Sorted Microbubble Populations

The specific procedure for sorting microbubbles is presented in this section as an item- ized list of steps and as a flow chart in Figure 4.4. The method used to create various size distributions is based on differences in buoyancy forces for different microbubble sizes and was recently described in detail by Feshitan and colleagues [41]. Using this technique, sorted distributions with mean diameters of ∼1.0 µm, ∼1.6 µm, and ∼3.5 µm may be obtained (Figure 4.3). It is important to note that the mode of the distri- bution is often larger than the mean diameter of the microbubble population. This is due to imperfections of the technique, which limit the amount of small microbubbles that are filtered during the isolation technique. Thus, the residual small populations that remain after centrifugation typically reduces the mean of the size distribution to a value that is less than the mode of the same distribution.

Microbubble Production

A large quantity of lipid solution is required (typically 90 mL or more at a minimum concentration of 1.0 _mLmg) to yield the appropriate number of sorted microbubbles. This lipid solution is prepared via the methods described in section 4.3.4. After the lipid solution is prepared, different diameter distributions are preferentially selected using a multi-step centrifugation procedure as follows:

1. Lipids are completely dissolved in the buffer solution by raising the solution tem- perature to 55◦C.

2. The beaker of lipids is transferred to the sonic dismembrator apparatus. The sonicator tip is placed such that it is ∼1 mm below the surface of the lipid solution.

3. The sonic dismembrator is set to a power of 70% and a time of 15 seconds. 4. A large collection of microbubbles is generated via acoustic emulsification by

turning the sonicator on while flowing decafluorobutane over the surface of the solution.

5. The resulting microbubbles (in solution) are collected using 30 mL syringes. 6. The 30 mL syringes are placed in a centrifuge for 10 minutes at 300 G.

7. After 10 minutes, a microbubble “cake” forms toward the plunger of the syringe. The buffer solution is slowly pushed out of the syringe (everything but the mi- crobubble “cake”) into a beaker for recycling.

8. Approximately 1 mL of buffer solution is added to the syringe containing the microbubble “cake” for every 1 mL of microbubbles produced.

9. Using the recycled lipid solution, steps 1 through 8 are repeated until at least 30 mL of microbubbles have been collected.

3.5 Micron Diameter Isolation

1. The well-mixed 30 mL syringe of microbubbles is placed into a centrifuge for 1 min at 30 G.

2. The resulting microbubble solution is slowly pushed out into another 30 mL syringe and the microbubble “cake” is discarded.

3. Filtered PBS is added to the syringe containing the solution until there is a final volume of 30 mL.

4. The 30 mL syringe of microbubbles is placed into a centrifuge for 1 min at 70 G. 5. The resulting microbubble solution is slowly pushed out into another 30 mL

syringe and the microbubble “cake” is discarded.

6. Filtered PBS is added to the syringe containing the solution until there is a final volume of 30 mL.

7. The 30 mL syringe of microbubbles is placed into a centrifuge for 1 min at 160 G.

8. The resulting microbubble solution is slowly pushed out into another 30 mL syringe for ∼1.0 µm and ∼1.6 µm selection.

9. The “cake” is transferred to a 5 mL syringe for the selection of∼3.5 µm diameter microbubbles.

10. Filtered PBS is added to the syringe until there is a final volume of 5 mL. 11. The 5 mL syringe is placed in centrifuge for 1 min at 120 G.

12. The resultant microbubble solution is slowly pushed out of the syringe while the microbubble “cake” is retained.

13. Steps 10 through 12 are repeated until the resultant solution is completely clear. 14. The final microbubble population is stored in a 3 mL syringe with no headspace.

The size distribution is described in Figure 4.3C. 1.6 Micron Diameter Isolation

1. Using the resultant microbubble solution from step 8 above, the 30 mL syringe is placed in the centrifuge for 1 min at 270 G.

2. The resulting microbubble solution is slowly pushed out into another 30 mL syringe for ∼1.0 µm size-selection.

3. Filtered PBS is added to the syringe containing the microbubble “cake” until there is a final volume of 30 mL.

4. The 30 mL syringe is placed in the centrifuge for 1 min at 270 G.

5. The resultant microbubble solution is slowly pushed out of the syringe and dis- carded while the microbubble “cake” is retained.

6. Steps 3 through 5 are repeated until the resultant solution is completely clear. 7. The final microbubble population is stored in a 3 mL syringe with no headspace.

The size distribution is described in Figure 4.3B. 1.0 Micron Diameter Isolation

1. Using the resultant microbubble solution from step 2 above, the 30 mL syringe is placed in the centrifuge for 10 min at 300 G.

2. The resulting microbubble solution is slowly pushed out and discarded.

3. The microbubble “cake” is retained and stored in a 3 mL syringe with no headspace. The size distribution is described in Figure 4.3A.

Figure 4.3: A: Resultan t 1.0 µ m Diameter Siz e Distribution. B: Resultan t 1.6 µ m Diameter Siz e Distribution. C: Resultan t 3.5 µ m Diameter Siz e Distribution. Note: The dotted lin es indicate the mo de of the distri bution, not the mean diamete r. 31

CHAPTER 5

Imaging and Animal Preparation