2025-04-03 オランダ・デルフト工科大学(TU Delft)
<関連情報>
- https://www.tudelft.nl/en/2025/tnw/revealing-capillaries-and-cells-in-living-organs-with-ultrasound
- https://www.science.org/doi/10.1126/science.ads1325
- https://www.nature.com/articles/nnano.2014.32
非線形サウンドシート顕微鏡: 毛細管および細胞スケールでの不透明器官のイメージング Nonlinear sound-sheet microscopy: Imaging opaque organs at the capillary and cellular scale
Baptiste Heiles, Flora Nelissen, Rick Waasdorp, Dion Terwiel, […], and David Maresca
Science Published:4 Apr 2025
Editor’s summary
Visualizing deep within living tissues is a perennial challenge. Heiles et al. developed a method to allow the generation of three-dimensional (3D) ultrasound images of gene expression and 2D ultrasound images of capillary vessels. The fundamental idea behind this method, which is called nonlinear sound-sheet microscopy, is simple. Biomolecules expressed in cells or microbubbles circulating in blood vessels are excited along thin ultrasound sheets that are swept across the sample. This method enables fast, deep, and volumetric imaging of living opaque organs labeled with echogenic reporters. Ultrasound sectioning, the ability to resolve structures in the z plane, was achieved by modulating acoustic pressure along nondiffractive beams to confine nonlinear echoes of biomolecules or microbubbles to a plane. —Stella M. Hurtley
Structured Abstract
INTRODUCTION
Enabling discoveries in the field of biology often requires new ways of visualization. One of the most informative methods for observing dynamic cellular processes in living organisms uses light-sheet microscopy to leverage genetically encoded fluorescent reporters. Unfortunately, optical microscopy is phototoxic to cells and remains restricted to the study of thin transparent specimens. The physics of high-frequency ultrasound is ideally suited to in vivo cellular imaging by providing a combination of deep penetration (~1 cm) and high spatiotemporal resolution (~100 μm, 1 ms). In addition, the recent introduction of genetically encoded gas vesicles (GVs) as the “green fluorescent protein for ultrasound” creates new opportunities for in vivo studies of cellular function. To equip ultrasound with capabilities analogous to those given to optical microscopy by fluorescent proteins, there is a need for fast high-resolution and volumetric ultrasound imaging methods capable of visualizing acoustic reporter genes and acoustic biosensors. If this can be achieved, the resulting capabilities will allow researchers to explore previously inaccessible cellular biology in vivo with unparalleled information content, resolution, coverage, and translatability to biological research and clinical development.
RATIONALE
We introduce the concept of nonlinear sound-sheet microscopy (NSSM), a method capable of detecting both genetically encoded GVs and synthetic lipid-shelled microbubbles (MBs) across thin living tissue sections. The fundamental idea behind this method is to modulate acoustic pressure along the main lobe of nondiffractive ultrasound beams to confine the nonlinear scattering of GVs and MBs to thin tissue sections. Because GVs and MBs respond to increasing acoustic pressure levels in a nonlinear way, they can be distinguished from surrounding tissues that respond to increasing pressure levels in a linear way. To maximize the volumetric field of view of NSSM, we developed our imaging method on a class of high-frequency ultrasound transducers called row-column–addressed (RCA) arrays. In our current implementation, the imaging field of view was approximately 1 cm3.
RESULTS
Firstly, we assessed the capacity of NSSM to detect bacterial acoustic reporter genes in three dimensions. Escherichia coli engineered to constitutively express nonlinearly scattering GVs were successfully detected along the two orthogonal directions of a 15-MHz RCA array. By sweeping electronically the sound-sheet plane along the two orthogonal directions of the RCA array, we captured volumetric images of bacterial acoustic reporter genes spanning 8.8 × 8.8 × 10 mm3. Secondly, we performed longitudinal NSSM of genetically labeled tumors and revealed three-dimensional (3D) patterns of GV expression over several days. We showed that NSSM can be used to track tumor growth but also to quantify both tumor and necrotic core volumes. Thirdly, we demonstrated that NSSM is capable of detecting synthetic lipid-shelled MBs, a class of resonant ultrasound contrast agents used as vascular reporters. Using NSSM at kilohertz frame rates in arbitrarily selected planes, we acquired nonlinear Doppler images of the rat brain vasculature across the entire brain. Lastly, the combination of NSSM with ultrasound localization microscopy allowed us to map cerebral blood flows below 3 mm/s, thereby revealing the capillary vasculature in living rat brains in 100-μm-thick tissue sections.
CONCLUSION
We demonstrated the ability of NSSM to confine nonlinear scattering of genetically encoded GVs and synthetic lipid-shelled MBs to wavelength-thin opaque tissue sections. NSSM is an imaging method that can either be tuned for speed or coverage. In two dimensions and at an ultrasound frequency of 15 MHz, NSSM can scan 1 cm deep with a theoretical frame rate of 25.6 kHz. In three dimensions, NSSM can acquire 8.8 × 8.8 × 10 mm3 volumes of tissue with a theoretical volume rate of 233 Hz. To use NSSM to the fullest, new generations of brighter acoustic reporter genes and faster biosensors will have to be developed. Additionally, the sensitivity of NSSM should be improved further to enable single-cell detection. If we are successful, NSSM will carry a wave of opportunities for dynamic imaging studies of biological processes across scales.
Nonlinear sound-sheet microscopy.
The ability to excite acoustic reporters one plane at a time enables molecular ultrasound imaging at the cellular and capillary scales. (i) In NSSM, the nonlinear scattering of acoustic reporters is confined to thin sound sheets spanning 0.1 × 10 × 9 mm3. (ii) Orthogonally swept sound-sheet imaging enables the 3D visualization of gene expression in opaque organs, whereas (iii) sound-sheet localization microscopy enables deep super-resolution imaging of brain capillaries. p, pitch of the RCA. p/2 was equal to 55 μm in this study.
Abstract
Light-sheet fluorescence microscopy has revolutionized biology by visualizing dynamic cellular processes in three dimensions. However, light scattering in thick tissue and photobleaching of fluorescent reporters limit this method to studying thin or translucent specimens. In this study, we applied nondiffractive ultrasound beams in conjunction with a cross-amplitude modulation sequence and nonlinear acoustic reporters to enable fast and volumetric imaging of targeted biological functions. We reported volumetric imaging of tumor gene expression at the cubic centimeter scale using genetically encoded gas vesicles and localization microscopy of cerebral capillary networks using intravascular microbubble contrast agents. Nonlinear sound-sheet microscopy provides a ~64× acceleration in imaging speed, ~35× increase in imaged volume, and ~4× increase in classical imaging resolution compared with the state of the art in biomolecular ultrasound.
超音波分子レポーターとしての生体ガスナノ構造 Biogenic gas nanostructures as ultrasonic molecular reporters
Mikhail G. Shapiro,Patrick W. Goodwill,Arkosnato Neogy,Melissa Yin,F. Stuart Foster,David V. Schaffer & Steven M. Conolly
Nature Nanotechnology Published:16 March 2014
DOI:https://doi.org/10.1038/nnano.2014.32
Abstract
Ultrasound is among the most widely used non-invasive imaging modalities in biomedicine, but plays a surprisingly small role in molecular imaging due to a lack of suitable molecular reporters on the nanoscale. Here, we introduce a new class of reporters for ultrasound based on genetically encoded gas nanostructures from microorganisms, including bacteria and archaea. Gas vesicles are gas-filled protein-shelled compartments with typical widths of 45–250 nm and lengths of 100–600 nm that exclude water and are permeable to gas. We show that gas vesicles produce stable ultrasound contrast that is readily detected in vitro and in vivo, that their genetically encoded physical properties enable multiple modes of imaging, and that contrast enhancement through aggregation permits their use as molecular biosensors.
