The authors declare there is no conflict of interest.

Different optical microscopy methods were combined to obtain information on the various steps in the delivery of nanoparticles from the surface of microbubbles to the surrounding medium. Imaging of the bubble oscillations was performed, as well as imaging of the release of the nanoparticles from the bubble shell, the extravasation, and the penetration through the extracellular matrix of tumors in vivo. In vitro imaging enables screening of many ultrasound parameters compared to the more complex in vivo setups. The benefit of combining this range of imaging modalities is the complementary information that can be obtained at different timescales - a feature that is crucial to characterize and optimize the microbubbles for successful delivery and to obtain therapeutic efficacy. This approach is useful to understand the delivery mechanisms for all microbubbles alike, including constructs with fluorescently-labeled nanoparticles and drugs.
The most critical steps in the microscopy methods used to study single microbubbles are as follows. For fluorescence microscopy, the nanoparticles should be fluorescently-labeled to enable visualization of the particle release. Furthermore, the sample solution should be diluted enough to isolate single microbubbles for analysis in confocal, bright-field, and fluorescence microscopy methods. In addition, it is important to choose an ultrasound driving frequency and acoustic pressure to excite the bubbles most efficiently, namely at their resonance. If the research question concerns delivery of the nanoparticle payload, the appropriate ultrasound parameters should be part of the investigation. Next to resonance, these bubbles should also be driven at or beyond their threshold for nanoparticle release, typically at relatively high acoustic pressure amplitudes (MI &#62; 0.3)51. For bright-field microscopy imaging, it is critical to choose a high-speed camera with a sufficiently high framerate to minimize motion blur and to avoid aliasing.
Bright-field microscopy is mainly limited by the imaging framerate and intensity of light sources available, as a higher framerate would give a more detailed time-resolved insight into the bubble dynamics, but requires more intense illumination due to shorter exposure times. To study particle release in more detail, the framerate for fluorescence imaging can, in principle, be increased by increasing the intensity of the laser light. However, absorption of the high-intensity laser light by the fluorescently-labeled microbubbles generates heat, even with high quantum yield dyes. This heat can interfere with the experiments at stake, and in extreme cases, induce photo-thermal cavitation52. Thus, in practice, there is a limit to the applied laser fluence. However, intense laser illumination can also be deliberately used to induce particle release from liposomes53. Temperature influences bubble dynamics and ultrasound response, depending on bubble type54. Therefore, if in vitro and intravital methods are to be compared objectively, the in vitro methods discussed in the protocol should be performed at 37 &#176;C. Another limitation of the in vitro methods discussed in the current paper is that the bubbles are not in a free-field environment, as microbubbles will float below the sample holder membrane. Furthermore, there is a selection bias when imaging single microbubbles. However, performing repeated experiments on single bubbles allows for the investigation of the effect of size and removal of the confounding factor-the size distribution. If the bubble response as a function of size can be understood while the concentration is not too high to prevent bubble-bubble interactions, the response of any arbitrary bubble population can be calculated.&#160;Finally, both bright-field and fluorescence microscopy methods provide insight into microbubbles convoluted in a two-dimensional (2D) image. If the research question requires more than 2D imaging, the 3D behavior of the bubbles can be resolved by combining the setup described in the protocol with a sideview setup for multiplane imaging55.
An alternative method to study microbubbles is acoustic characterization56. However, measuring the echo of a single microbubble requires locating and isolating a single microbubble within the ultrasound beam56, which poses a challenge typically tackled by the use of a narrow tube or optical or acoustical tweezers57,58. To size bubbles acoustically, the microbubbles can be insonified in the geometrical scattering regime at frequencies much higher than their resonance frequency, which does not induce volumetric microbubble oscillations59. The use of an &#34;acoustical camera&#34; is such a method to image the radial dynamics of single microbubbles in response to ultrasound, wherein a high frequency ultrasound probe is used to determine the radial response of the bubble to a low-frequency driving wave60. The disadvantage of this method is that it can only be used to determine the relative change of the microbubble radius; hence, another method is needed to determine the absolute bubble radius, e.g., through optical imaging61,62. The disadvantage of methods wherein microbubbles are exposed to ultrasound at frequencies higher than their resonance frequency is that at such high frequencies, the penetration depth is decreased59, limiting the usability for in vivo applications. Other forms of microscopy may also be used to study microbubbles such as scanning electron microscopy, atomic force microscopy, and transmission electron microscopy63. The achievable spatio-temporal resolution of these alternative microscopy techniques, however, is generally more limited, and these techniques have the disadvantage that imaging is performed either before or after ultrasound exposure by off-line analysis and typically present a low throughput63. Another alternative is to use a light scattering method, which can be used to study radial dynamics of single microbubbles in real-time, but has a low signal to noise ratio as compared to acoustic scattering methods64.
Real-time intravital microscopy during ultrasound exposure is a powerful method to acquire new insight on the vasculature, behavior of microbubbles, nanoparticles, or other molecules (such as dextran in this case) during ultrasound exposure. A general limitation when performing real-time intravital microscopy is that only a small area of the tissue is imaged, and the penetration depth of the light into tissue is limited. If the imaged vessels contain very few microbubbles and/or nanoparticles within the field of view, little or no information on the nanoparticle behavior and extravasation can be obtained. In addition, because of the limited field of view, a proper alignment between the light and ultrasound paths is crucial. If the ultrasound pressure is high enough to induce bubble destruction, it is also important to choose a pulse repetition frequency that allows fresh bubbles to reperfuse into the field of view between ultrasound pulses. Moreover, as the ultrasound will be reflected from the cover glass in the window chamber and the objective, placing the transducer at an angle is important to reduce reflections as to prevent the formation of standing waves, which distort the calibrated pressure field. Another practical issue is that the setup needs to have sufficient space to mount the ultrasound transducer and waveguide above or below the objective in the microscope setup. The tumors in the dorsal window chamber will have a limited thickness due to the confining chamber and the cover slip; however, if needed, other models could be used. Examples are skinfold tumors, for instance, in the mammary fat pad65 or abdominal intravital imaging of tumors in the various organs66. Such tumors can be grown orthotopically in the appropriate microenvironment, and as such, present a more clinically relevant case.
The methods described in this work enlighten the potential of fluorescently-labeled microbubbles to study the fundamentals of drug delivery applications using bubbles and ultrasound. This combination of microscopy methods provides valuable insight into the microbubble response to ultrasound insonation and its associated acoustic parameter space and presents a clear view of the microbubble and payload behavior over a relevant range of time and length scales.

Ultrasound is the most widely used medical imaging technique. It is non-invasive, fast, safe, cost-effective, and portable1,2,3. However, blood is a poor ultrasound scatterer, and the contrast of the blood pool can be enhanced by an intravenous injection of ultrasound contrast agents3. This enhanced blood-pool contrast enables the quantification of organ perfusion for diagnostic purposes, e.g., in the detection of coronary artery disease4 and metastatic liver disease5. Indeed, tumor vasculature was proven to be an important prognostic factor6. A major research effort is now directed towards microbubble-assisted, targeted molecular imaging and tailoring contrast agents for therapeutic use.
Commercially available ultrasound contrast agents typically consist of a suspension of coated microbubbles7,8 with diameters ranging from 1 &#181;m to 10 &#181;m9. Since ultrasound contrast agent microbubbles are slightly smaller than red blood cells7, the microbubbles can safely reach even the smallest capillaries without creating an occlusion3. Microbubbles have a dramatically increased ultrasound backscattering coefficient compared to tissue10, owing to their compressible gas core11. Furthermore, the microbubble echo is highly nonlinear, i.e., its spectrum contains harmonics and subharmonics of the driving frequency. In addition, the echo strength is strongly dependent on the resonant response of the bubble12. While tissue scatters only linearly, a small number of microbubbles is sufficient to achieve a high detection sensitivity in harmonic imaging13,14. This nonlinear contrast generation can even be strong enough to track single bubbles in the body15.
The shell of the ultrasound contrast agent stabilizes the bubbles against dissolution and coalescence, thereby increasing their circulation time in the blood pool16. The shell can consist of lipids, polymers, or denatured proteins3,8. It decreases the interfacial tension, thereby limiting the effect of Laplace pressure-driven dissolution17 and creates a resistive barrier against gas diffusion18. To further increase stability, the contrast microbubbles are typically filled with a high-molecular weight gas with low solubility in blood11. The microbubble shell dramatically changes the response of the microbubbles to ultrasound insonation11. Uncoated&#160;gas bubbles have a characteristic resonance frequency that is inversely proportional to their size and the addition of a lipid coating increases the resonance frequency with respect to that of an uncoated buble owing to the intrinsic stiffness of the shell3. Furthermore, the shell dissipates energy through dilatational viscosity, which constitutes the dominant source of damping for coated bubbles3. The stabilizing shell has the additional advantage that it can be functionalized, e.g., by binding targeting ligands to the surface of microbubbles. This targeting enables many applications for these bubbles and, in particular, molecular imaging with ultrasound14,19.
Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Microbubbles oscillating in the confinement of a blood vessel can cause microstreaming as well as local normal and shear stresses on the capillary wall3. At high acoustic pressures, large amplitude oscillations may lead to microbubble collapse in a violent process termed inertial cavitation, which, in turn, may lead to rupture or invagination of the blood vessel20. These violent phenomena can induce bioeffects such as sonopermeation21, enhancing the extravasation of therapeutic drugs into the interstitium across the endothelial wall, either paracellularly or transcellularly. It may also improve the penetration of therapeutic agents through the extracellular matrix of stroma-rich tumors21,22 and biofilms23,24, although this mechanism is still poorly understood26.
Ultrasound-mediated drug delivery has shown promising results both preclinically27,28 and in clinical trials22. Moreover, when used with relatively low-frequency ultrasound (~1 MHz), microbubbles have been reported to locally and transiently increase the blood-brain barrier permeability, thereby enabling drugs to enter the brain parenchyma, both in preclinical and clinical studies29,30,31,32,33,34.
There are generally two approaches to ultrasound-mediated drug delivery: the therapeutic material can be co-administered with the bubbles, or it can be attached to or loaded in the bubble shell28,35,36. The second approach has been shown to be more efficient in terms of drug delivery37. Microbubbles can be loaded with drugs or genetic material encapsulated in nanoparticles (liposomes or polymeric nanoconstructs) attached to the shell or incorporated directly in the microbubble shell35,36. Nanoparticle-loaded microbubbles can be activated by (focused) ultrasound to locally release the nanoparticle payload28,33,38,39,40. If such a microbubble is in direct contact with a cell, it has been shown in vitro that the payload can even be deposited onto the cell cytoplasmic membrane in a process called sonoprinting34,35.
The ultrasound parameter space for microbubble insonation is extensive, and the in vivo biological conditions further add complexity. Thus, the combination of focused ultrasound and nanoparticle-loaded microbubbles poses a challenge in the field of targeted therapeutics.
The aim of this work is to provide protocols that can be used to image, in detail, the response of microbubbles as a function of the ultrasound parameters and to study the mechanisms leading to shell rupture and subsequent release of the fluorescently-labeled shell material. This set of protocols is applicable to microbubbles with shells that contain a fluorescent dye. Figure 1 shows a schematic representation of the polymeric-nanoparticle-and-protein-stabilized microbubbles developed at SINTEF (Trondheim, Norway). These bubbles are filled with perfluoropropane gas (C3F8) and the nanoparticles that stabilize the shell contain NR668, which is a lipophilic derivative of Nile Red fluorescent dye38,43. The nanoparticles consist of poly(2-ethyl-butyl cyanoacrylate) (PEBCA) and are PEGylated. Functionalization with polyethylene glycol (PEG) reduces opsonization and phagocytosis by the mononuclear phagocyte system, thereby extending the circulation time14,44. As a result, PEGylation increases the amount of nanoparticles reaching the target site, thereby improving the efficacy of the treatment16.&#160;Figure 2 illustrates how the use of four microscopy methods allows researchers to cover all relevant time and length scales. It should be noted that the spatial resolution achievable in optical microscopy is determined by the diffraction limit, which depends on the wavelength of the light and numerical aperture (NA) of the objective and that of the object illumination source45. For the systems at hand, the optical resolution limit is typically 200 nm. Additionally, intravital microscopy can be used to image on the subcellular level46. For the nanoparticle-and-protein-stabilized microbubbles used in this work, the minimum length scale relevant for intravital microscopy is the size of small capillaries (&#8805;10 &#181;m). In vitro high-speed optical imaging (10 million frames per second) and high-speed fluorescence imaging (500,000 frames per second) experiments are described for single microbubbles. High-speed bright-field imaging at nanosecond timescales is suitable to study the time-resolved radial dynamics of the vibrating bubbles. In contrast, high-speed fluorescence microscopy allows for direct visualization of the release of the fluorescently-labeled nanoparticles. Furthermore, the structure of the microbubble shell can be investigated using Z-stack three-dimensional (3D) confocal microscopy, and scanning electron microscopy (the protocol for the latter is not included in the current work). Intravital microscopy consists in using multiphoton microscopy to image tumors growing in dorsal window chambers to provide real-time information on local blood flow and on the fate of fluorescently-labeled nanoparticles in vivo47. The combination of these microscopy methods ultimately provides detailed insight into the behavior of therapeutic microbubble agents in response to ultrasound, both in vitro and in vivo.

<table><tbody><tr><td>100 MS/s Dual-Channel Arbitrary Waveform Generator model 8026</td><td>Tabor Electronics</td><td></td><td>Arbitrary waveform generator (programmable)</td></tr><tr><td>2100 L</td><td>ENI</td><td></td><td>Amplifier, used in window chamber setup</td></tr><tr><td>2 MDa dextran</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>33522 A</td><td>Agilent Technologies</td><td></td><td>Arbitrary wave form generator, used in window chamber setup</td></tr><tr><td>A1R</td><td>Nikon Instruments</td><td></td><td>Confocal microscope</td></tr><tr><td>ACE I</td><td>SCHOTT</td><td></td><td>Dimmable AC halogen light source</td></tr><tr><td>Atipemazol</td><td>Orion Pharma</td><td></td><td>Antidote to wake animal</td></tr><tr><td>Baytril</td><td>Bayer</td><td></td><td>Enrofloxacin</td></tr><tr><td>BD Neoflon 24 G</td><td>Becton Dickinson &amp;amp; Company</td><td></td><td>Tail vein catheter</td></tr><tr><td>BNC model 575</td><td>Berkely Nucleonics Corporation</td><td></td><td>Pulse/delay generator</td></tr><tr><td>Branson 2510 Ultrasonic Cleaner</td><td>Branson</td><td></td><td>Ultrasonic bath</td></tr><tr><td>Channel slide</td><td>Ibidi</td><td></td><td></td></tr><tr><td>CLINIcell 25</td><td>Laboratoires Mabio International</td><td></td><td>Cell culture casette (volume 10 mL, membrane area 25 cm&lt;sup&gt;2&lt;/sup&gt;, membrane thickness 175 &amp;micro;m)</td></tr><tr><td>Cohlibri</td><td>Lightline</td><td></td><td>Laser (5 W, excitation wavelength 532 nm)</td></tr><tr><td>DP03014 Digital Phosphor Oscilloscope</td><td>Tektronix</td><td></td><td>Oscilloscope</td></tr><tr><td>Fentanyl</td><td>Actavis Group HF</td><td></td><td>Anaesthesia of mouse</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td></td><td>Supplement for cell culture medium</td></tr><tr><td>Fiber-optic hydrophone</td><td>Precision Acoustics</td><td></td><td>Used for alignment</td></tr><tr><td>Flumanezil</td><td>Fresenius Kabi</td><td></td><td>Antidote to wake animal</td></tr><tr><td>Heated animal holder</td><td>Custom design</td><td></td><td>A steel holder where the mouse is positioned on its side in a cavity fitting the size of a mouse, with the window chamber lying flat and immobilized with screws on each side. Below the chamber there is a hole in the holder to secure acoustic contact between the transducer and the skin. The holder is heated to a maximum temperature of 37&amp;deg;C, and the temperature is controlled by feedback from a rectal temperature probe in the mouse. The holder is mounted to an XY positioning stage so the animal can be moved independently to image different areas of the window chamber</td></tr><tr><td>Hyper Vision HPV-X2</td><td>Shimadzu</td><td></td><td>High-speed camera</td></tr><tr><td>ImageJ</td><td>National Institutes of Health&amp;nbsp;and the Laboratory for Optical and Computational Instrumentation, University of Wisconsin</td><td></td><td>open source image processing program</td></tr><tr><td>In vivo SliceScope</td><td>Scientifica</td><td></td><td>Multiphoton microscope</td></tr><tr><td>Isoflurane</td><td>Baxter</td><td></td><td></td></tr><tr><td>ISOTON</td><td>Beckman Coulter</td><td></td><td>Filtered, phosphate-buffered saline solution</td></tr><tr><td>LUMPLFLN60XW</td><td>Olympus</td><td></td><td>Water immersion objective (magnification 60x, working distance 2 mm)</td></tr><tr><td>MaiTai DeepSee</td><td>Spectra-Physics</td><td></td><td>Pulsed laser</td></tr><tr><td>MATLAB</td><td>Mathworks</td><td></td><td>Programming environment</td></tr><tr><td>Medetomidine</td><td>Orion Pharma</td><td></td><td>Anesthesia of mouse</td></tr><tr><td>Midazolam</td><td>Accord Healthcare Limited</td><td></td><td>Anesthesia of mouse</td></tr><tr><td>Milli-Q</td><td>Merck</td><td></td><td>Ultrapure water</td></tr><tr><td>MVS 7010 High Intensity Xenon Strobe</td><td>PerkinElmer</td><td></td><td>Strobe light</td></tr><tr><td>Panametrics-NDT C305</td><td>Olympus</td><td></td><td>Single-element focused immersion transducer (center frequency 2.25 MHz, focal distance 1&quot;, diameter 1&quot;)</td></tr><tr><td>Panametrics-NDT V304</td><td>Olympus</td><td></td><td>Single-element focused immersion transducer (center frequency 2.25 MHz, focal distance 1.88&quot;, diameter 1.25&quot;)</td></tr><tr><td>Penicillin</td><td>Sigma-Aldrich</td><td></td><td>Addition to cell culture medium before implantation of tumor in animals</td></tr><tr><td>Perfluoropropane gas</td><td>F2 Chemicals</td><td></td><td></td></tr><tr><td>Roswell Park Memorial Institute 1640</td><td>Gibco Thermo-Fisher</td><td></td><td>Cell culture medium</td></tr><tr><td>Safe-Lock tube</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Streptomycin</td><td>Sigma-Aldrich</td><td></td><td>Addition to cell culture medium before implantation of tumor in animals</td></tr><tr><td>T 25 basic ULTRA-TURRAX</td><td>IKA laboratory technology</td><td></td><td>Dispersion tool</td></tr><tr><td>TDS 210</td><td>Tektronix</td><td></td><td>Oscilloscope, used in window chamber setup</td></tr><tr><td>Transducer</td><td>Precision Acoustics Ltd</td><td></td><td>Used in window chamber setup</td></tr><tr><td>U-TLU</td><td>Olympus</td><td></td><td>Tube lens</td></tr><tr><td>VBA100-200</td><td>Vectawave</td><td></td><td>Amplifier</td></tr><tr><td>Window chambers</td><td>Custom made</td><td></td><td>Used in window chamber setup</td></tr><tr><td>XLUMPLFLN20 XW</td><td>Olympus</td><td></td><td>20x water dipping objective</td></tr><tr><td>XY(Z) translation stages</td><td>Thorlabs</td><td></td><td></td></tr></tbody></table>

multi-timescale microscopy methods for the characterization of fluorescently-labeled microbubbles for ultrasound-triggered drug release

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

The microbubbles, produced as described in the protocol, were analyzed using various microscopy methods and at various timescales.
The fluorescence of the nanoparticles in confocal microscopy (Figure 6A) indicates that the shell has a non-uniform particle distribution. Other microscopy methods can be used for bubble characterization. For example, Figure 6B shows the overall structure of the microbubble using scanning electron microscopy, as presented in previous work34.
Radial dynamics and phenomenological bubble behavior can be studied using the described in vitro bright-field microscopy method wherein microbubbles were imaged at 10 million frames per second. The radius of single microbubbles was extracted over time using a script written in-house. An example of such a radial response is shown in Figure 7.
An image sequence of typical successful nanoparticle delivery, as described in section 2.3.6, is shown in Figure 8A. The nanoparticles embedded in the microbubble shell can be seen to light up due to fluorescence when the laser light reaches the bubble. Driven by ultrasound insonation, the fluorescent nanoparticles detach from the gas core of the microbubbles and are deposited on the membrane of the sample holder. Finally, the laser is turned off, and the fluorescent nanoparticles are no longer excited. Unsuccessful delivery of the fluorescently-labeled payload of the microbubbles typically looks like the image sequence shown in Figure 8B, where the fluorescent nanoparticles light up on the shell of the microbubble that stays intact during ultrasound exposure.
Real-time intravital multiphoton microscopy during ultrasound was used to investigate the effects of ultrasound and microbubbles on nanoparticle behavior in the blood, enhancement of the permeability of tumor blood vessels, and improvement of the delivery of nanoparticles. The extent and kinetics of penetration into the extracellular matrix as a function of acoustic pressure, frequency, and pulse lengths can be characterized. The effect of the ultrasound treatment may vary with respect to the size and morphology of the vessels and resulting confinement of the bubble. How the ultrasound treatment affects the blood flow and direction can be determined. An example experiment showing the extravasation of nanoparticles over time is shown in Figure 9 at a mechanical index (MI) of 0.826. Results of intravital multiphoton microscopy elucidate the spatial and temporal extravasation of nanoparticles during ultrasound exposure, which is highly beneficial for the complete understanding of the mechanisms underlying ultrasound-mediated delivery of nanoparticles and to optimize such technologies26.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62251/62251fig01.jpg" /> 
Figure 1: Schematic representation of a microbubble with a shell of fluorescently-labeled polymeric nanoparticles in denatured casein. The microbubbles are typically between 1 &#181;m and 10 &#181;m in diameter. The nanoparticles have a diameter mostly between 100 nm and 200 nm38. Abbreviation: C3F8 = perfluoropropane gas. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62251/62251fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62251/62251fig02.jpg" /> 
Figure 2: Schematic overview showing the relevant time and length scales for bright-field, fluorescence, confocal, and intravital microscopy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62251/62251fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62251/62251fig03.jpg" /> 
Figure 3: Schematic representation of bright-field microscopy experiments. (A)&#160;Experimental setup, (B) the timing diagram, and (C) a typical recorded frame. Scale bar in (C) = 10 &#181;m. Abbreviation: fps = frames per second. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62251/62251fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62251/62251fig04.jpg" /> 
Figure 4:&#160;Schematic representation of fluorescence microscopy experiments. (A) Experimental setup, (B) the timing diagram, and (C) a typical recorded frame.&#160;Scale bar in (C) = 10 &#181;m. Abbreviation: fps = frames per second. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62251/62251fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62251/62251fig05.jpg" /> 
Figure 5: Schematic representation of intravital microscopy experiments. (A) Experimental setup, (B) the timing diagram, and (C) a typical recorded frame.&#160;Scale bar in (C) = 50 &#181;m. Green corresponds to dextran-FITC and red to nanoparticles. Abbreviation: GaAsP = gallium arsenide phosphide. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62251/62251fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62251/62251fig06.jpg" /> 
Figure 6: 3D structure of a single nanoparticle-and-protein-stabilized microbubble. (A) Using confocal microscopy to show the nanoparticles, and (B) using a scanning electron microscope to show the 3D structure. (B) has been reproduced with permission from34. Scale bar in (A) = 5 &#181;m; scale bar in (B) = 2 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62251/62251fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62251/62251fig07.jpg" /> 
Figure 7: Typical spherical oscillations of a 2.89 &#181;m radius nanoparticle-and-protein-stabilized microbubble insonified at an ultrasound frequency of 1 MHz and an acoustic pressure amplitude of 142 kPa. (A-D) Images from the high-speed recording and the corresponding bubble radius over time curve (bottom). Scale bars = 5 &#181;m, and the red line indicates the initial radius. The illumination profile (arbitrary units) is indicated by yellow. The magnification is 120x. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62251/62251fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62251/62251fig08.jpg" /> 
Figure 8: Image sequence from high-speed fluorescence microscopy. (A) Successful delivery of fluorescently-labeled nanoparticles of a nanoparticle-and-protein-stabilized microbubble insonified at an ultrasound frequency of 2 MHz and an acoustic pressure amplitude of 600 kPa. (B) Unsuccessful delivery of fluorescently-labeled nanoparticles of a nanoparticle-and-protein-stabilized microbubble insonified at an ultrasound frequency of 2 MHz and an acoustic pressure amplitude of 210 kPa. Scale bars = 10 &#181;m. The magnification is 120x. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62251/62251fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/62251/62251fig09.jpg" /> 
Figure 9: Intravital microscopy after insonation of nanoparticle-and-protein-stabilized microbubbles at an ultrasound frequency of 1 MHz and an acoustic pressure amplitude of 800 kPa. (A) Nanoparticles within the vessel, and (B) an image sequence of the area indicated by the white dashed square in (A) depicting the extravasation of dextran (green) and nanoparticles (red). Scale bars = 50 &#181;m. The magnification is 20x. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62251/62251fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release at JoVE.com

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

The presented protocols can be used to characterize the response of fluorescently-labeled microbubbles designed for ultrasound-triggered drug delivery applications, including their activation mechanisms as well as their bioeffects. This paper covers a range of in vitro and in vivo microscopy techniques performed to capture the relevant length and timescales.

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic ...

multi-timescale-microscopy-methods-for-characterization-fluorescently

Nanyang Technological University

Research

JoVE Journal

Bioengineering

3.8K Views.  University of Twente. Dit protocol kan worden gebruikt om de respons te karakteriseren van fluorescerend gelabelde microbubbels die zijn ontworpen voor echografie-geactiveerde medicijnafgiftetoepassingen die de activeringsmechanismen en bio-effecten omvatten. De sleutel ligt in de combinatie van beeldvormingstechnieken die ons in staat stelt om het multi-schaal probleem van ultrasoon getriggerde medicijnafgifte met bubbels te ontrafelen, zowel op verschillende ruimtelijke schalen als op verschillende tijdschalen. ...

Video: Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

Developmental Biology

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using sonication, mechanical agitation, microfluidic devices, or homogenization. Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver oxygen intravenously during medical emergencies, reversing life-threatening hypoxemia, and preventing subsequent organ injury, cardiac arrest, and death. We present methods for scaled-up production of highly oxygenated microbubbles using a closed-loop high-shear homogenizer. The process can produce 2 L of concentrated LOMs (90% by volume) in 90 min. Resulting bubbles have a mean diameter of ~2 &#956;m, and a rheologic profile consistent with that of blood when diluted to 60 volume %. This technique produces LOMs in high capacity and with high oxygen purity, suggesting that this technique may be useful for translational research labs.

We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using ...

Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely manipulate neural firing, hormonal secretion and genetically-reprogrammed cells. However, the translation of this technology for medical applications is currently hampered by a lack of biophysical mechanisms by which targeted tissues sense and respond to LIPUS. A suitable approach to identify these mechanisms would be to use optical biosensors in combination with LIPUS to determine underlying signaling pathways. However, implementing LIPUS to a fluorescence microscope may introduce undesired mechanical artefacts due to the presence of physical interfaces that reflect, absorb and refract acoustic waves. This article presents a step-by-step procedure to incorporate LIPUS to commercially-available upright epi-fluorescence microscopes while minimizing the influence of physical interfaces along the acoustic path. A simple procedure is described to operate a single-element ultrasound transducer and to bring the focal zone of the transducer into the objective focal point. The use of LIPUS is illustrated with an example of LIPUS-induced calcium transients in cultured human glioblastoma cells measured using calcium imaging.

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) is a modality for non-invasive mechanical stimulation of endogenous or engineered cells with high spatial and temporal resolution. This article describes how to implement LIPUS to an epi-fluorescence microscope and how to minimize acoustic impedance mismatch along the ultrasound path to prevent unwanted mechanical artefacts.

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely ...

Engineering

Induction of Microstreaming by Nonspherical Bubble Oscillations in an Acoustic Levitation System

When located near biological barriers, oscillating microbubbles may increase cell membrane permeability, allowing for drug and gene internalization. Experimental observations suggest that the temporary permeabilization of these barriers may be due to shear stress that is exerted on cell tissues by cavitation microstreaming. Cavitation microstreaming is the generation of vortex flows which arise around oscillating ultrasound microbubbles. To produce such liquid flows, bubble oscillations must deviate from purely spherical oscillations and include either a translational instability or shape modes. Experimental studies of bubble-induced flows and shear stress on nearby surfaces are often restricted in their scope due to the difficulty of capturing shape deformations of microbubbles in a stable and controllable manner. We describe the design of an acoustic levitation chamber for the study of symmetry-controlled nonspherical oscillations. Such control is performed by using a coalescence technique between two approaching bubbles in a sufficiently intense ultrasound field. The control of nonspherical oscillations opens the way to a controlled cavitation microstreaming of a free surface-oscillating microbubble. High-frame rate cameras allow investigating quasi-simultaneously the nonspherical bubble dynamics at the acoustic timescale and the liquid flow at a lower timescale. It is shown that a large variety of fluid patterns may be obtained and that they are correlated to the modal content of the bubble interface. We demonstrate that even the high-order shape modes can create large-distance fluid patterns if the interface dynamics contain several modes, highlighting the potential of nonspherical oscillations for targeted and localized drug delivery.

A fast and reliable technique is proposed to control the shape oscillations of a single, trapped acoustic bubble that is based on coalescence technique between two bubbles. The steady-state, symmetry-controlled bubble shape oscillations allow analysis of the fluid flow generated in the vicinity of the bubble interface.

When located near biological barriers, oscillating microbubbles may increase cell membrane permeability, allowing for drug and gene internalization. ...

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

Fabricating and Labeling Microbubbles with Fluorescent and Radioactive Tracers

Microbubbles are lipid-shelled, gas-filled particles that have evolved from vascular ultrasound contrast agents into revolutionary cancer therapy platforms. When combined with therapeutic focused ultrasound (FUS), they can safely and locally overcome physiological barriers (e.g., blood-brain barrier), deliver drugs to otherwise inaccessible cancers (e.g., glioblastoma and pancreatic cancer), and treat neurodegenerative diseases. The therapeutic arsenal of microbubble-FUS is advancing in new directions, including synergistic combination radiotherapy, multimodal imaging, and all-in-one drug loading and delivery from microbubble shells.
Labeling microbubbles with radiotracers is key to establishing these expanded theranostic capabilities. However, existing microbubble radiolabeling strategies rely on purification methodologies known to perturb microbubble physicochemical properties, use short-lived radioisotopes, and do not always yield stable chelation. Collectively, this creates ambiguity surrounding the accuracy of microbubble radioimaging and the efficiency of tumor radioisotope delivery.
This protocol describes a new one-pot, purification-free microbubble labeling methodology that preserves microbubble physicochemical properties while achieving &#62;95% radioisotope chelation efficiency. It is versatile and can be applied successfully across custom and commercial microbubble formulations with differing acyl lipid chain length, charge, and chelator/probe (porphyrin, DTPA, DiI) composition. It can be adaptively applied during ground-up microbubble fabrication and to pre-made microbubble formulations with modular customizability of fluorescence and multimodal fluorescence/radioactive properties. Accordingly, this flexible method enables the production of tailored, traceable (radio, fluorescent, or radio/fluorescent active) multimodal microbubbles that are useful for advancing mechanistic, imaging, and therapeutic microbubble-FUS applications.

This protocol outlines the fabrication of lipid microbubbles and a compatible one-pot microbubble radiolabeling method with purification-free &gt;95% labeling efficiency that conserves microbubble physicochemical properties. This method is effective across diverse lipid microbubble formulations and can be tailored to generate radioactive and/or fluorescent microbubbles.

Microbubbles are lipid-shelled, gas-filled particles that have evolved from vascular ultrasound contrast agents into revolutionary cancer therapy ...

Cancer Research

Imaging and Quantification of the Area of Fast-Moving Microbubbles Using a High-Speed Camera and Image Analysis

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental technique and image analysis protocol presented here can also be applied for imaging microscopic bubbles in other fields of research; therefore, it has a wide range of applications. We apply this to image cavitation around dental ultrasonic scalers. It is important to image cavitation to characterize it and to understand how it can be exploited for various applications. Cavitation occurring around dental ultrasonic scalers can be used as a novel method of dental plaque removal, which would be more effective and cause less damage than current periodontal therapy techniques. We present a method for imaging the cavitation bubble clouds occurring around dental ultrasonic scaler tips using a high-speed camera and a zoom lens. We also calculate the area of cavitation using machine learning image analysis. Open source software is used for image analysis. The image analysis presented is easy to replicate, does not require programming experience, and can be modified easily to suit the application of the user.

Cavitation microbubbles are imaged using a high-speed camera attached to a zoom lens. The experimental setup is explained, and image analysis is used to calculate the area of the cavitation. Image analysis is done using ImageJ.

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental ...

Studying Cavitation Enhanced Therapy

Interest in the therapeutic applications of ultrasound is significant and growing, with potential clinical targets ranging from cancer to Alzheimer&#8217;s disease. Cavitation - the formation and subsequent motion of bubbles within an ultrasound field - represents a key phenomenon underpinning many of these treatments. There remains, however, considerable uncertainty regarding the detailed mechanisms of action by which cavitation promotes therapeutic effects and there is a need to develop reliable monitoring techniques that can be implemented clinically. In particular, there is significant variation between studies in the exposure parameters reported as successfully delivering therapeutic effects and the corresponding acoustic emissions. The aim of this paper is to provide design guidelines and an experimental protocol using widely available components for performing studies of cavitation-mediated bioeffects, and&#160;include real-time acoustic monitoring. It is hoped that the protocol will enable more widespread incorporation of acoustic monitoring into therapeutic ultrasound experiments and facilitate easier comparison across studies of exposure conditions and their correlation to relevant bio-effects.

The presented experimental protocol can be used to perform real time measurements of cavitation activity in a cell culture device with the aim of enabling investigation of the conditions required for successful drug delivery and/or other bioeffects.

Interest in the therapeutic applications of ultrasound is significant and growing, with potential clinical targets ranging from cancer to ...

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain.
Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.

This protocol describes the surgical and technical procedures that enable real-time in vivo multiphoton fluorescence imaging of the rodent brain during focused ultrasound and microbubble treatments to increase blood-brain barrier permeability.

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...

Preparation and Characterization of Targeted Microbubbles

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of microbubble preparation and targeting ligand attachment are cumbersome, complicated, and lengthy. Therefore, there is a need to simplify the targeted microbubble preparation procedure to bring it closer to clinical translation. The purpose of this publication is to provide a detailed description and explanation of the steps necessary for targeted microbubble preparation, functional characterization and testing. A sequence of the optimized and simplified procedures is presented for two systems: a biotin-streptavidin targeting pair model, and a cyclic RGD peptide targeting the recombinant &#945;v&#946;3 protein, which is overexpressed on the endothelial lining of the tumor neovasculature.
Here, we show the following: covalent coupling of the targeting ligand to a lipid anchor, assessment of the reagent quality, and tests that confirm the successful completion of the reaction; preparation of the aqueous precursor medium containing microbubble shell components, followed by microbubble preparation via amalgamation; assessment of the efficacy of lipid transfer onto the microbubble stabilizer shell; adjustment of microbubble size distribution by flotation at normal gravity to remove larger microbubbles that might be detrimental for in vivo use; assessment of microbubble size distribution by electrozone sensing; evaluation of targeted binding of the microbubbles to receptor-coated surface in a static binding assay test (in an inverted dish); and evaluation of targeted binding of the microbubbles to receptor-coated surface in a parallel plate flow chamber test.

The goal of this protocol is to prepare, purify and characterize gas-filled microbubbles (targeted contrast agents for ultrasound molecular imaging). Two targeting systems are described: biotinylated bubbles adherent to streptavidin, and cyclic RGD peptide microbubbles that bind to &#945;v&#946;3, a known tumor neovasculature biomarker.

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

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