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

NOTE: All experimental procedures were approved by the Norwegian Animal Research Authorities. Details of materials that were used in the protocol can be found in the Table of Materials.

1. Production of microbubbles

NOTE: In this work, the microbubbles of interest are protein-and-nanoparticle-stabilized microbubbles, for which the production protocol has been described previously^28,33,48. Therefore, the fabrication protocol has been briefly summarized here. 

1. First, using a pipette, mix ultrapure water with 0.5 wt% of casein in phosphate-buffered saline (PBS) and 1 wt% of the nanoparticles labeled with 0.21 wt% of the fluorescent dye, NR668 (modified Nile Red), in a sterile glass crimp top vial (10 mL, diameter of 2 cm). The polymeric nanoparticles are prepared using the mini-emulsion polymerization method as described by Mørch et al.³⁸.
   NOTE: Here, the dye functions as a model drug to enable visualization of nanoparticle release. When working with the nanoparticle solution, wear a lab coat, goggles, and gloves. Wipe away any spills of the nanoparticle solution immediately with 100% acetone.
2. Cap the vial with the rubber cap, mix slightly, and place the vial in an ultrasonic bath for 10 min at room temperature to eliminate possible aggregates. Place a dispersion tool with the tip of the stirrer ~0.5 cm from the bottom of the glass vial. Using a glass pipette connected to the gas container, add the perfluoropropane gas to the head space of the vial containing the solution until the solution starts bubbling slightly.
   NOTE: Wrap self-sealing film around the base of the dispersion tool to prevent slipping of the glass vial during stirring.
3. Stir the solution vigorously at 1935 × g (24,000 rpm with a radius of rotation of 3 mm) for 4 min using the dispersion tool. Close the vial with the rubber cap, and seal the vial for further use.
   NOTE: The stirring entraps the gas in the liquid. The microbubble-shell subsequently self-assembles without requiring any active step.
4. Store the excess casein and nanoparticle solution at 4 °C, and clean the dispersion tool with 100% acetone.

2. Imaging single bubbles

1. Confocal microscopy
   1. Sample preparation
      1. Dilute the bubble solution to image single microbubbles as follows. Place a venting needle (19 G-21 G) in a glass crimp top vial containing the microbubbles produced by following the procedure described in section 1. Turn the vial upside down to allow large bubbles to move away from the seal of the vial.
      2. Insert another needle tip (19 G) of a small (~1 mL) syringe into the vial, while the vial is still upside down. Remove a small amount of the bubble suspension, and transfer the contents of the syringe into a small tube for easier pipetting in the next step.
         NOTE: The volume of the suspension to extract directly depends on the type and concentration of the bubble suspension. In this case, 0.2 mL was extracted.
      3. Using a pipette, dilute the microbubble suspension (from section 1) in filtered PBS to achieve a concentration of approximately 2 × 10⁵ to 6 × 10⁵ microbubbles/mL to enable single-bubble imaging.
         ​NOTE: Depending on the bubble type, it is recommended to wash the bubble suspension to remove free fluorescent dye. This is particularly important with bubbles for which the fluorescent dye is infused in the shell. To wash bubbles, dilute the bubble suspension (e.g., by taking 100 µL of the bubble solution in 10 mL of PBS), and centrifuge it (typically at speeds of the order of 100 × g). Finally, remove the supernatant containing the microbubbles with a pipette for further analysis. The remaining solution contains the free fluorescent particles and can be discarded. The washing step should be repeated as necessary.
      4. Add glycerol to the mixture to increase the viscosity of the medium and eliminate the movement induced by Brownian motion that would otherwise interfere with the rather slow confocal Z-stack imaging.
         ​NOTE: The amount of glycerol depends on the type of bubble that is imaged (here, ~50%). For some types of bubbles, glycerol might have an adverse effect on stability⁴⁹. However, no noticeable change was observed in the bubbles over approximately 30 min under confocal imaging. Furthermore, glycerol may change the acoustic response of microbubbles and can therefore only be used with imaging methods where the microbubbles are not insonified.
      5. Place the microbubble suspension in a chamber with thin walls for optimal imaging such as a channel slide.
   2. Imaging protocol
      1. Switch on the confocal microscope, and select a suitable objective and the desired laser and scanner to use during confocal microscopy.
         NOTE: Here, use a 60x water immersion objective for a resolution of 0.08 µm/pixel and depending on the bubble size, image a region of 256 x 256 pixels or 128 x 128 pixels. In these specific experiments, use a 488 nm laser and a Galvano scanner. The emission wavelength depends on the fluorescent dye and is typically broadband.
      2. Find a microbubble in bright-field, and switch to confocal microscopy. Set the desired top and bottom planes in between which the confocal microscope will scan. Acquire a Z-stack to observe the 3D structure; use a step size of 100 nm in the Z-direction.
2. Bright-field microscopy
   1. Assembly of the optical system
      NOTE: A schematic representation of the bright-field microscopy setup is shown in Figure 3A. To ensure undisturbed ultrasound propagation, the water bath contains two openings: one for a light source and one for an ultrasound transducer. The optical system consists of a (modular) microscope, a high-speed camera, and matching optics. As the period of microbubble oscillations is typically of the order of 1 µs (using 1 MHz ultrasound), the camera should be set to record at a framerate of at least 5 million frames per second. Here, the camera is to be set to record at 10 million frames per second (256 x 400 pixels) for 256 frames (25.6 µs) to capture all details of the bubble dynamics including higher harmonics.
      1. Attach a water-immersion objective with an appropriate magnification, working distance, and NA to the microscope.
         ​NOTE: A water-immersion objective was used to provide a stable working distance despite gradual evaporation of the water. Here, a water-immersion objective with a magnification of 60x, a working distance of 2 mm, and an NA of 1 was selected.
      2. Use a strobe light with a peak power output of at least 1 kW for illumination and a tube lens between the microscope and the camera to ensure that as little ambient light as possible reaches the sensor of the high-speed camera.
      3. Use a dimmable halogen light source for focusing on single microbubbles and alignment of the optical and acoustical system for real-time imaging.
   2. Assembly of the acoustical system
      1. Use a programmable arbitrary waveform generator and a power-amplifier (56 dB gain) to drive the transducer with a smooth envelop and waveform. Connect an oscilloscope to the arbitrary waveform generator to check the signal. Connect a personal computer to the arbitrary waveform generator to program the incoming acoustic pressure wave, using a script written in-house.
      2. Use a pulse/delay generator as master trigger to synchronize the optical and acoustical systems. Set the trigger delays on the pulse/delay generator and the camera software such that the recording starts 16 µs after ultrasound transmission to allow the ultrasound wave to reach the bubbles. Trigger the light source 1.5 µs before the start of the recording to ensure proper illumination during the bubble oscillations (see Figure 3B for the timing diagram).
      3. Choose a suitable transducer with an appropriate center frequency. Place it in an opening of the water bath, so that it is at an angle with respect to the optical axis to minimize reflections from the sample holder membranes and to reduce standing wave formation. 
         NOTE: Here, a single-element focused immersion transducer with a center frequency of 2.25 MHz, a focal distance of 1" and element diameter of 0.75" was placed at an angle of 35° with respect to the optical axis. The calibration of the transfer function needs to be performed using the same amplifier that is used in the acoustical system. Calibrate the transfer function from voltage amplitude to pressure amplitude of the transducer using a fiber optic hydrophone as a function of the ultrasound transmit frequency.
   3. Choosing the sample holder
      1. Use a sample holder with optically and acoustically transparent membranes and a volume that is large enough to allow for imaging of several single microbubbles within the same sample.
         NOTE: Here, a cell culture cassette with a volume of 10 mL, membrane areas of 25 cm², and membrane thickness of 175 µm was used. Due to acoustic reflections on the lower membrane, and interference from waves reflected by the microscope objective and the upper membrane, the in situ acoustic pressure might differ from that programmed on the arbitrary waveform generator. Placing the transducer at an angle with respect to the sample holder membranes reduces standing wave formation, but can increase reflections from the membranes.
      2. Ensure that the sample can be fully submerged and brought within the focus of both the transducer and the microscope objective. Use an aluminum support attached to a 3D micropositioning stage to move the sample holder independently.
   4. Alignment of the optical and acoustical systems
      1. For 3D translation to align the setup, attach the water bath to an XY-translation stage, and attach the stage to an optical table to ensure it does not move during experiments. Then, fill the water bath with water, and switch on the dimmable halogen light source. During alignment, move the microscope objective to the side to prevent ultrasound reflections.
      2. Attach a needle hydrophone (0.2 mm) to the sample holder arm, and place the needle hydrophone in the water bath, with the tip in the field of view of the objective. Turn on the amplifier and the arbitrary waveform generator; use single pulses of 5 to 10 ultrasound cycles and a pulse repetition frequency of 15 Hz. Make sure that the hydrophone tip is centered and in focus on the microscope image. Move the tank in the XY-direction and the needle in the Z-direction until the maximum pressure amplitude is reached.
      3. Adjust the focus of the microscope to refocus on the tip of the hydrophone.
         NOTE: This protocol ensures the alignment between the microscope focus and the transducer focus. Do not change the position of the microscope and the transducer after alignment.
   5. Sample preparation
      1. Repeat steps 2.1.1.1 through 2.1.1.3 to prepare the sample solution. Dilute the bubble solution to enable single-bubble imaging and to rule out acoustic interactions of neighboring bubbles.
      2. Open the outlet of the sample holder. Using a syringe, inject the sample solution into the other opening of the sample holder until it is completely filled. Ensure that there are no air bubbles inside the sample holder to prevent unwanted interactions with the ultrasound field.
      3. Close both valves of the sample holder, and place the sample holder perpendicular to the optical axis.
         NOTE: Keep the filled sample holder level to prevent shifting of the bubbles to one side of the sample holder during moving.
   6. Imaging protocol
      1. Program the desired ultrasound driving frequency and acoustic pressure in the arbitrary waveform generator through the aforementioned in-house written script.
         NOTE: Here, the acoustic pressure wave was a single burst of 40 cycles, with an 8-cycle Gaussian-tapered pulse. Ultrasound frequencies used in these experiments were 1 MHz, 2 MHz, or 3 MHz, with acoustic pressure amplitudes ranging from 81 kPa to 1200 kPa.
      2. Move the sample holder containing the sample solution using the XYZ-stage to locate single microbubbles in the focus of the microscope. Start with a field of view at a corner of the sample holder, and ensure that the edge of the microbubbles is clearly visible and in focus (see Figure 3C for an ideal camera view).
      3. Attach the end of an optical fiber that was previously connected to the halogen light to a strobe light, so that the other end is still connected to the water bath. Trigger the recording.
      4. Repeat steps 2.2.6.2 through 2.2.6.3 as many times as desired per ultrasound setting (frequency and acoustic pressure), moving the cell culture cassette containing the microbubbles at least 2 mm (in the focal plane) from the previous location to ensure that the microbubbles in the field of view are not insonified in previous experiments.
         NOTE: Here, each experiment was repeated ~20 times. When the whole sample holder is insonified, empty the sample holder, and refill it with fresh sample solution for subsequent experiments.
   7. Data analysis
      1. Adopt a programming environment to perform data analysis according to the research question, and perform edge detection after processing the images. Using a function that measures properties of image regions, find the centroid of a bubble and the derivative of the intensity profile around each bubble to detect the contour of the bubble (and thus, the bubble radius R). Extract relevant parameters from the radius over time for single microbubbles.
         NOTE: In the present study, a programming environment was used for image processing to binarize and filter recordings of single microbubbles. An in-house script was used to find the derivative of the intensity profile around each bubble.
3. Fluorescence microscopy
   1. Assembly of the optical system
      1. Build the setup for fluorescence microscopy (Figure 4A), with the same base used in the bright-field microscopy described in section 2.2.
         NOTE: The setup described in section 2.3 can be combined with that described for bright-field microscopy in section 2.2. Combining both fluorescence microscopy and bright-field microscopy enables visualization of the microbubble gas core while imaging the nanoparticle release.
      2. Set the high-speed camera to record at 500,000 frames per second (400 x 250 pixels) for 128 frames (256 µs).
         ​NOTE: The imaging time is longer than in the bright-field experiments as the light intensity is limited in fluorescence, and because the timescale over which particle delivery occurs is longer than that of the bubble dynamics.
      3. Select a laser with a power high enough to supply sufficient light and that has a suitable excitation wavelength, and ensure that it is coupled with an acousto-optic modulator to avoid bleaching the sample.
         ​NOTE: In this study, a 5 W continuous wave laser with an excitation wavelength of 532 nm was used to excite the fluorescence of the nanoparticles.
      4. Place a beam splitter, dichroic mirror and notch filter between the laser and the microscope objective to direct the excitation light towards the sample while allowing the fluorescence emission to reach the camera.
   2. Assembly of the acoustical system
      1. To insonify the microbubbles, use the same acoustical setup as in section 2.2.2. Change the transducer in these specific experiments to a single-element, focused immersion transducer with a center frequency of 2.25 MHz, a focal distance of 1.88", and element diameter of 1". Place it at an angle of 35° with respect to the optical axis to minimize reflections from the sample holder membranes and reduce standing wave formations.
   3. Alignment of the optical and acoustical systems
      1. Repeat steps described in section 2.2.4.
   4. Sample preparation
      1. Prepare the sample solution as described in section 2.2.5.
   5. Imaging protocol
      1. Set the desired ultrasound driving frequency and acoustic pressure amplitude on the arbitrary waveform generator through the aforementioned in-house written script.
         NOTE: Here, the acoustic pressure wave was programmed to be a single burst of ultrasound of 140 cycles, with a 10-cycle Gaussian-tapered pulse. Longer pulse durations are generally required to induce bio-effects compared to those required to study bubble dynamics. Ultrasound frequencies used in these experiments were 1 MHz, 2 MHz, or 3 MHz, with acoustic pressure amplitudes ranging from 81 kPa to 1200 kPa.
      2. On the pulse/delay generator, set the trigger delay for the laser for fluorescent excitation of the nanoparticles from the microbubbles during the recording.
         NOTE: For these specific experiments, the trigger delay was between 20 µs and170 µs for a total duration of 150 µs. The timing diagram is shown in Figure 4B.
      3. Move the sample holder containing the sample solution using the XYZ-stage to locate single microbubbles in the focus of the microscope. Start with a field of view of a corner of the sample holder; see Figure 4C for an ideal camera view in which the interface of the microbubbles is clearly visible and in focus. Trigger the recording.
      4. Repeat step 2.3.5.3 as many times as desired per ultrasound setting (frequency and acoustic pressure), moving the cell culture cassette containing the microbubbles at least 2 mm (in the optical plane) from the previous location to ensure microbubbles in the field of view are not sonicated in previous experiments.
         ​NOTE: In this study, each experiment was repeated ~10-20x. When the whole sample holder is insonified, empty the sample holder, and refill it with fresh sample solution for subsequent experiments. Which distance to move the sample holder between experiments depends on the acoustic beam size.
   6. Data analysis
      1. Analyze the fluorescence microscopy recordings according to the research question. For every microbubble, visually determine whether delivery of the nanoparticles in the fluorescence microscopy experiments occurred. If detachment and deposition of the nanoparticles from the gas core onto the sample holder membrane is observed for a single microbubble, manually enter that delivery occured in the programming environment.

3. Intravital microscopy

1. Dorsal skinfold window chamber surgery (described previously^26,47,50)
   1. Acclimate the animals for one week before placing the window chambers. Although both female and male mice can be used, and age is unimportant, ensure that the weight of the mice is at least 22-24 g so that the skin is sufficiently flexible.
   2. Perform the surgery under general anesthesia with intraoperative and postoperative analgesic treatment. Anesthetize the animal by a subcutaneous injection of fentanyl (0.05 mg/kg)/medetomidine (0.5 mg/kg)/midazolam (5 mg/kg)/water (2:1:2:5) at a dose of 0.1 mL per 10 g weight. Use a heating pad or a heating lamp to maintain the body temperature of the animal.
   3. Pull gently on the double layer of skin on the back of the animal so that the skin is sandwiched between two symmetric polyoxymethylene frames of the window chamber. Fix the chamber by placing two screws extending through the double skin layer and suturing along the upper edge of the chamber.
   4. Remove the skin within the circular frame of the chamber on one side of the skin fold. Place a cover glass with a diameter of 11.8 mm within the frame where the skin is removed to form a window into the tissue.
   5. Use a subcutaneous injection of atipemazole (2.5 mg/kg), flumazenil (0.5 mg/kg), and water (1:1:8) at a dose of 0.1 mL per 10 g as antidote to terminate the anesthesia. Place the animal in a heated recovery rack overnight. Supplement the water for the animals with 25 mg/mL of enrofloxacin to prevent infection in the surgical site.
2. Tumor model creation
   1. Maintain cancer cells at 37 °C and in a 5% CO₂ atmosphere in appropriate culture medium supplemented with 10% fetal bovine serum and 100 U/mL penicillin and 100 mg/mL streptomycin.
      NOTE: The human osteosarcoma (OHS) cell line was used in this protocol, but other cell lines can also be used.
   2. On the day after step 3.1.5, anesthetize the animal by isoflurane (5% during induction and 1-2% during maintenance) for a couple of minutes. Remove the cover glass, apply 5 × 10⁶ cancer cells in 30 µL of cell culture medium, and replace the cover glass.
   3. Allow the tumors to grow for 2 weeks before imaging, and monitor the weight and health status of the animals at least 3 times per week during this period.
3. Assembly of the optical system
   1. Perform intravital imaging during ultrasound treatment (as described in previous work²⁶) with a suitable microscope and objective depending on the research question at stake. See Figure 5A for a schematic representation of the experimental setup.
      ​NOTE: For this specific experiment, a multiphoton microscope was used, equipped with a 20x water dipping objective (NA of 1.0 and working distance of 2 mm) and a pulsed laser. Images were acquired in resonant scanning mode at 31 frames per second (512 x 512 pixels) with a field of view of 400 x 400 µm². The excitation wavelength was 790 nm. The filters in front of the two gallium arsenide phosphide detectors were long-pass 590 nm and band-pass 525/50 nm for the detection of fluorescence.
4. Assembly of the acoustical system
   1. Mount a suitable ultrasound transducer in a waveguide (custom made) positioned below the objective at an angle of 45° with respect to the optical axis to minimize reflections from the cover glass of the dorsal skinfold window chamber and to reduce standing wave formations. Fill the waveguide with distilled and degassed water. Apply ultrasonic coupling gel on top of the waveguide.
5. Alignment of the optical and acoustical systems
   1. Align the optical axis with the focus of the ultrasound. Position a fiber-optic hydrophone in the focus of the objective. Then, turn on the amplifier and the arbitrary waveform generator to excite the transducer with short bursts (5-10 cycles) with a pulse repetition frequency of 100 Hz, and move the ultrasound transducer to the position where the highest pressure is detected with the hydrophone signal on the oscilloscope.
      ​NOTE: Do not change the position of the transducer after alignment.
6. Imaging protocol
   1. Position the heated animal holder (custom designed) connected to an XY-positioning stage between the waveguide and the objective, and add more coupling gel. Anesthetize the animal, and place a tail vein catheter. Place the mouse in the heated holder, and fix the window chamber in the holder. Add a water droplet on top of the cover slip in the window chamber, and move the objective in place to image the tumor tissue.
   2. Figure 5B shows the timing diagram of the experiments depicting the order of events. Inject fluorescently-labelled 2 MDa dextran intravenously (30 µL, 4 mg/mL diluted in saline) to visualize the vasculature, and move the mouse using the XY-translation stage to find a position with suitable blood vessels. Record baseline images before the ultrasound treatment. Adjust frame rate, field of view, and length of recording depending on the research question and the specifics of the microscope and dyes to be imaged.
      ​NOTE: In these experiments, 31 frames per second were recorded with a field of view of 400 x 400 µm², and imaging was done continuously for 5 min.
   3. Set the desired ultrasound driving frequency, pulse length, and acoustic pressure amplitude on the arbitrary waveform generator.
      NOTE: For these experiments, a frequency of 1 MHz was used with a pulse length of 10 ms and peak negative pressure amplitudes between 0.2 MPa and 0.8 MPa. A pulse repetition frequency of 0.5 Hz or 0.1 Hz was used to allow new microbubbles to reperfuse into the treated area between ultrasound pulses.
   4. Inject 50 µL microbubbles (2 × 10⁸ to 5 × 10⁸ microbubbles/mL) intravenously, and apply ultrasound while imaging, as described in²⁶.
7. Data analysis
   1. Depending on the research question, analyze images with (open source) image processing software and a programming environment, as described in²⁶, to determine blood vessel parameters (diameter, branching, flow speed, and direction), accumulation of nanoparticles in the vessels, and kinetics and penetration depth of extravasation of dextran and nanoparticles into tumor tissue.