Name: imaging and quantification of the area of fast-moving microbubbles using a high-speed camera and image analysis
Uploaded: 2020-09-05T13:00:00.000+00:00
Duration: 5 min 31 s
Description: Watch this Scientific Journal Video about Imaging and Quantification of the Area of Fast-Moving Microbubbles Using a High-Speed Camera and Image Analysis at JoVE.com

The authors are grateful for funding from the Engineering and Physical Sciences Research Council EP/P015743/1.

1. Instrument setup
<ol>
	<li>Select the instrument or object to be imaged. In this experiment an ultrasonic scaler was imaged. Cavitation bubbles occur around the tips of ultrasonic scalers in water.</li>
	<li>Select a micro positioning stage for the instrument to be imaged with XYZ translation and rotation. Place on a laboratory jack. Attach the instrument handle to the micro positioning stage</li>
	<li>Select an optically transparent water container for imaging. The container used in these experiments was created with glass microscope slides.</li>
	<li>Select an XY stage with a rotation platform. Place on a laboratory jack. Place the water container on the stage and fill with filtered water (reverse osmosis or distilled).</li>
</ol>
2. High-speed camera setup
<ol>
	<li>Select a high-speed camera with the desired frame rate and resolution and a high intensity light source with a fibre light guide.</li>
	<li>Attach a micropositioning sliding plate to the high-speed camera body and connect it to a tripod stand.</li>
	<li>Select a lens with the desired resolution and focal length and attach this to the camera. For this experiment a zoom lens was used at a resolution of 8.4 &#181;m/pixel.</li>
	<li>Fill the imaging tank with water and position the tip of the instrument to be imaged in the water tank in the desired orientation.</li>
	<li>After connecting the camera and loading the live view in the software, use low magnification to focus on the tip of the ultrasonic scaler, repositioning the light source if necessary. Position the instrument and the light source in front of the camera and focus. Adjust to the desired frame rate and brightness. 
	NOTE: A higher light intensity is required for imaging at high frames rates, short shutter speeds and/or high magnifications. Illumination can be provided in reflection mode or transmission mode. In this protocol the illumination is provided in transmission mode (bright field) using a high intensity cold illumination device.</li>
	<li>Set an optimal frame rate and shutter speed for the high-speed camera. In this experiment the frame rate was 6400 fps with a shutter speed of 262 nanoseconds. A short shutter speed is required for fast moving bubbles such as cavitation bubbles to ensure that they are in focus.</li>
	<li>Adjust the magnification of the zoom lens and the intensity of the light source so the background is white without being overexposed.</li>
</ol>
3. Calibration
<ol>
	<li>Record the position of the tip (rotation in x-y stage, rotation angle of instrument for reproducibility).</li>
	<li>To ensure the field of view is consistent for each repeat, choose a reference point and note down the coordinates. In this case the reference point was the tip of the ultrasonic scaler. It can then be repositioned in future experiments in the same place within the field of view.</li>
	<li>If the pixel size is unknown, image a graticule with 10 &#181;m markings at the set magnification and use image analysis software such as Fiji to calculate the resolution.</li>
</ol>
4. High-speed video recording
<ol>
	<li>Image the instrument without cavitation. This will be subtracted from the cavitation images in image analysis when calculating the area of the cavitation bubbles. Save the videos in a format such as TIFF so no image quality is lost.</li>
	<li>Image the instrument operating with cavitation. Ensure there are sufficient frames for accurate analysis, for example 5 repeats with 500 frames each.</li>
</ol>
5. Image processing
<ol>
	<li>Download Fiji19 from the ImageJ website (https://imagej.net/Fiji). An ImageJ macro code has been provided which automatically does the image analysis steps described below and can also be changed to suit the application. The individual steps of the macro are described in steps 5.3-5.5.</li>
	<li>Crop the image to remove any darker areas resulting from uneven illumination, if needed. Ensure that all images are cropped to the same size and at the identical point in the image.</li>
	<li>Convert the images to binary by automatically thresholding using one of the auto thresholds. In this example the minimum auto threshold is used.</li>
	<li>Run the fill holes command to remove any black pixels from inside the bubbles which were falsely segmented.</li>
	<li>Calculate the histogram of the stack to show the number of pixels corresponding to the scaler and the cavitation in each frame.</li>
	<li>In this case the pixels corresponding to the bubbles are white and have value 255. Save these measurements.</li>
	<li>Repeat steps 5.3-5.6 for the video of the instrument operating without the bubbles.</li>
	<li>Calculate the mean area of the ultrasonic scaler tip only from the results of the histogram.</li>
	<li>Subtract the mean area of the instrument from each of the areas calculated from the videos of the bubbles around the scaler. The area of the bubbles is left to measure.</li>
	<li>Visualize by subtracting the binary image of the scaler from the binary image of the scaler with bubbles using the image calculator in Fiji.</li>
	<li>Calculate the mean and standard deviation of the area of the bubbles.</li>
	<li>Convert the values from number of pixels to area (in this case &#181;m2) by multiplying by the pixel size squared. Calculate the size of each pixel by imaging a graticule with the high-speed camera at the same magnification as was used for imaging and use ImageJ to set the scale.</li>
	<li>Plot the data. It is also possible to conduct statistical analysis to show any significant difference in the area of bubbles if comparing different conditions.</li>
</ol>
6. ImageJ macro
<ol>
	<li>In the ImageJ/Fiji menu, go to Plugins &#62; New &#62; Macro. Ensure IJ1 Macro is checked under the language menu and copy and paste the following code. Click run to execute the macro (Supplementary File).</li>
</ol>

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The authors have nothing to disclose.

The technique described in this paper enables imaging of fast-moving microbubbles with high spatial and temporal resolution. It can potentially benefit a wide range of scientific disciplines such as chemical engineering, dentistry and medicine. Engineering applications include imaging cavitation bubbles for cleaning surfaces, or for imaging bubbles in fluidized bed reactors. Biomedical applications include imaging cavitation around medical and dental instruments and imaging biofilm debridement from hard and soft tissue using cavitation bubbles. In this study we demonstrated the technique by imaging cavitation around two different dental ultrasonic scaler tips. The amount of cavitation varies between the two tips tested in this study, with more cavitation clouds observed around the free end of tip 10P. This has previously been linked to vibration amplitude20. The high-speed videos show that the FSI 1000 tip has less vibration, which is likely to be why there is less cavitation around this tip.
One limitation of the image analysis method is that the image subtraction technique to remove the area of the scaler is not completely accurate because the scaler is oscillating and therefore the subtraction may leave some areas of the scaler falsely segmented as bubbles. However, this has been accounted for by averaging the area from a large number of frames (n=2000). This would not be a problem for applications where the object to be subtracted is stationary. For studies where the moving object to be subtracted has a much higher variance, we recommend synchronizing the movements in both videos before subtracting for accurate results. In the current study, we did not synchronize the oscillations but since the vibration was low, we can assume that the oscillations correspond well to each other in these two measurements.
The image thresholding is accurate because the brightfield illumination provides a uniform background with good contrast. It is critical to ensure that the background is uniform and does not contain any other objects which could be falsely segmented. The thresholding method can be modified by using other automatic thresholds to suit the application. Manual thresholding, where the user sets the threshold value, is also possible but is not recommended as it reduces the reproducibility of the results, since different users will select different threshold values.
Image analysis has been used for many other bubble imaging studies. These also use a similar method of backlighting to get optimum contrast between the bubbles and the background, and thresholding to segment the bubbles21,22,23,24. The method shown in the current study can also be generalized to be used for many different bubble imaging applications, which are not limited to only high-speed imaging. High-speed imaging has been used for cavitation bubbles generated in water and also around instruments such as endodontic files and ultrasonic scalers12,25,26,27,28. For example Rivas et al. and Macedo et al. used a high-speed camera attached to a microscope, with illumination provided by a cold light source to image cleaning with cavitation, and to image cavitation around an endodontic file17,29. Bright field illumination provides more contrast between the background and the bubbles, making it possible to use simple segmentation techniques such as thresholding, as demonstrated by Rivas et al. for imaging and quantifying cavitation erosion and cleaning over time29. Dark field illumination makes thresholding more difficult due to the higher variation in grey scales4,30. Image analysis has been used in other studies to gather more information about bubbles1,2. Vyas et al. used a machine learning approach to segment cavitation bubbles around an ultrasonic scaler20. The method described in the current paper is quicker because it uses simple thresholding so it is less computationally intensive, and bubbles occurring above and below the scaler can be analysed. However, the thresholding method used in the current paper is only accurate if the background is uniform. If it is not possible to obtain a uniform background during imaging, other image processing techniques can be used such as the use of background subtraction using a rolling ball radius to correct for uneven illumination, filtering using median or Gaussian filters to remove noise, or also using machine learning based techniques20,31.
In conclusion, we present a high-speed imaging and analysis protocol to image and calculate the area of a microscopic moving object. We have demonstrated this method by imaging cavitation bubbles around an ultrasonic scaler. It can be used for imaging cavitation around other dental instruments such as endodontic files and it can be easily adapted for other non-dental bubble imaging applications.

Imaging the motion of bubbles is important for various applications because it controls the hydrodynamics of a system. There are many applications where this can be useful: in fluidized bed reactors1,2, or for cleaning with cavitation bubbles3,4. The purpose of imaging bubbles is to understand more about the bubble dynamics or about the direction and motion of a cloud of bubbles. This can be done through observing structures imaged and also by using image analysis to obtain quantitative information, such as the size of the bubbles.
Cavitation bubbles are gas or vapor entities that occur in a fluid when the pressure drops below the saturated pressure value5. They can occur when an acoustic field is applied to a fluid at ultrasonic frequencies. They repeatedly grow and collapse, and upon collapse can release energy in the form of high-speed micro-jets and shockwaves6,7. These can dislodge particles on a surface through shear forces and cause surface cleaning8. Cavitation bubbles are being investigated for surface cleaning in different industries, such as for semiconductors, food, and wound cleaning9,10,11,12. They could also be used to clean dental plaque from teeth and biomaterials such as dental implants12,13. Cavitation occurs around currently used dental instruments such as ultrasonic scalers and endodontic files and shows potential as an additional cleaning process with these instruments14.
The oscillation of cavitation bubbles occurs over a few microseconds and therefore a high-speed camera is required to capture their motion by imaging at thousands of frames per second8. We demonstrate a method of imaging microbubble cavitation around dental ultrasonic scalers. The aim is to understand how cavitation varies around different ultrasonic scalers, so it can be optimised as a novel way to clean dental plaque.
Previous methods used to investigate the cavitation include sonochemiluminesence, which uses luminol to detect where cavitation has occurred15,16. However, this is an indirect technique and it is not able to visualize the cavitation bubbles in real time. Therefore, it is not able to accurately determine exactly where it happens on the instrument, and no information can be gained on the bubble dynamics, unless it is combined with other imaging techniques17. High-speed imaging can image not only the cavitation bubbles growing and collapsing but also the type of cavitation occurring: cavitation clouds, microstreamers and micro-jets6,7,18. These give more information about how the cavitation can clean surfaces.
We present a method of imaging cavitation microbubbles using a high-speed camera and calculating the mean area of cavitation occurring. This method is demonstrated using an example of cavitation occurring around different dental ultrasonic scaler tips, although the experimental and image analysis steps can be used for other applications, such as for imaging other macro and microbubbles.

<table><tbody><tr><td>0.25x attachment</td><td>Navitar</td><td>1-50011</td><td></td></tr><tr><td>12x with 12mm fine focus&lt;br/&gt; Long distance microscope zoom lens</td><td>Navitar</td><td>1-50486</td><td></td></tr><tr><td>2x adaptor with f mount</td><td>Navitar</td><td>1-62922</td><td></td></tr><tr><td>Cavitron Plus Ultrasonic Scaler</td><td>Dentsply Sirona</td><td>8184003</td><td></td></tr><tr><td>Cavitron Ultrasonic Insert FSI 1000FSI 1000</td><td>Dentsply Sirona</td><td>UCAFTHD</td><td></td></tr><tr><td>Fibre light guide. 8mm fibre bundle 1500mm length. Focussing lens assembly for Hayashi light, 1/4&quot;-20 tripod&lt;br/&gt; thread for mounting.</td><td>Hayashi</td><td>LGC1-&lt;br/&gt; 8L1500</td><td></td></tr><tr><td>Geared head</td><td>Manfrotto</td><td>MN405</td><td>7.5kg load capacity</td></tr><tr><td>HDF7010 High-Power LED Endoscope light&lt;br/&gt; source. 150W LED provides cold output equivalent to 250W&lt;br/&gt; Xenon.</td><td>Hayashi</td><td>LA-HDF710</td><td></td></tr><tr><td>Heavy weight Tripod</td><td>Manfrotto</td><td>MN475B</td><td>Geared centre column, 12kg load capacity</td></tr><tr><td>High Speed Camera</td><td>Photron</td><td>103526</td><td>FASTCAM Mini AX200 900K M3 (16GB memory)</td></tr><tr><td>High-Precision Rotation&amp;nbsp;Stage</td><td>Thorlabs</td><td>PR01/M</td><td></td></tr><tr><td>Laboratory jacks</td><td>Camlab</td><td>1194083</td><td></td></tr><tr><td>Micropositioning sliding plate</td><td>Manfrotto</td><td>SKU 454</td><td></td></tr><tr><td>Micropositioning stage 3D</td><td>Thorlabs</td><td>PT3/M</td><td></td></tr><tr><td>Micropositioning stage rotation</td><td>Thorlabs</td><td>OCT-XYR1/M</td><td>OCT-XYR1/M - XY Stage with Solid Top Plate</td></tr><tr><td>NEWTRON P5 XS Ultrasonic Scaler&amp;nbsp;</td><td>Acteon</td><td>F62118</td><td></td></tr><tr><td>Ultrasonic Insert 10P</td><td>Acteon</td><td>F00253</td><td></td></tr></tbody></table>

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.

The image analysis steps can be seen in Figure 1 for one of the ultrasonic scaler tips tested. A FSI 1000 tip and a 10P tip were imaged inside a water tank with the cooling water turned off (Figure 2). Cavitation occurred near the bend of tip FSI 1000 at maximum power, and near the free end in tip 10P (Figure 3 and Figure 4). The mean area of cavitation was 0.1 &#177; 0.07 mm2 for the FSI 1000 tip and 0.50 &#177; 0.25 mm2 for the 10P tip (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61509/61509fig1.jpg" /> 
Figure 1:&#160;High-speed imaging setup and image analysis steps (a) Schematic of the high-speed imaging setup used in the study. (b) Schematic of the image analysis steps used in the study, showing the raw images on the left of the scaler tip only and with cavitation, which were then binarized and subtracted from each other to calculate the area of the cavitation clouds. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61509/61509fig1large.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/61509/61509fig2.jpg" /> 
Figure 2: Comparison between different tips High-speed image stills showing cavitation occurring around the two ultrasonic scaler tips tested (a) FSI 1000 (b) 10P. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61509/61509fig2large.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/61509/61509fig3.jpg" /> 
Figure 3: Tip 10P high-speed images: High-speed image stills of tip 10P, from a video taken at 6400 frames per second. Cavitation can be seen around the free end of the tip. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61509/61509fig3large.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/61509/61509fig4.jpg" /> 
Figure 4: Tip FSI1000 high-speed images: High-speed image stills of tip FSI 1000, from a video taken at 6400 frames per second. Cavitation can be seen around the middle of the tip. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61509/61509fig4large.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/61509/61509fig5.jpg" /> 
Figure 5: Cavitation area image analysis results. The mean area of cavitation occurring around the FSI 1000 and 10P ultrasonic scaler tips calculated using the image analysis technique described. The error bars represent the standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61509/61509fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary&#160;File. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61509/Jove_macro.ijm" target="_blank">Please click here to download this file.</a>

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

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 ...

imaging-quantification-area-fast-moving-microbubbles-using-high-speed

Nanyang Technological University

Research

JoVE Journal

Bioengineering

5.8K Views.  University of Birmingham. High-speed imaging kan worden gebruikt om beeld snelle processen, zoals cavitatie bubble dynamics. We demonstreren een methode van beeldvorming cavitatie bellen rond een tandheelkundige ultrasone scaler tip. Cavitatie rond ultrasone scalers wordt onderzocht voor het reinigen van tandplak, maar de hier getoonde methode kan worden gebruikt voor veel verschillende toepassingen.We hebben ook een beeldanalyseprotocol ontwikkeld met behulp van open source-software waarmee de gebruiker het ge...

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

Determining 3D Flow Fields via Multi-camera Light Field Imaging

In the field of fluid mechanics, the resolution of computational schemes has outpaced experimental methods and widened the gap between predicted and observed phenomena in fluid flows. Thus, a need exists for an accessible method capable of resolving three-dimensional (3D) data sets for a range of problems. We present a novel technique for performing quantitative 3D imaging of many types of flow fields. The 3D technique enables investigation of complicated velocity fields and bubbly flows. Measurements of these types present a variety of challenges to the instrument. For instance, optically dense bubbly multiphase flows cannot be readily imaged by traditional, non-invasive flow measurement techniques due to the bubbles occluding optical access to the interior regions of the volume of interest. By using Light Field Imaging we are able to reparameterize images captured by an array of cameras to reconstruct a 3D volumetric map for every time instance, despite partial occlusions in the volume. The technique makes use of an algorithm known as synthetic aperture (SA) refocusing, whereby a 3D focal stack is generated by combining images from several cameras post-capture 1. Light Field Imaging allows for the capture of angular as well as spatial information about the light rays, and hence enables 3D scene reconstruction. Quantitative information can then be extracted from the 3D reconstructions using a variety of processing algorithms. In particular, we have developed measurement methods based on Light Field Imaging for performing 3D particle image velocimetry (PIV), extracting bubbles in a 3D field and tracking the boundary of a flickering flame. We present the fundamentals of the Light Field Imaging methodology in the context of our setup for performing 3DPIV of the airflow passing over a set of synthetic vocal folds, and show representative results from application of the technique to a bubble-entraining plunging jet.

A technique for performing quantitative three-dimensional (3D) imaging for a range of fluid flows is presented. Using concepts from the area of Light Field Imaging, we reconstruct 3D volumes from arrays of images. Our 3D results span a broad range including velocity fields and multi-phase bubble size distributions.

In the field of fluid mechanics, the resolution of computational schemes has outpaced experimental methods and widened the gap between predicted and ...

Engineering

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

Fast Imaging Technique to Study Drop Impact Dynamics of Non-Newtonian Fluids

In the field of fluid mechanics, many dynamical processes not only occur over a very short time interval but also require high spatial resolution for detailed observation, scenarios that make it challenging to observe with conventional imaging systems. One of these is the drop impact of liquids, which usually happens within one tenth of millisecond. To tackle this challenge, a fast imaging technique is introduced that combines a high-speed camera (capable of up to one million frames per second) with a macro lens with long working distance to bring the spatial resolution of the image down to 10 &#181;m/pixel. The imaging technique enables precise measurement of relevant fluid dynamic quantities, such as the flow field, the spreading distance and the splashing speed, from analysis of the recorded video. To demonstrate the capabilities of this visualization system, the impact dynamics when droplets of non-Newtonian fluids impinge on a flat hard surface are characterized. Two situations are considered: for oxidized liquid metal droplets we focus on the spreading behavior, and for densely packed suspensions we determine the onset of splashing. More generally, the combination of high temporal and spatial imaging resolution introduced here offers advantages for studying fast dynamics across a wide range of microscale phenomena.

Drop impact of non-Newtonian fluids is a complex process since different physical parameters influence the dynamics over a very short time (less than one tenth of a millisecond). A fast imaging technique is introduced in order to characterize the impact behaviors of different non-Newtonian fluids.

In the field of fluid mechanics, many dynamical processes not only occur over a very short time interval but also require high spatial resolution for ...

A Microfluidic Model of Biomimetically Breathing Pulmonary Acinar Airways

Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards optimizing drug inhalation techniques as well as predicting deposition patterns of potentially toxic airborne particles in the pulmonary alveoli. Here, soft-lithography techniques are used to fabricate complex acinar-like airway structures at the truthful anatomical length-scales that reproduce physiological acinar flow phenomena in an optically accessible system. The microfluidic device features 5 generations of bifurcating alveolated ducts with periodically expanding and contracting walls. Wall actuation is achieved by altering the pressure inside water-filled chambers surrounding the thin PDMS acinar channel walls both from the sides and the top of the device. In contrast to common multilayer microfluidic devices, where the stacking of several PDMS molds is required, a simple method is presented to fabricate the top chamber by embedding the barrel section of a syringe into the PDMS mold. This novel microfluidic setup delivers physiological breathing motions which in turn give rise to characteristic acinar air-flows. In the current study, micro particle image velocimetry (&#181;PIV) with liquid suspended particles was used to quantify such air flows based on hydrodynamic similarity matching. The good agreement between &#181;PIV results and expected acinar flow phenomena suggest that the microfluidic platform may serve in the near future as an attractive in vitro tool to investigate directly airborne representative particle transport and deposition in the acinar regions of the lungs.

Soft-lithography was utilized to produce a representative true-scale model of pulmonary alveolated airways that expand and contract periodically, mimicking physiological breathing motion. This platform recreates respiratory acinar flows on a chip, and is anticipated to facilitate experimental investigation of inhaled aerosol dynamics and deposition in the pulmonary acinus.

Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards ...

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

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 ...

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 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 ...

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.

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

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