µL of the sample after dilution was placed in the well on a 25 × 75 × 1 mm microscope slide covered with a glass slide cover (Fisher Scientific, Waltham, MA).

About 10 μl of cell samples were mounted on a number 1.5, 24 × 50 mm (thickness of 0.16–0.19 mm) cover glass slide (Fisher or VWR). Cells were cushioned with a 3% (w/v) agarose gel pad to restrict the movement of the live cells. Cells were optically imaged using a Nikon Eclipse Ti inverted microscope equipped with crossed polarizers and a Photometrics CoolSNAP HQ2 CCD camera using a Nikon 100X oil objective lens. Phase-contrast images of bacterial cells were recorded with an exposure time of 50 ms using Nikon NIS Elements software. Multiple snapshots were collected for each experiment. All images were analyzed to measure the cell length in Oufti (54 (link)) using one single optimized parameter set and manual verification. Confidence intervals of the fraction of elongated cells for each mutant were computed from 1000 replicates of bootstrap resampling (55 ).