Extended Data Fig. 7: Effects of BURST imaging on cell viability.
From: Ultrasensitive ultrasound imaging of gene expression with signal unmixing
(a) Darkfield optical image of ARG-expressing E. coli Nissle colonies on an agar plate 15 h after seeding. Width of 1 square is 12.7 mm. To assay the effects of BURST and BURST+ imaging on bacterial population growth and confirm ARG re-expression after imaging, we cultured ARG Nissle as colonies embedded in soft hydrogel media and applied BURST+ to half the sample. (b) Image of the same plate 23 h after application of BURST+ to the bottom half. (c) Representative magnified images of colonies from the top half of the plate in (a) (left) and (b) (right). (d) Representative magnified images of colonies from the bottom half of the plate in (a) (left) and (b) (right). (e) Area of colonies exposed or not exposed to BURST+ at the 38-hour time point, 23 hours after application of BURST+. Error bars: SEM. N = 36 independent colonies in the –Ultrasound condition and N = 48 independent colonies in the +Ultrasound condition. Two-sided two-sample t-test. p = 0.10. (f) Illustration of the experimental setup for single-cell viability. An acoustic cuvette with mylar windows is filled with 1% agarose and submerged in a water tank. A 2 mm diameter cylindrical inclusion in the agarose is filled with a suspension of GV-expressing cells (1×105 ARG Nissle cells/ml or 2.5×105 mARG HEK cells/ml) and imaged with BURST, BURST+, or 0.3 MPa B-mode as a control to assess the impact of BURST and BURST+ imaging on cells in liquid suspension. (g) Representative BURST and BURST+ images of ARG Nissle samples overlaid on a grayscale B-mode image. The edges of the cylindrical inclusion are indicated with dashed white lines. Scale bars: 2 mm. (h) Colony forming units of ARG-expressing E. coli Nissle cells for the samples exposed to BURST and BURST+ relative to B-mode controls. After imaging, the bacteria were plated on selective solid media and the number of colonies formed after 20 hours was counted. Error bars: SEM. N = 12 samples from 6 biological replicates. One-sided approximate permutation test with 107 permutations. p = 0.021 for BURST vs. control and p = 0.0015 for BURST+ vs. control. (i) Viable mARG-expressing HEK cells, as measured by flow cytometry, after exposure to BURST and BURST+, relative to B-mode controls. We exposed liquid suspensions of mARG-expressing HEK cells to these imaging modes in the same apparatus as described above for bacteria. Following ultrasound exposure, we counted the number of live (metabolically active) and dead cells using flow cytometry. We observed no significant difference in the viability of cells exposed to either BURST or BURST+ relative to the low-pressure controls. Error bars: SEM. N = 3 biological replicates. One-sided exact permutation test. p = 0.83 for BURST vs. control and p = 0.48 for BURST+ vs. control.
