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Ultrasensitive ultrasound imaging of gene expression with signal unmixing

Abstract

Acoustic reporter genes (ARGs) that encode air-filled gas vesicles enable ultrasound-based imaging of gene expression in genetically modified bacteria and mammalian cells, facilitating the study of cellular function in deep tissues. Despite the promise of this technology for biological research and potential clinical applications, the sensitivity with which ARG-expressing cells can be visualized is currently limited. Here we present burst ultrasound reconstructed with signal templates (BURST)—an ARG imaging paradigm that improves the cellular detection limit by more than 1,000-fold compared to conventional methods. BURST takes advantage of the unique temporal signal pattern produced by gas vesicles as they collapse under acoustic pressure above a threshold defined by the ARG. By extracting the unique pattern of this signal from total scattering, BURST boosts the sensitivity of ultrasound to image ARG-expressing cells, as demonstrated in vitro and in vivo in the mouse gastrointestinal tract and liver. Furthermore, in dilute cell suspensions, BURST imaging enables the detection of gene expression in individual bacteria and mammalian cells. The resulting abilities of BURST expand the potential use of ultrasound for non-invasive imaging of cellular functions.

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Fig. 1: BURST paradigm.
Fig. 2: BURST imaging of ARG-expressing cells.
Fig. 3: Detection sensitivity of BURST imaging.
Fig. 4: BURST imaging of orally gavaged cells.
Fig. 5: Dynamic BURST imaging of systemically injected probiotics.
Fig. 6: Single-cell imaging using BURST.

Data availability

Primary image data are available on GitHub (https://github.com/shapiro-lab/burst-imaging-public). Source data are provided with this paper.

Code availability

MATLAB code is available on GitHub (https://github.com/shapiro-lab/burst-imaging-public).

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Acknowledgements

We thank P. Dutka for assistance with electron microscopy, M. Swift for assistance with animal protocols and B. Jin for the supine mouse illustration. D.P.S. is supported by the NSF graduate research fellowship (award number 1745301). A.B.-Z. is supported by the European Union’s Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement no. 792866. A.F. was supported by the NSERC graduate fellowship. This research was funded by the National Institutes of Health (grant no. R01-EB018975 (to M.G.S.)). Related research in the Shapiro laboratory is also supported by the Chan-Zuckerberg Initiative, Heritage Medical Research Institute, Burroughs Wellcome Career Award at the Scientific Interface, the Pew Scholarship in the Biomedical Sciences and the Packard Fellowship for Science and Engineering.

Author information

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Authors

Contributions

D.P.S. and M.G.S. conceived and designed the study. D.P.S., A.F. and A.B.-Z. designed the BURST pulse sequence. D.P.S. designed the BURST+ pulse sequence and the signal unmixing algorithm. D.P.S. wrote the MATLAB scripts for ultrasound imaging and data processing. A.B.-Z. prepared genetic constructs in S. Typhimurium and E. coli Nissle 1917. A.F. and S.S. prepared genetic constructs in HEK293 cells. S.S. performed flow cytometry measurements. D.P.S. performed the in vitro ultrasound experiments. D.P.S., A.L.-G. and B.L. performed in vivo ultrasound experiments. D.P.S. and M.G.S. analyzed the data. D.P.S. and M.G.S. wrote the manuscript with input from all authors. M.G.S. supervised the research.

Corresponding author

Correspondence to Mikhail G. Shapiro.

Ethics declarations

Competing interests

The California Institute of Technology has filed a patent application (US 16/736,581) related to the imaging method described in this article.

Additional information

Peer review information Nature Methods thanks Junjie Yao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Nina Vogt was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 BURST background signal in phantoms.

(a) The same image data from Fig. 2e displayed with a lower limit of 70 dB on color scale. (b) Fig. 2e, shown for comparison. All scalebars: 2 mm.

Extended Data Fig. 2 Collapse signal generation mechanism.

The imaging target for all panels is ARG E. coli Nissle at 103 cells/ml in suspension. All images are displayed in dB scale with the same colormap shown in the bottom right of panel (g) (min: 0 dB, max: 80 dB). All scalebars are 2 mm. (a) Distribution of BURST ray line peak intensities (that is maxima over columns of pixels) for PPP = 4.3 MPa. N = 650. (b) Distribution of BURST+ ray line peak intensities for PPP = 4.3 MPa. N = 650. (c) BURST pressure ramp images with PPP ranging from 3.7 MPa to 4.3 MPa. (d) BURST+ pressure ramp with the same pressures as in (c). (e) Peak image intensity vs PPP for BURST and BURST+. Error bars: SEM. N = 10 BURST acquisitions. (f) Image time series acquired with an ultrafast implementation of BURST+, with 1 frame/100 µsec, at 4.3 MPa. (g) Cycle ramp images with the number of transmit waveform cycles ranging from 0.5 cycles to 10.5 cycles and PPP held constant at 4.0 MPa. (h) Mean intensity of cycle ramp images vs. depth. Traces are averaged over 10 replicates. Error bars not shown for clarity. (i) Proposed mechanism to account for the presence of dim signals, but not bright signals, in BURST. (j) Proposed mechanism to account for the presence of both dim and bright sources in BURST+ and pulse sequences with more than one cycle. (k) BURST (top) and BURST+ (bottom) images at 4.3 MPa with arrows indicating the punctate signal whose RF data is plotted in the following panels. (l-m) Beamformed RF waveforms for the BURST (top) and BURST+ (bottom) ray lines in (k) indicated by the arrows. The waveforms are acquired from the pre-collapse, low-pressure frame (l), the first high-pressure frame (m), and the second high-pressure frame (n) of the BURST or BURST+ pulse sequence for arrival times corresponding to a depth range of 18 mm to 23 mm. (o) N = 464 RF waveforms corresponding to dim (20–60 dB) BURST signals aligned by peak envelope intensity. (p) N = 304 RF waveforms corresponding to bright (60–80 dB) BURST+ signals aligned by peak envelope intensity. (q) N = 308 RF waveforms corresponding to dim (20–60 dB) BURST+ signals aligned by peak envelope intensity. (r) Full width at half maximum (FWHM) for the envelopes of the RF waveforms shown in panels (o-q). N = 464 ray lines for BURST, N = 308 ray lines for dim (20–60 dB) BURST+, and N = 304 ray lines for bright (60–80 dB) BURST+. Error bars represent ± SEM.

Source data

Extended Data Fig. 3 Transmission electron micrographs of ARG-expressing E. coli Nissle cells after imaging.

(a) Representative cell from a sample not imaged with BURST or BURST+. A total of 14 micrographs of cells in this condition were acquired with similar results. (b) Representative cell from a sample imaged with BURST. A total of 13 micrographs of cells in this condition were acquired with similar results. (c) Representative cell from a sample imaged with BURST+. A total of 16 micrographs of cells in this condition were acquired with similar results. All scalebars: 500 nm. GVs are visible inside the control cell as lighter objects, and are absent in the imaged cells.

Extended Data Fig. 4 BURST thresholds for different types of gas vesicles.

Normalized BURST+ signal as a function of peak positive pressure in BURST+ images of different types of GVs suspended in liquid buffer at concentrations of 10−5 OD500nm. Halo GVs were purified from Halobacterium salinarium, Ana GVs were purified from Anabaena flos-aquae, and Mega GVs were purified from E. coli. N = 5 ray line acquisitions. Error bars represent ± SEM.

Source data

Extended Data Fig. 5 Acoustic shielding in BURST sequence at high ARG-expressing cell concentration.

(a) Images from the high-pressure frames (frames 2–5) of a BURST+ sequence applied to a 1% agarose phantom with wells containing tissue-mimicking scatterers mixed with 108 cells/ml RFP-expressing E. coli Nissle (left) and ARG-expressing E. coli Nissle (right). Scale bars: 2 mm. (b) Mean pixel intensity vs. frame number for the ARG well, corresponding to the ROI of the same color in Frame 5 of the previous panel.

Extended Data Fig. 6 In vitro BURST imaging of ARG-expressing bacteria in plain agarose gel.

(a-c) Array of ultrasound images of a cross section of rectangular wells containing Nissle E. coli embedded in 1% agarose wells within an agarose phantom. Each image contains a pair of wells, the left well containing RFP-expressing Nissle, the right well containing ARG-expressing Nissle. Rows correspond to cell concentrations, which range over six orders of magnitude. (a) B-mode images. (b) BURST images. (c) BURST+ images. The top edge of each image corresponds to a depth of 17.5 mm, the bottom to a depth of 23 mm. The left edge of each image corresponds to a lateral coordinate of −7 mm, the right to +7 mm. Scalebars: 2 mm. (d) Mean CTR vs log cell concentration for BURST and BURST+ on agarose-embedded cells. N = 12 wells, 4 from each of 3 biological replicates. CTR values represent the mean intensity of the ARG well relative to the mean intensity of the RFP well. Error bars: SEM.

Source data

Extended Data Fig. 7 Effects of BURST imaging on cell viability.

(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.

Source data

Extended Data Fig. 8 ARG-expressing Nissle colonies continue to grow and re-express GVs after exposure to BURST+.

23 hours after initial exposure to BURST+, strong GV-specific BURST+ signal was observed from ARG-expressing Nissle colonies, confirming GV re-expression. Similar results were obtained with two additional plates. (a) Darkfield optical images of the half of the plate exposed to BURST+ after incubation at 30 °C for a total of 15 h (top) and 38 h (bottom). (b) BURST+ composite ultrasound images of the plate after 15 h (top) and 38 h (bottom), with the first collapse frame removed prior to template unmixing to reduce BURST signal area and allow comparison of signal spatial distribution with the optical image. The composite image was formed by taking the maximum of each BURST image plane along the axial dimension and concatenating the resulting rows of pixels to form 2D composite image. Prior to dB scaling, a 3×3 median filter was applied to the composite image, followed by a Gaussian filter with σ = 1. (c) The same BURST+ ultrasound images with all frames included in template unmixing. Scale bars: 10 mm.

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Sawyer, D.P., Bar-Zion, A., Farhadi, A. et al. Ultrasensitive ultrasound imaging of gene expression with signal unmixing. Nat Methods 18, 945–952 (2021). https://doi.org/10.1038/s41592-021-01229-w

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