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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#38; 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 cm2, membrane thickness 175 &#181;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&#176;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&#160;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&#34;, diameter 1&#34;)</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&#34;, diameter 1.25&#34;)</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/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/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/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/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/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/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/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/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/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 ...

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Research

JoVE Journal

Bioengineering

3.7K Views.  University of Twente. 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.

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

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Here we describe a protocol for the automated segmentation of fluorescently labeled tissues on slides using a widefield high-content analysis system (WHCAS). This protocol has wide-ranging applications in any field which involves the quantitation of fluorescent markers in biological tissues including the biological sciences, medical engineering, and health sciences.

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Production of Membrane-Filtered Phase-Shift Decafluorobutane Nanodroplets from Preformed Microbubbles

There are many methods that can be used for the production of vaporizable phase-shift droplets for imaging and therapy. Each method utilizes different techniques and varies in price, materials, and purpose. Many of these fabrication methods result in polydisperse populations with non-uniform activation thresholds. Additionally, controlling the droplet sizes typically requires stable perfluorocarbon liquids with high activation thresholds that are not practical in vivo. Producing uniform droplet sizes using low-boiling point gases would be beneficial for in vivo imaging and therapy experiments. This article describes a simple and economical method for the formation of size-filtered lipid-stabilized phase-shift nanodroplets with low-boiling point decafluorobutane (DFB). A common method of generating lipid microbubbles is described, in addition to a novel method of condensing them with high-pressure extrusion in a single step. This method is designed to save time, maximize efficiency, and generate larger volumes of microbubble and nanodroplet solutions for a wide variety of applications using common laboratory equipment found in many biological laboratories.

This protocol describes a method of generating large volumes of lipid encapsulated decafluorobutane microbubbles using probe-tip sonication and subsequently condensing them into phase-shift nanodroplets using high-pressure extrusion and mechanical filtration.

There are many methods that can be used for the production of vaporizable phase-shift droplets for imaging and therapy. Each method utilizes different ...