Contrast Agents For Ultrasound Imaging Research Articles

Harmonic imaging for nonlinear detection of acoustic biomolecules.

Gas vesicles (GVs) based on acoustic reporter genes have emerged as potent contrast agents for cellular and molecular ultrasound imaging. These air-filled, genetically encoded protein nanostructures can be expressed in a variety of cell types in vivo to visualize cell location and activity or injected systemically to label and monitor tissue function. Distinguishing GV signal from tissue deep inside intact organisms requires imaging approaches such as amplitude modulation (AM) or collapse-based pulse sequences. However, these approaches have limitations either in sensitivity or require the destruction of GVs, restricting the imaging of dynamic cellular processes. To address these limitations, we developed harmonic imaging to enhance the sensitivity of nondestructive GV imaging. We hypothesized that harmonic imaging, integrated with AM, could significantly elevate GV detection sensitivity by leveraging the nonlinear acoustic response of GVs. We tested this hypothesis by imaging tissue-mimicking phantoms embedded with purified GVs, mammalian cells genetically modified to express GVs, and mice liver in vivo post-systemic infusion of GVs. Our findings reveal that harmonic cross-propagating wave AM (HxAM) imaging markedly surpasses traditional xAM in isolating GVs' nonlinear acoustic signature, demonstrating significant (p < 0.05) enhancements in imaging performance. HxAM imaging improves detection of GV producing cells up to three folds in vitro, enhances in vivo imaging performance by over 10 dB, while extending imaging depth by up to 20%. Investigation into the backscattered spectra further elucidates the advantages of harmonic imaging. These advancements bolster ultrasound's capability in molecular and cellular imaging, underscoring the potential of harmonic signals to improve GV detection.

Suppression of the Tissue Component With the Total Least‐Squares Algorithm to Improve Second Harmonic Imaging of Ultrasound Contrast Agents

ABSTRACTThe second harmonic (SH) of ultrasound contrast agents (UCAs) is widely used in contrast‐enhanced ultrasound imaging; however, is affected by the nonlinearity of surrounding tissue. Suppression of the tissue component based on the total least‐squares (STLS) algorithm is proposed to enhance the SH imaging of UCAs. The image blocks of pulse‐inversion‐based SH images before and after UCA injections are set as the reference and input of the total least‐squares model, respectively. The optimal coefficients of the model are obtained by minimizing the Frobenius norm of perturbations in the input and output signals. After processing all image blocks, the complete SH image of UCAs is obtained by subtracting the optimal output of the model (i.e., the estimated tissue SH image) from the SH image after UCA injection. Simulation and in vivo experiments confirm that the STLS approach offers clearer capillaries. For in vivo experiments, the STLS‐based contrast‐to‐tissue ratios and contrasts increase by 26.90% and 56.27%, as well as 26.99% and 56.43%, respectively, compared with those based on bubble‐echo deconvolution and pulse inversion bubble‐wavelet imaging methods. The STLS approach enhances the SH imaging of UCAs by effectively suppressing more tissue SH components, having the potential to provide more accurate diagnostic information for clinical applications.

Ultrasound protein-copolymer microbubble library engineering through poly(vinylpyrrolidone-co-acrylic acid) structure

HypothesisWhile albumin-coated microbubbles are routine contrast agents for ultrasound imaging, their short duration of contrast enhancement limits their use, yet can be improved by incorporating protein-copolymer hybrids into microbubble shells. The incorporation of N-vinyl-2-pyrrolidone and acrylic acid copolymer (P(VP–AA)) has been shown to enhance the performance of bovine serum albumin (BSA)-coated microbubbles. However, the impact of the copolymer structural properties on key microbubble characteristics (i.e., concentration, mean diameter and acoustic response) remains poorly understood. Therefore, we hypothesize that the copolymer structure affects its capacity to form micelle-like nanoaggregates, protein-copolymer hybrids, and microbubble shells, ultimately influencing the physicochemical and acoustic properties of the microbubbles. ExperimentsHere we evaluate the production and performance of BSA@P(VP–AA) microbubbles synthesized using a series of P(VP–AA) copolymers with –C8H17 and –C18H37 end groups and molecular weight cutoffs between 3.5 and 15 kDa. Both simulation and experimental data demonstrate that interactions between BSA and the copolymers significantly influence the performance of the resulting microbubbles across the library of 60 formulations. FindingsThe introduction of –C8H17 terminated copolymers into microbubble shells resulted in up to 200-fold higher concentration, 7-fold greater acoustic response, and 5-fold longer ultrasound contrast enhancement compared to plain BSA microbubbles. The enhanced acoustic performance was sustained during in vivo cardiac ultrasound imaging, without altering liver accumulation after copolymer introduction. These findings underscore how optimizing copolymer structure (specifically the terminal end group and molecular weight) can tailor the formation and performance of protein-copolymer-coated microbubbles, offering valuable insights for designing ultrasound contrast agents.

Characterisation of Gas Vesicles as Cavitation Nuclei for Ultrasound Therapy using Passive Acoustic Mapping.

Genetically encodable gas filled particles known as gas vesicles (GVs) have shown promise as a biomolecular contrast agent for ultrasound imaging and have the potential to be used as cavitation nuclei for ultrasound therapy. In this study, we used passive acoustic mapping techniques to characterize GV-seeded cavitation, utilizing 0.5 and 1.6 MHz ultrasound over peak rarefactional pressures ranging from 100 to 2200 kPa. We found that GVs produce cavitation for the duration of the first applied pulse, up to at least 5000 cycles, but that bubble activity diminishes rapidly over subsequent pulses. At 0.5 MHz the frequency content of cavitation emissions was predominantly broadband in nature, whilst at 1.6 MHz narrowband content at harmonics of the main excitation frequency dominated. Simulations and high-speed camera imaging suggest that the received cavitation emissions come not from individual GVs but instead from the coalescence of GV-released gas into larger bubbles during the applied ultrasound pulse. These results will aid the future development of GVs as cavitation nuclei in ultrasound therapy.

Polymeric Microbubble Shell Engineering: Microporosity as a Key Factor to Enhance Ultrasound Imaging and Drug Delivery Performance.

Microbubbles (MB) are widely used as contrast agents for ultrasound (US) imaging and US-enhanced drug delivery. Polymeric MB are highly suitable for these applications because of their acoustic responsiveness, high drug loading capability, and ease of surface functionalization. While many studies have focused on using polymeric MB for diagnostic and therapeutic purposes, relatively little attention has thus far been paid to improving their inherent imaging and drug delivery features. This study here shows that manipulating the polymer chemistry of poly(butyl cyanoacrylate) (PBCA) MB via temporarily mixing the monomer with the monomer-mimetic butyl cyanoacetate (BCC) during the polymerization process improves the drug loading capacity of PBCA MB by more than twofold, and the in vitro and in vivo acoustic responses of PBCA MB by more than tenfold. Computer simulations and physisorption experiments show that BCC manipulates the growth of PBCA polymer chains and creates nanocavities in the MB shell, endowing PBCA MB with greater drug entrapment capability and stronger acoustic properties. Notably, because BCC can be readily and completely removed during MB purification, the resulting formulation does not include any residual reagent beyond the ones already present in current PBCA-based MB products, facilitating the potential translation of next-generation PBCA MB.

Bioactive Nanotherapeutic Ultrasound Contrast Agent for Concurrent Breast Cancer Ultrasound Imaging and Treatment.

Contrast-enhanced ultrasound (CEUS) plays a crucial role in cancer diagnosis. The use of ultrasound contrast agents (UCAs) is inevitable in CEUS. However, current applications of UCAs primarily focus on enhancing imaging quality of ultrasound contrast rather than serving as integrated platforms for both diagnosis and treatment in clinical settings. In this study, a novel UCA, termed NPs-DPPA(C3F8), is innovatively prepared using a combination of nanoprecipitation and ultrasound vibration methods. The DPPA lipid possesses inherent antiangiogenic and antitumor activities, and when combined with C3F8, it functions as a theranostic agent. Notably, the preparation of NPs-DPPA(C3F8) is straightforward, requiring only one hour from raw materials to the final product due to the use of a single material, DPPA. NPs-DPPA(C3F8) exhibits inherent antiangiogenic and biotherapeutic activities, effectively inhibiting triple-negative breast cancer (TNBC) angiogenesis and reducing VEGFA expression both in vitro and in vivo. Clinically, NPs-DPPA(C3F8) enables simultaneous real-time imaging, tumor assessment, and antitumor activity. Additionally, through ultrasound cavitation, NPs-DPPA(C3F8) can overcome the dense vascular walls to increase accumulation at the tumor site and facilitate internalization by tumor cells. The successful preparation of NPs-DPPA(C3F8) offers a novel approach for integrating clinical diagnosis and treatment of TNBC.

History of contrast enhanced ultrasound (CEUS).

The 50th year of the European Federation of Societies in Ultrasound in Medicine and Biology (EFSUMB) has been celebrated 2022 publishing articles on the history of US. Contrast enhanced ultrasound (CEUS) allows to visualize blood flow and tissue perfusion. CEUS has proven to be safe without risk of nephrotoxicity. The availability of a contrast agent (tracer) for ultrasound imaging allows for the first time a dynamic assessment of tissue perfusion (blood flow and wash-in/wash-out pattern) which is an essential part for the detection and characterisation of pathological tissue and abnormal organ function. It was an outstanding achievement of academic centers in close cooperation with EFSUMB to investigate and validate the clinical potential of this new technology for the diagnosis and monitoring of various diseases and to develop clinical guidelines based on an in-depth assessment of the existing scientific publications. An important part of the implementation of CEUS in clinical practice was the development of contrast-specific imaging modes on the ultrasound scanners (in close cooperation with the machine manufacturers), the optimization of the machine setups for contrast imaging and the education provided to clinical users in form of workshops, webinars, textbooks and scientific congresses.

Genetic Modulation of Biosynthetic Gas Vesicles for Ultrasound Imaging.

Gas vesicles (GVs) from microorganisms are genetically air-filled protein nanostructures, and serve as a new class of nanoscale contrast agents for ultrasound imaging. Recently, the genetically encoded GV gene clusters have been heterologously expressed in Escherichia coli, allowing these genetically engineered bacteria to be visualized in vivo in a real-time manner by ultrasound. However, most of the GV genes remained functionally uncharacterized, which makes it difficult to regulate and modify GVs for broad medical applications. Here, the impact of GV proteins on GV formation is systematically investigated. The results first uncovered that the deletions of GvpR or GvpU resulted in the formation of a larger proportion of small, biconical GVs compared to the full-length construct, and the deletion of GvpT resulted in a larger portion of large GVs. Meanwhile, the combination of gene deletions has resulted in several genotypes of ultrasmall GVs that span from 50 to 20nm. Furthermore, the results showed that E. coli carrying the ΔGvpCRTUmutant can produce strong ultrasound contrast signals in mouse liver. In conclusion, the study provides new insights into the roles of GV proteins in GV formation and produce ultrasmall GVs with a wide range of in vivo research.

Hydrophobin-Coated Perfluorocarbon Microbubbles with Strong Non-Linear Acoustic Response

Gas-filled microbubbles are well-established contrast agents for ultrasound imaging and widely studied as delivery systems for theranostics. Herein, we have demonstrated the promising potential of the hydrophobin HFBII—a fungal amphiphilic protein—in stabilizing microbubbles with various fluorinated core gases. A thorough screening of several experimental parameters was performed to find the optimized conditions regarding the preparation technique, type of core gas, HFBII initial concentration, and protein dissolution procedure. The best results were obtained by combining perfluorobutane (C4F10) gas with 1 mg/mL of aqueous HFBII, which afforded a total bubble concentration higher than 109 bubbles/mL, with long-term stability in solution (at least 3 h). Acoustic characterization of such microbubbles in the typical ultrasound frequency range used for diagnostic imaging showed the lower pressure resistance of HFBII microbubbles, if compared to conventional ones stabilized by phospholipid shells, but, at the same time, revealed strong non-linear behavior, with a significant harmonic response already at low acoustic pressures. These findings suggest the possibility of further improving the performance of HFBII-coated perfluorinated gas microbubbles, for instance by mixing the protein with other stabilizing agents, e.g., phospholipids, in order to tune the viscoelastic properties of the outer shell.

From Nanovesicles to Nanobubbles Based on Repeated Compression Method.

Nanobubbles have been increasingly applied in biomedicine, which is attributed to their ability to work as ultrasound imaging contrast agents and powerful gene/drug carriers. Different production techniques or approaches have been developed to generate uniform and stable shelled nanobubbles. However, these shelled nanobubbles are usually prepared based on disordered shell materials, such as free phospholipids and polymers. In recent years, the continuous repeated compression method for a gas-liquid mixture has been developed to produce free and lipid-shelled nanobubbles. In this study, to explore the response of well-organized nanostructures to this method, the repeated compression method was used to treat preprepared liposomes and polymeric nanovesicles. Size distribution, morphologies, and ultrasound image contrast enhancement of these nanovesicles were determined before and after repeated compression. Results demonstrate that the presence of a phospholipid bilayer is vital to form liposome-based nanobubbles. And the low elastic modulus of the polymeric membrane is key to encapsulate gases into polymeric nanovesicles. Overall, it demonstrated the advantages of well-organized nanostructures to produce nanobubble structures, giving new insights into the preparation and understanding of nanobubbles.

BR55 Ultrasound Molecular Imaging of Clear Cell Renal Cell Carcinoma Reflects Tumor Vascular Expression of VEGFR-2 in a Patient-Derived Xenograft Model.

Standard imaging cannot reliably predict the nature of renal tumors. Among malignant renal tumors, clear cell renal cell carcinoma (ccRCC) is the most common histological subtype, in which the vascular endothelial growth factor 2 (VEGFR-2) is highly expressed in the vascular endothelium. BR55, a contrast agent for ultrasound imaging, consists of gas-core lipid microbubbles that specifically target and bind to the extracellular portion of the VEGFR-2. The specific information provided by ultrasound molecular imaging (USMI) using BR55 was compared with the vascular tumor expression of the VEGFR-2 by immunohistochemical (IHC) staining in a preclinical model of ccRCC. Patients' ccRCCs were orthotopically grafted onto Nod-Scid-Gamma (NSG) mice to generate patient-derived xenografts (PdX). Mice were divided into four groups to receive either vehicle or axitinib an amount of 2, 7.5 or 15 mg/kg twice daily. Perfusion parameters and the BR55 ultrasound contrast signal on PdX renal tumors were analyzed at D0, D1, D3, D7 and D11, and compared with IHC staining for the VEGFR-2 and CD34. Significant Pearson correlation coefficients were observed between the area under the curve (AUC) and the CD34 (0.84, p < 10-4), and between the VEGFR-2-specific signal obtained by USMI and IHC (0.72, p < 10-4). USMI with BR55 could provide instant, quantitative information on tumor VEGFR-2 expression to characterize renal masses non-invasively.

Effect of Anesthetic Carrier Gas on In Vivo Circulation Times of Intravenously Administered Phospholipid Oxygen Microbubbles in Rats.

For the treatment of tumor hypoxia, microbubbles comprising oxygen as a majority component of the gas core with a stabilizing shell may be used to deliver and release oxygen locally at the tumor site through ultrasound destruction. Previous work has revealed differences in circulation half-life in vivo for perfluorocarbon-filled microbubbles, typically used as ultrasound imaging contrast agents, as a function of anesthetic carrier gas. These differences in circulation time in vivo were likely due to gas diffusion as a function of anesthetic carrier gas, among other variables. This work has motivated studies to evaluate the effect of anesthetic carrier gas on oxygen microbubble circulation dynamics. Circulation time for oxygen microbubbles was derived from ultrasound image intensity obtained during longitudinal kidney imaging. Studies were constructed for rats anesthetized on inhaled isoflurane with either pure oxygen or medical air as the anesthetic carrier gas. Results indicated that oxygen microbubbles were highly visible via contrast-specific imaging. Marked signal enhancement and duration differences were observed between animals breathing air and oxygen. Perhaps counterintuitively, oxygen microbubbles disappeared from circulation significantly faster when the animals were breathing pure oxygen compared with medical air. This may be explained by nitrogen counterdiffusion from blood into the bubble, effectively changing the gas composition of the core, as has been observed in perfluorocarbon core microbubbles. Our findings suggest that the apparent longevity and persistence of oxygen microbubbles in circulation may not be reflective of oxygen delivery when the animal is anesthetized breathing air.

Ultrasonic Characterization of Ibidi μ-Slide I Luer Channel Slides for Studies With Ultrasound Contrast Agents.

Understanding and controlling the ultrasound contrast agent (UCA)'s response to an applied ultrasound pressure field are crucial when investigating ultrasound imaging sequences and therapeutic applications. The magnitude and frequency of the applied ultrasonic pressure waves affect the oscillatory response of the UCA. Therefore, it is important to have an ultrasound compatible and optically transparent chamber in which the acoustic response of the UCA can be studied. The aim of our study was to determine the in situ ultrasound pressure amplitude in the ibidi μ -slide I Luer channel, an optically transparent chamber suitable for cell culture, including culture under flow, for all microchannel heights (200, 400, 600, and [Formula: see text]). First, the in situ pressure field in the 800- [Formula: see text] high channel was experimentally characterized using Brandaris 128 ultrahigh-speed camera recordings of microbubbles (MBs) and a subsequent iterative processing method, upon insonification at 2 MHz, 45° incident angle, and 50-kPa peak negative pressure (PNP). Control studies in another cell culture chamber, the CLINIcell, were compared with the obtained results. The pressure amplitude was -3.7 dB with respect to the pressure field without the ibidi μ -slide. Second, using finite-element analysis, we determined the in situ pressure amplitude in the ibidi with the 800- [Formula: see text] channel (33.1 kPa), which was comparable to the experimental value (34 kPa). The simulations were extended to the other ibidi channel heights (200, 400, and [Formula: see text]) with either 35° or 45° incident angle, and at 1 and 2 MHz. The predicted in situ ultrasound pressure fields were between -8.7 and -1.1 dB of the incident pressure field depending on the listed configurations of ibidi slides with different channel heights, applied ultrasound frequencies, and incident angles. In conclusion, the determined ultrasound in situ pressures demonstrate the acoustic compatibility of the ibidi μ -slide I Luer for different channel heights, thereby showing its potential for studying the acoustic behavior of UCAs for imaging and therapy.

Enhancing Structural Stability of Oil-Shell Microbubbles via Incorporation of a Gold Nanoparticle Protective Shell for Theranostic Applications

Phospholipid-stabilized microbubbles are utilized as contrast agents in medical ultrasound imaging, and researchers are currently investigating their potential as theranostic agents. Due to the inadequate water solubility and poor stability of numerous new therapeutics, the development of stable microbubbles with the capacity to encapsulate hydrophobic therapeutics is necessary. Herein, we proposed a flow-focusing microfluidic device to generate highly monodispersed, phospholipid-stabilized dual-layer microbubbles for theranostic applications. The stability and microstructural evolution of these microbubbles were investigated by microscopy and machine-learning-assisted segmentation techniques at different phospholipid and gold nanoparticle concentrations. The double-emulsion microbubbles, formed with the combination of phospholipids and gold nanoparticles, developed a protective gold nanoparticle shell that not only acted as a steric barrier against gas diffusion and microbubble coalescence but also alleviated the progressive dewetting instability and the subsequent cascade of coalescence events.

A review of high‐speed optical imaging technology for the analysis of ultrasound contrast agents in an acoustic field

AbstractStabilized microbubbles were commercialized over 30 years ago for use as contrast agents in ultrasound imaging. In recent years, interest in microbubble–acoustic interactions has expanded to applications not only in ultrasound imaging but also in drug and gene delivery. To understand the interaction of a microbubble and ultrasonic field, scientists optically observe the behavior of microbubbles during acoustic excitation. Because of the fast oscillations of microbubbles in ultrasound fields, the application of ultra‐high‐speed photography is required to capture bubble behavior. This manuscript reviews the approaches, challenges, and progress in high‐speed imaging systems utilized for microbubble analysis, focusing on innovations in camera technology.

Compressibility and porosity modulate the mechanical properties of giant gas vesicles

Gas vesicles used as contrast agents for noninvasive ultrasound imaging must be formulated to be stable, and their mechanical properties must be assessed. We report here the formation of perfluoro-n-butane microbubbles coated with surface-active proteins that are produced by filamentous fungi (hydrophobin HFBI from Trichoderma reesei). Using pendant drop and pipette aspiration techniques, we show that these giant gas vesicles behave like glassy polymersomes, and we discover novel gas extraction regimes. We develop a model to analyze the micropipette aspiration of these compressible gas vesicles and compare them to incompressible liquid-filled vesicles. We introduce a sealing parameter to characterize the leakage of gas under aspiration through the pores of the protein coating. Utilizing this model, we can determine the elastic dilatation modulus, surface viscosity, and porosity of the membrane. These results demonstrate the engineering potential of protein-coated bubbles for echogenic and therapeutic applications and extend the use of the pipette aspiration technique to compressible and porous systems.

Real-time imaging of nanobubble ultrasound contrast agent flow, extravasation, and diffusion through an extracellular matrix using a microfluidic model.

Lipid shell-stabilized nanoparticles with a perfluorocarbon gas-core, or nanobubbles, have recently attracted attention as a new contrast agent for molecular ultrasound imaging and image-guided therapy. Due to their small size (∼275 nm diameter) and flexible shell, nanobubbles have been shown to extravasate through hyperpermeable vasculature (e.g., in tumors). However, little is known about the dynamics and depth of extravasation of intact, acoustically active nanobubbles. Accordingly, in this work, we developed a microfluidic chip with a lumen and extracellular matrix (ECM) and imaging method that allows real-time imaging and characterization of the extravasation process with high-frequency ultrasound. The microfluidic device has a lumen and is surrounded by an extracellular matrix with tunable porosity. The combination of ultrasound imaging and the microfluidic chip advantageously produces real-time images of the entire length and depth of the matrix. This captures the matrix heterogeneity, offering advantages over other imaging techniques with smaller fields of view. Results from this study show that nanobubbles diffuse through a 1.3 μm pore size (2 mg mL-1) collagen I matrix 25× faster with a penetration depth that was 0.19 mm deeper than a 3.7 μm (4 mg mL-1) matrix. In the 3.7 μm pore size matrix, nanobubbles diffused 92× faster than large nanobubbles (∼875 nm diameter). Decorrelation time analysis was successfully used to differentiate flowing and extra-luminally diffusing nanobubbles. In this work, we show for the first time that combination of an ultrasound-capable microfluidic chip and real-time imaging provided valuable insight into spatiotemporal nanoparticle movement through a heterogeneous extracellular matrix. This work could help accurately predict parameters (e.g., injection dosage) that improve translation of nanoparticles from in vitro to in vivo environments.

A New Nanoscale Ultrasound Phase-Variant Contrast Agent for Phase Variant Low-Frequency Medical Ultrasound Imaging That Can Scavenge Reactive Oxygen Species

Nanoscale phase-variant ultrasound contrast agents have attracted the interest of many researchers. However, it is a challenge to design nanobubbles that are activated by low-frequency medical ultrasound that do not cause damage to normal tissues. In this study, we designed a new type of nanoparticle consisting of perfluoropentane and iron polyphthalocyanine loaded into liposomes. These nanoparticles can be activated by a low-frequency medical ultrasound imager at a frequency of 5 MHz for ultrasound imaging and clear reactive oxygen species at a rate of more than 50%. This ability to scavenge excess reactive oxygen species can alleviate the damage these species cause and protect macrophages. Moreover, these nanoparticles can effectively enhance ultrasound contrast imaging for real-time visualization in the diagnosis and treatment of diseases.

Microbubble-Assisted Ultrasound for Imaging and Therapy of Melanoma Skin Cancer: A Systematic Review.

Recent technological developments in ultrasound (US) imaging and ultrasound contrast agents (UCAs) have improved diagnostic confidence in echography. In the clinical management of melanoma, contrast-enhanced ultrasound (CEUS) imaging complements conventional US imaging (i.e., high-resolution US and Doppler imaging) for clinical examination and therapeutic follow-up. These developments have set into motion the combined use of ultrasound and UCAs as a new modality for drug delivery. This modality, called sonoporation, has emerged as a non-invasive, targeted and safe method for the delivery of therapeutic drugs into melanoma. This review focuses on the results and prospects of using US and UCAs as dual modalities for CEUS imaging and melanoma treatment.

Recent Advances in the Study of Gas Vesicle Proteins and Application of Gas Vesicles in Biomedical Research.

The formation of gas vesicles has been investigated in bacteria and haloarchaea for more than 50 years. These air-filled nanostructures allow cells to stay at a certain height optimal for growth in their watery environment. Several gvp genes are involved and have been studied in Halobacterium salinarum, cyanobacteria, Bacillus megaterium, and Serratia sp. ATCC39006 in more detail. GvpA and GvpC form the gas vesicle shell, and additional Gvp are required as minor structural proteins, chaperones, an ATP-hydrolyzing enzyme, or as gene regulators. We analyzed the Gvp proteins of Hbt. salinarum with respect to their protein–protein interactions, and developed a model for the formation of these nanostructures. Gas vesicles are also used in biomedical research. Since they scatter waves and produce ultrasound contrast, they could serve as novel contrast agent for ultrasound or magnetic resonance imaging. Additionally, gas vesicles were engineered as acoustic biosensors to determine enzyme activities in cells. These applications are based on modifications of the surface protein GvpC that alter the mechanical properties of the gas vesicles. In addition, gas vesicles have been decorated with GvpC proteins fused to peptides of bacterial or viral pathogens and are used as tools for vaccine development.