Ultra-fast bright field and fluorescence imaging of the dynamics of micrometer-sized objects

Chen, X.C.; Wang, J.J.; Versluis, Michel; Jong, Nico de; Villanueva, Flordeliza S.

doi:10.1063/1.4809168

Cited by 38 publications

(31 citation statements)

References 29 publications

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“…(B) Synchronous triggering between the ultrasound transducer and ultrafast microscopy system was achieved to collect an ultrafast frame rate bright-field recording of microbubble behavior (∼8-25 μs), a fluorescence video recording of the corresponding PI uptake before, during, and after ultrasound transmission (∼2-3 min), and a set of bright-field and calcein pre-and postultrasound still frames. The UPMC-Cam (26), currently the only imaging system of its kind in North America, is based on a rotating mirror framing camera. A mirror prism, rotated by a gas (helium) turbine, achieves up to 20,000 rotations per second to direct the incoming photons through a bank of relay lenses and beam splitters, projecting two images on a single CCD camera.…”

Section: Resultsmentioning

confidence: 99%

Biophysical insight into mechanisms of sonoporation

Helfield

Chen

Watkins

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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223

177

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This study presents a unique approach to understanding the biophysical mechanisms of ultrasound-triggered cell membrane disruption (i.e., sonoporation). We report direct correlations between ultrasound-stimulated encapsulated microbubble oscillation physics and the resulting cellular membrane permeability by simultaneous microscopy of these two processes over their intrinsic physical timescales (microseconds for microbubble dynamics and seconds to minutes for local macromolecule uptake and cell membrane reorganization). We show that there exists a microbubble oscillation-induced shear-stress threshold, on the order of kilopascals, beyond which endothelial cellular membrane permeability increases. The shear-stress threshold exhibits an inverse squareroot relation to the number of oscillation cycles and an approximately linear dependence on ultrasound frequency from 0.5 to 2 MHz. Further, via real-time 3D confocal microscopy measurements, our data provide evidence that a sonoporation event directly results in the immediate generation of membrane pores through both apical and basal cell membrane layers that reseal along their lateral area (resealing time of ∼<2 min). Finally, we demonstrate the potential for sonoporation to indirectly initiate prolonged, intercellular gaps between adjacent, confluent cells (∼>30-60 min). This real-time microscopic approach has provided insight into both the physical, cavitation-based mechanisms of sonoporation and the biophysical, cell-membrane-based mechanisms by which microbubble acoustic behaviors cause acute and sustained enhancement of cellular and vascular permeability.ultrasound therapy | microbubble contrast agent | endothelial membrane | gene delivery | sonoporation D elivery vehicles for therapeutic nucleic acids (e.g., siRNA, mRNA, plasmids, oligonucleotides), including nanoparticles or viruses, are intended to increase the local effectiveness of the therapeutic within a target tissue while reducing off-target effects. A major barrier to the successful delivery of molecular therapeutics in this manner is the endothelial cell membrane. Viral vectors, although able to efficiently deliver genetic material to target cells via their intracellular trafficking machinery, may elicit specific inflammatory and nonspecific antiviral immune responses (1, 2). As an alternative, nonviral vectors--for example, localized needle injection of naked therapeutic nucleic acids or lipofection--use physical forces or compounds, respectively, to deliver a genetic payload into a cell and are generally less toxic and immunogenic than viral vectors (3). However, direct needle injection into the target poses challenges in achieving homogeneous tissue distribution of the payload (4), and clinical implementation is limited by the impractical requirements of repetitive needle injections into sites which may be difficult to access. Systemically injected liposomes face the endothelial barrier, are vulnerable to intravascular destruction and/or renal excretion (depending on size), and, when endocytosed b...

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Section: Resultsmentioning

confidence: 99%

Biophysical insight into mechanisms of sonoporation

Helfield

Chen

Watkins

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

223

177

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“…Optical methods based on direct measurement of the bubble radius versus time can be used due to availability of ultra-high-speed imaging systems 9,10 such as the Brandaris 128 fast-framing (0.5-25 Mfps) camera. 11 This system enables optical characterization studies by imaging single microbubble dynamical phenomena occurring at multiple time scales.…”

Section: Introductionmentioning

confidence: 99%

Impulse response method for characterization of echogenic liposomes

Raymond

Luan²,

Rooij³

et al. 2015

The Journal of the Acoustical Society of America

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An optical characterization method is presented based on the use of the impulse response to characterize the damping imparted by the shell of an air-filled ultrasound contrast agent (UCA). The interfacial shell viscosity was estimated based on the unforced decaying response of individual echogenic liposomes (ELIP) exposed to a broadband acoustic impulse excitation. Radius versus time response was measured optically based on recordings acquired using an ultra-highspeed camera. The method provided an efficient approach that enabled statistical measurements on 106 individual ELIP. A decrease in shell viscosity, from 2.1 Â 10 À8 to 2.5 Â 10 À9 kg/s, was observed with increasing dilatation rate, from 0.5 Â 10 6 to 1 Â 10 7 s À1 . This nonlinear behavior has been reported in other studies of lipid-shelled UCAs and is consistent with rheological shear-thinning. The measured shell viscosity for the ELIP formulation used in this study [j s ¼ (2.1 6 1.0) Â 10 À8 kg/s] was in quantitative agreement with previously reported values on a population of ELIP and is consistent with other lipid-shelled UCAs. The acoustic response of ELIP therefore is similar to other lipid-shelled UCAs despite loading with air instead of perfluorocarbon gas. The methods described here can provide an accurate estimate of the shell viscosity and damping for individual UCA microbubbles.

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“…1). The tank was filled with deionized water and placed under a 60Â water-coupled objective lens (LUMPLFL, 100X/WI, Olympus), coupled to a 2Â magnifier and to the UPMC-Cam (Chen et al, 2013), an ultrafast frame rate microscopy imaging system capable of recording up to 25 Â 10 6 frames per second (25 Mfps) for 128 frames. Optical-acoustical co-alignment was performed with a pulse-echo approach after which suspensions of microbubbles were diluted in either 1 or 4 cP fluid and injected in an Opticell TM chamber (Thermo Scientific, Waltham, MA) placed on an XY positioning stage.…”

Section: Optical-acoustical Apparatusmentioning

confidence: 99%

Individual lipid encapsulated microbubble radial oscillations: Effects of fluid viscosity

Helfield

Chen

Qin

et al. 2016

The Journal of the Acoustical Society of America

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Ultrasound-stimulated microbubble dynamics have been shown to be dependent on intrinsic bubble properties, including size and shell characteristics. The effect of the surrounding environment on microbubble response, however, has been less investigated. In particular, microbubble optimization studies are generally conducted in water/saline, characterized by a 1 cP viscosity, for application in the vasculature (i.e., 4 cP). In this study, ultra-high speed microscopy was employed to investigate fluid viscosity effects on phospholipid encapsulated microbubble oscillations at 1 MHz, using a single, eight-cycle pulse at peak negative pressures of 100 and 250 kPa. Microbubble oscillations were shown to be affected by fluid viscosity in a size-and pressure-dependent manner. In general, the oscillation amplitudes exhibited by microbubbles between 3 and 6 lm in 1 cP fluid were larger than in 4 cP fluid, reaching a maximum of 1.7-fold at 100 kPa for microbubbles 3.8 lm in diameter and 1.35-fold at 250 kPa for microbubbles 4.8 lm in diameter. Simulation results were in broad agreement at 250 kPa, however generally underestimated the effect of fluid viscosity at 100 kPa. This is the first experimental demonstration documenting the effects of surrounding fluid viscosity on microbubble oscillations, resulting in behavior not entirely predicted by current microbubble models.

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Ultra-fast bright field and fluorescence imaging of the dynamics of micrometer-sized objects

Cited by 38 publications

References 29 publications

Biophysical insight into mechanisms of sonoporation

Biophysical insight into mechanisms of sonoporation

Impulse response method for characterization of echogenic liposomes

Individual lipid encapsulated microbubble radial oscillations: Effects of fluid viscosity

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