2.1. Description of the integrated device

A schema of the developed multi-functional setup is presented in Fig. 1.

Top: Schema of the integrated experimental setup (PBS: Polarizing beam splitter; BS: Beam sampler; KTP: Frequency doubling KTP crystal; KG3: Filter glass (Bandpass: 315 - 710 nm); PD: Si Photodetector (Detector, 200 - 1100 nm); λ/2: Achromatic half-wave plate). Bottom: Cross-section (left) and 3D view (right) of the FUS/PTT/PA probe.

The device can be decomposed into three parts:

- The FUS/PTT/PA probe (Fig. 1, Bottom) comprises a single-element focused transducer (diameter 25 mm, focal depth 20±2 mm, central frequency 1.5 MHz, Imasonic, France), presenting a hole in its center (diameter of central hole 6 mm, specifically manufactured by Imasonic, France). The measured focal volume of the transducer was 1×1×6 mm3 at −6 dB. The output pressure of the transducer were measured in a degassed water tank, using a 0.5 mm needle hydrophone (Precision Acoustics, Dorchester, UK) mounted on a positioning stage. The transducer was driven by a built-in signal generator connected via a 20 W power amplifier (Image Guided Therapy, France) (Fig. 2). The acquired signal from the hydrophone was sampled by the oscilloscope (Picoscope 5243A, Pico Technology, UK). After scanning the whole focal spot, the hydrophone was placed in the center on the focal spot to record the relationship between the electrical input and acoustic pressure. The ultrasound transducer was then coupled to the head of the animal via the water balloon filled with deionized and degassed water to reach 7 to 10 mm thickness, depending on the targeted depth, and covered with degassed echographic gel (70% v/v in water). Electrical power sent to the transducer was monitored during the BBB opening session. As a coupling medium, a thin and transparent latex-based membrane was fixed to the transducer and a water filling circuit was connected in order to fill or empty this balloon up to the desired thickness. This flexibility permits a scanning in depth up to 13±2 mm of the examined organ. The water was degassed with built-in pumps in order to avoid any acoustic impedance mismatch within the balloon. The hermeticity is guaranteed by an O-ring. The transducer can be used in two modes: active mode as emitter for the BBB opening with FUS, or passive mode as receiver for the temperature monitoring with PA. The PA signal is collected through a digital oscilloscope (Picoscope 5243A, 2 channels, 100 MHz, 500 MSamples/s, rise time 5.8 ns, Pico Technology, UK). A hermetic glass window has been mounted and positioned on the top of the transducer mount in order to receive an optical fiber (MHP910L02, Core Ø910 μm, 0.22 NA, Thorlabs, Germany) through which the light sources are delivered to the sample.

Calibration of ultrasound transducer. Top: Experimental setup Bottom left: axial slice view of the focal spot. Bottom right: cross-section view of the focal spot, measurement performed at 0.65 MPa.

- The light delivery part includes two types of lasers, whose beams were piped through the same optical fiber: a CW laser diode for the PTT (LD830-ME-2W, 830 nm, 2 W, Thorlabs, Germany), controlled in current and temperature (controller TEC,5A/225W, ITC4005, Thorlabs, Germany) and a pulsed laser for the PA (Brilliant EaZy, Nd:Yag, 1064 nm, 330 mJ, 5 ns, 10 Hz, Quantel, France). The pulsed laser was frequency doubled with a KTP crystal at 532 nm (Eksma Optics, Lithuania). Optical components (neutral densities, linear polarizers and half-wave plates) were placed on the pulsed laser path in order to control the energy delivered through the fiber. A combination of mirrors and lens enabled to align both CW and pulsed lasers and direct both beams through the fiber. An additional focusing lens was added at the exit of the fiber in order to lower the beams diameter to 4 mm after the water balloon. Pulsed laser energy and CW power were controlled right after the water balloon before each experiment. Typical values were 150-300 μJ for the pulsed laser and 100-300 mW for the CW. An additional red light pencil laser (632 nm) is also added for alignment.

- A platform (Image Guided Therapy, France) comprising a small animal holder, a transducer holder mounted on fully programmable 3D scanning stages and a programmable ultrasound single channel amplifier. This system allows performing a fast raster scan (10 mm/s) during the FUS-BBB opening and possibly PTT. Hot water is conveyed to the animal bed via plastic tubes to keep the body temperature of the animal stable during the therapy. The temperature of the circulating water is stabilized (Corio CP-BC4, Circulating themostat, Julabo, Germany).

The different acoustic and optical emissions and data acquisition were controlled with a computer. Prior PA signal recording, a band pass filter was applied [0.04-3.5 MHz] to the signal and averaged over a certain number of measurements. In the present experiments, as the temperature rise during the therapy was usually fast (less than 30 s), the PA signal was averaged over only two data acquisitions, therefore the pulsed laser fluence was adapted in order to perform all the measurements with a sufficiently high signal-to-noise ratio. The fluence was simultaneously monitored and recorded with a photodiode (PDA10A2, Thorlabs, Germany), for normalization of the PA signal.

One of the difficulties that could be encountered was the resistance of the water balloon to high optical power during PTT combined with PA thermometry. A series of tests were specifically performed to measure a possible fluence or power attenuation through the water balloon during 15 min measurements. No specific damage was noticed on the water balloon, that could be used for the whole experiment campaign. Optical attenuation remained sufficiently low through latex to be able to perform both PTT and PA measurements.

The proposed FUS/PTT protocol includes three consecutive steps: i) systemic administration of efficient photoabsorbers (NPs or others); ii) injection of intravascular microbubbles followed by the application of FUS, by using the transducer in its active mode, with calibrated sonication parameters allowing BBB opening; iii) application of the PTT with PA temperature monitoring with a calibrated optical irradiation dose inducing tumor cell death. At the present stage, the FUS-induced BBB opening is a process well controlled in our setup and validated by SPECT imaging (results presented hereafter). Work is in progress to identify the best photoabsorber candidate and, despite of the large number of publications on preclinical PTT [23], a balance is still to be found between light thermal dose that induces tumor cell death and minimum damages caused to healthy surrounding tissues. The present setup is developed to that purpose.