Acoustic Behavior of Halobacterium salinarum Gas Vesicles in the High-Frequency Range: Experiments and Modeling

Abstract

Gas vesicles are a new and unique class of biologically derived ultrasound contrast agents with sub-micron size whose acoustic properties have not been fully elucidated. In this study, we investigated the acoustic collapse pressure and behavior of Halobacterium salinarum gas vesicles at transmit center frequencies ranging from 12.5 to 27.5 MHz. The acoustic collapse pressure was found to be above 550 kPa at all frequencies, 9 fold higher than the critical pressure observed in hydrostatic conditions. We show that gas vesicles behave non-linearly when exposed to ultrasound at incident pressure ranging from 160 kPa to the collapse pressure, and generate second harmonic amplitudes of −2 to −6 dB below the fundamental in media with viscosities ranging from 0.89 to 8 mPa.s. Simulations performed using a Rayleigh-Plesset type model accounting for buckling, and a dynamic finite element analysis, suggest that buckling is the mechanism behind the generation of harmonics. We found good agreement between the level of second harmonic relative to the fundamental measured at 20 MHz and the Rayleigh-Plesset model predictions. Finite element simulations extended these findings to a non-spherical geometry, confirmed that the acoustic buckling pressure corresponds to the critical pressure in hydrostatic conditions, and support the hypothesis of limited gas flow across the GV shell during the compression phase in the frequency range investigated. From simulations, estimates of GV bandwidth-limited scattering indicate that a single GV has a scattering cross-section comparable to that of a red blood cell. These findings will inform the development of GV-based contrast agents and pulse sequences to optimize their detection with ultrasound.

Preparation of biogenic gas vesicle nanostructures for use as contrast agents for ultrasound and MRI

“…Furthermore, we found that GVs’ unique physical properties enable them to produce harmonic ultrasound signals useful for contrast specificity in vitro and in vivo 9 , 10 . We also showed that GVs from different species can be imaged in multiplex based on their differential responses to acoustic pressure, and that conditional GV clustering leads to contrast enhancement, allowing them to be used as molecular sensors 8 .…”

“…The ultrasound contrast from Halo GVs and their utility for nonlinear imaging in vitro and in vivo has been demonstrated in our previous work 8 . The acoustic behavior of Halo GVs at ultrasound frequencies of 12.5–27.5 MHz has also been investigated through modeling and experiments, suggesting acoustic buckling as the mechanism underlying generation of non-linear signals 10 . In parallel, we recently developed an amplitude modulation scheme taking advantage of the nonlinear pressure dependence of backscattered signals in engineered Ana GVs, allowing selective imaging of these nanostructures 9 .…”

“…Although this protocol presents methods for producing three different types of GVs – Ana, Halo and Mega – one of these types may be most appropriate for a given application (see Table 1). For ultrasound, unmodified Halo GVs can be used directly in ultrasound imaging to obtain non-linear signals 8 , 10 . Ana GVs are the system of choice if one wishes to genetically tune the properties of GVs for multiplexing, multimodal imaging and targeting applications 9 , 14 .…”

Proteins, air and water: reporter genes for ultrasound and magnetic resonance imaging

“…Indeed, this ability was demonstrated using GVs isolated from cyanobacteria and haloarchaea [6]. Building on this initial discovery, several other studies have been undertaken to understand the acoustic properties of GVs [9], to engineer them through genetic and biochemical modifications [10,11] (*ref. 10), to devise ultrasound imaging techniques tailored to distinguish their signal from background [12], and to characterize their in vivo biodistribution as purified, injectable agents [13].…”

Introductory Chapter: A Brief Overview of Archaeal Applications

“…Potential applications of Archaea were subdivided into four ields (commercial enzymes and/or molecules (stars), environment (circles), food (triangles) and health (squares)) based on the reference(s) listed following each species. Thirty eight (n=38) archaeal species were integrated into the above phylogenetic tree (one DPANN species (white color), 21 Euryarchaeota species (dark grey color), 16 TACK species (light grey color)): Acidianus hospitalis W1 (NC_015518) [34,35], Acidilobus saccharovorans 345-15 (NC_014374) [36,37], Aeropyrum camini JCM 12091 (NC_121692) [38], Aeropyrum pernix K1 (NC_000854) [39], Archaeoglobus fulgidus DSM 4304 (NC_000917) [40,41], Caldivirga maquilingensis IC-167 (NC_009954) [42], Desulfurococcus fermentans DSM 16532 (NC_018001) [43], Desulfurococcus mobilis DSM 2161 (NC_014961) [44], Ferroglobus placidus DSM 10642 (NC_013849) [45], Fervidicoccus fontis Kam940 (NC_017461) [46], Halobacterium salinarum R1 (NC_010364) [47], Halobacterium sp. NRC-1 (NC_002607) [48], Haloferax mediterranei ATCC 33500 (NC_017941) [49], Halogeometricum borinquense DSM 11551 (NC_014729) [50], Halorhabdus utahensis JCM 11049 (NC_013158) [51], Halostagnicola larsenii XH-48 (NZ_CP007055) [52], Haloterrigena turkmenica DSM 5511 (NC_013743) [53], Metallosphaera cuprina Ar-4 (NC_015435) [54], Metallosphaera sedula DSM 5348 (NC_009440) [55], Methanocaldococcus jannaschii DSM 2661 (NC_000909) [27,56], Methanotorris igneus DSM 5666 (NC_015562) [57], Natrialba magadii ATCC 43099 (NC_013922) [58], Nanoarchaeum equitans Kin4-M (NC_005213) [59,60], Pyrobaculum aerophilum IM2 (NC_041958) [61,62], Pyrobaculum calidifontis JCM 11548 (NC_009073) [63], Pyrobaculum sp.…”