Multisizer 4e coulter counter ms4

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).