2.1.

System Description

The optoacoustic mouse imaging system consists of four main components: fiber-optic light delivery, a mouse holder with translation and rotation, an array of transducers (detectors), and data acquisition and imaging electronics. We used the following two sources of optical illumination: (1) an alexandrite laser (Ta-2, Light Age, Inc., Somerset, New Jersey), operating at a $755\text{-}\mathrm{nm}$ wavelength, a $10\text{-}\mathrm{Hz}$ pulse repetition rate, energy of $\sim 100\mathrm{mJ}$ per pulse, and a pulse duration of $\sim 75\mathrm{ns}$ ; and (2) Nd:YAG laser (Brilliant, Quantel, Bozeman, Montana), operating at a $1064\text{-}\mathrm{nm}$ wavelength, a $10\text{-}\mathrm{Hz}$ pulse repetition rate, $\sim 100\mathrm{mJ}$ of energy per pulse, and pulse duration of $\sim 15\mathrm{ns}$ . Light delivery was performed orthogonally to the array of acoustic transducers by a bifurcated, randomized fiber bundle (Schott AG, Germany) of $48\mathrm{in.}$ in length. The diameter on the input side was $0.4\mathrm{in.}$ , while the diameter on the bifurcated output side was $0.25\mathrm{in.}$ . Transmission loss within the fiber bundle was about 50%. After leaving the fiber, the two light beams, $E=25\mathrm{mJ}$ each, were expanded to a diameter of $8\mathrm{cm}$ at the target. Figure 1 shows a mouse in the tank during the scan.

Fig. 1

Picture of a mouse during a scan, showing the placement of the mouse and illumination with respect to the array of wideband ultrasonic transducers.

Imaging was performed in a water tank with dimensions of $20\mathrm{in.}$ $\mathrm{L}\times 16\mathrm{in.}$ $\mathrm{W}\times 17\mathrm{in.}$ H. The temperature in the water tank was maintained at $36°\mathrm{C}$ by heating elements placed underneath. These heat pads were controlled by a proportional-integral-derivative controller (PID temperature controller) (Watlow Inc., Columbia, Missouri) with a precision of $0.1°\mathrm{C}$ . The tight control of the temperature ensured a stable speed of sound in the water surrounding mouse, which is necessary for accurate reconstruction of the 3-D mouse volumes. The rotation of the mouse was performed by a dc motor with an optical encoder (Faulhaber Gmbh, Schoenaich, Germany), while the position of the array was controlled by three motorized linear stages (IAI Inc., Torrance, California).

The optoacoustic signals were acquired with a 64-element arc-shaped piezocomposite array designed in our lab and custom built by Imasonic, SAS (France). The center frequency of ultrasonic transducers was $3.1\mathrm{MHz}$ , and the bandwidth was $⩾80\%$ at the $-6\text{-}\mathrm{dB}$ point. The piezocomposite elements had square dimension of $2\mathrm{mm}$ and were spaced $0.7\mathrm{mm}$ apart. The focal length of the array was $65\mathrm{mm}$ and the overall angular aperture between element 1 and 64 was $152\mathrm{deg}$ , which gave an angle of $2.4\mathrm{deg}$ between elements. The step size for the rotation was set to $2.4\mathrm{deg}$ to ensure even detector spacing in the equatorial and meridianal directions. The data acquisition system consisted of three custom-designed 48 channel analog amplifiers featuring an input impedance of $750\mathrm{k}\Omega$ , a variable gain between 0 and $70\mathrm{dB}$ , a bandwidth of $25\mathrm{kHz}\text{to}13\mathrm{MHz}$ at the $-3\text{-}\mathrm{dB}$ point, and an input noise of $2\mathrm{nV}∕\sqrt{\mathrm{Hz}}$ . For the mouse scan, the amplifiers were set to $60\text{-}\mathrm{dB}$ amplification. Data digitalization was performed with two in-house-designed 32-channel boards containing analog-to-digital converters with a sampling rate of up to $40\mathrm{MHz}$ , a field programmable gate array (Stratix FPGA, Altera (San Jose, California) for signal processing and data transfer to a personal computer through the Ethernet protocol. A standard personal computer (Intel/Pentium IV, $3\mathrm{GHz}$ ) running our custom-made LOIS software was used to capture the data and reconstruct optoacoustic images.

2.2.

Signal Processing and Image Reconstruction

Multiple data acquisitions with the mouse rotating about the central axis of the arc-shaped acoustic array are equivalent to a single acquisition with acoustic transducers distributed over the corresponding spherical surface (see Fig. 2 ). In our studies, a full circle $\left(360\text{-}\mathrm{deg}\right)$ rotation was performed with 150 steps of $2.4\mathrm{deg}$ for each step, thus representing data acquisition with 9600 virtual transducers placed on the reconstruction sphere with an animal enclosed in its center [Fig. 2a]. The captured optoacoustic signals were filtered before undergoing reconstruction. We routinely used a second-order Butterworth filter with a bandpass between $250\mathrm{kHz}$ and $5\mathrm{MHz}$ . This filter worked best for processing of optoacoustic signals in preparation for reconstruction of mouse organs and major blood vessels. A more sophisticated signal filtering was required for visualization of smaller vasculature or a combination of larger and smaller tissue structures presented on the same image. For this purpose, we used previously developed multiscale wavelet filter based on a wavelet resembling typical N-shaped optoacoustic signals.³³ Later we optimized this wavelet to replicate the third derivative of the Gaussian wavelet, which can be used to enhance sharpness of the boundaries while significantly reducing low-frequency acoustic trends for optoacoustic sources of arbitrary shapes. ³⁴ Nine scales of this wavelet covering a wide range of frequencies from 4 to 1024 digital samples enabled flexibility in the complex signal processing. An advantage inherent in the multiscale wavelet processing is that the wavelet filter simultaneously serves as an integrator, resulting in conversion of bipolar optoacoustic pressure profiles into monopolar profiles of the absorbed optical energy.³⁵

Fig. 2

(a) Side view and (b) top view of the reconstruction sphere showing the geometric representation of the detector placement during one full rotation of the mouse. The blue area represents the location of the mouse. The blue area represents the location of the mouse. The detector density is higher at the poles in comparison to the equator. (Color online only.)

Three-dimensional tomographic reconstruction was performed using a 3-D reconstruction-algorithm-based radial back projection of filtered and simultaneously integrated optoacoustic signals,²⁵ where the integration was achieved through a multiscale wavelet transform. ³² Such reconstruction in the 3-D case uses the complete data set and, therefore, is rigorous. Since rotation of an arc of transducers around a vertical axis generates a variable density of the detector elements continuously increasing toward the poles of the reconstruction sphere, we normalized the optoacoustic signals by the local transducer density employing a weighting factor of $A$ , which compensated for distortions caused by the transducer density [Fig. 2b]:

2.3.

Postprocessing of Tomographic Images

A commercial 3-D image visualization package, VolView-2.0 (Kitware, Clifton Park, New York), was used for the image postprocessing. We designed a postprocessing routine for the 3-D optoacoustic image based on the assumption that the majority of the voxel data is comprised of noise, situated around the principal mode of the distribution of the image intensity. The principal mode was determined from the maximum of the voxel brightness histogram. For voxels with values less than this mode, the opacity was set to 0. Of the remaining voxel values, the bottom 60% of values were made transparent to limit noise from being displayed. This kind of manipulation can be done in the setting of the Scalar Opacity Map of Volview. A 0.5 intensity value was placed in the Scalar Opacity Map at the 90% mark, and an intensity value of 1 was placed at the 100% mark to make the relevant voxels brighter and more easily seen. The opacity of the remaining data was allowed to linearly increase toward these marks of the intensity histogram.

The image palette was set to gray scale for those values with nonzero opacity according to the following algorithm. The brightness value of 0, i.e., black color, was set to the values that were most proximal to the intensity histogram mode. The next 70% of the data with nonzero opacity was linearly scaled between 0 and 0.5 color values. The brightness value of 1, i.e., white color, was assigned to the most opaque 10% of the data, with the remaining data linearly scaled between 0.5 and 1 color values in the order of increasing opacity. We also applied the Strong Edge Detection processing available in Volview, which can be found in the Gradient Opacity Mapping, to emphasize large intensity changes between neighboring voxels. The gradient opacity mapping assigns an opacity multiplier to the gradient magnitudes. It calculates the difference in intensity between the voxels in all three dimensions and generates a gradient intensity histogram. This enables clearing the regions with similar voxel intensities, and enhances the areas of sharp intensity change, effectively performing 3-D edge detection during rendering.

2.4.

Mouse Holder

All procedures of the mouse study were in compliance with our Institutional Animal Use and Care Committee (IACUC) protocol. The mouse was anesthetized with isoflurane in pure oxygen using an isoflurane distribution unit (Summit Anesthesia Solutions, Bend, Oregon) and then placed into a custom-made mouse holder consisting of two main parts (see Fig. 1). The top part is a hollow cylinder with one end drilled in a conical manner so as to be able to fit the mouse’s nose and face. The bottom part consisted of an acrylic disk with a center hole for the tail to pass through. The top and bottom part of the mouse holder were connected with pretensioned fiber glass rods. The mouse was held in place with tape. The mouse’s arms were attached on the outside of the top part of the mouse holder and the legs of the mouse were attached to the disk at the bottom of the mouse holder so as to minimize shifts and movements from the mouse rotation. The soft tape ensured that the mouse was held in place with minimal pressure so as not to restrict blood flow into the extremities. The anesthesia gas was delivered to the mouse holder via a central freely rotating shaft inserted into the upper cylindrical part of the mouse holder. The upper portion of the mouse holder’s conical opening created a “diving bell” for the head of the mouse with the anesthesia gas pumped into it and enabled the mouse to breathe freely while placed in the tank under water with excess gas bubbling into the tank. The breathing cone simultaneously shielded the eyes of the mouse from the laser exposure. The flow rate of oxygen was $2\mathrm{L}∕\min$ with an isoflurane concentration of 2% for initial anesthesia. Isoflurane concentration was later varied between 1 and 2% on continuous inspection of the animal. Euthanasia was performed after the experiment through spinal dislocation under 5% isoflurane in oxygen.

3.1.

Resolution of the Optoacoustic Array

The resolution of the imaging system was determined using a cross made from two horse hairs. The thickness of the hairs was $200\mu \mathrm{m}$ . The two hairs placed at an angle of $20\mathrm{deg}$ between them. After placement in the tank, the hair cross was illuminated orthogonally to the acoustic array with the dual fiber bundle and the data were collected. A 3-D volume was reconstructed with a voxel size of $0.1\mathrm{mm}$ . Figure 3 shows the optoacoustic image of the hair cross. Two horizontal slices orthogonal to the image depicted in Fig. 3 were selected from the volume and are presented in Figs. 4a and 4c . The first slice [Fig. 5a ] was at the location of the intersection of the hair cross where the area of the pixel intensity is the smallest. Figure 4b shows the normalized intensity profile through the center of the hair cross. The thickness of the hair cross is $\sim 0.5\mathrm{mm}$ at full width at half maximum (FWHM). Taking the width of the hair into account we get $\sim 0.3\text{-}\mathrm{mm}$ resolution, which corresponds to a frequency of $4.5\mathrm{MHz}$ , well within the ultrasonic frequency band of the probe. Subsequent slices were analyzed below the hair intersection. We determined a slice where the intensity between the two maxima decreased to 50% of the values of the maxima. An image of this second slice is shown in Fig. 4c. The distance between the two maxima was $\sim 0.8\mathrm{mm}$ [Fig. 4d]. This shows that after subtraction of the width of the hairs the resolvable distance between two points is $0.6\mathrm{mm}$ .

Fig. 3

Reconstructed optoacoustic volume of a hair cross made from $200\text{-}\mu \mathrm{m}$ -diam horse hair at a $26\text{-}\mathrm{deg}$ angle used for resolution studies.

Fig. 4

(a) Vertical slice of the hair cross at the location of the intersection of the two horse hairs, (b) normalized intensity profile through the maximum of the $x\text{-}y$ slice, (c) vertical slice of the hair cross $1.7\mathrm{mm}$ below the location of the intersection of the two horse hairs, (d) normalized intensity profile through the two maxima of the $x\text{-}y$ slice. The normalized intensity value drops to 50% of the maxima at a separation of $\sim 0.5\mathrm{mm}$ between the two maxima.

Fig. 5

(a) Photograph of the $0.75\text{-}\mathrm{mm}$ -inner-diameter silicone tube maze used as a phantom to simulate blood vessels and (b) optoacoustic volumetric image of silicone tubes filled with $\mathrm{Cu}\mathrm{S}{\mathrm{O}}_4$ solution adjusted to an absorption coefficient of $9{\mathrm{cm}}^{-1}$ .

3.2.

Phantoms Used in Studies of Optoacoustic Image Contrast and Resolution

For the phantom studies, a cupric sulfate solution with an absorption coefficient of $9{\mathrm{cm}}^{-1}$ contained in $0.75\text{-}\mathrm{mm}$ internal-diam silicone tubing was used to simulate blood vessels. The tubes were wrapped and entangled within a holder made from acrylic plates and fiberglass rods. Fiberglass is a glass-cloth laminate with an epoxy resin binder (Professional Plastics, Fullerton, California). In our test, it showed excellent strength, no water absorption, and minimal optoacoustic signals were generated by the material. Figure 5a shows a picture of the tube setup used in the phantom studies, while Fig. 5b shows a projection of the optoacoustic volume (also see Videos 1 ). Visual comparisons show good agreement between the photograph and reconstructed volume, indicating the ability to image and visualize complex structures with sufficient clarity, a requirement for whole body mouse imaging.

Video 1

This video corresponds to Fig. 5, showing a 3-D optoacoustic volumetric image of silicone tubes filled with $\mathrm{Cu}\mathrm{S}{\mathrm{O}}_4$ solution adjusted to an absorption coefficient of $9{\mathrm{cm}}^{-1}$ (QuickTime, 3.8 MEG). .

3.3.

Mouse Images

Figure 6 shows the optoacoustic volume of a mouse with the alexandrite laser at $755\mathrm{nm}$ and a vertically elongated oval beam (also see Videos 2 ). The Gaussian output beam from the fiber bundle was elongated with cylindrical lenses into an oval beam shape to facilitate a more uniform illumination over the mouse body. This allowed the imaging of a larger section of the mouse, resulting in the visualization of the abdominal aorta and its bifurcation into the femoral veins. Additionally, both kidneys are visualized as well as the spleen and a partial lobe of the liver. The imaged mouse was female and the ovarian vessels were visible.

Fig. 6

Three-dimensional optoacoustic volume of a nude mouse illuminated at $755\mathrm{nm}$ with optical fluence of $1\mathrm{mJ}∕{\mathrm{cm}}^2$ , showing an area of the mouse with bifurcation of the inferior vena cava extended into the gonadal (ovarian) veins. Both kidneys are visualized as well as the spleen and a partial lobe of the liver.

Video 2

This video corresponds to Fig. 6, showing a 3-D optoacoustic volume of a nude mouse illuminated at $755\mathrm{nm}$ with optical fluence of $1\mathrm{mJ}∕{\mathrm{cm}}^2$ . The rotating image shows an area of the mouse with bifurcation of the inferior vena cava extended into the gonadal (ovarian) veins. Both kidneys are visualized as well as the spleen and a partial lobe of the liver (QuickTime, 2 MEG). .

The results from a male mouse scan are shown in Fig. 7 (also see Videos 3 ). This mouse was also illuminated with the alexandrite laser $\left(755\mathrm{nm}\right)$ , and similarly to Fig. 6, it shows the descending aorta with bifurcation. These features of mouse anatomy are similar in both images (Figs. 6 and 7), indicating that key features are reproducible in different subjects. However, only an obscured view of the kidneys is displayed in Fig. 7. On the other hand, Fig. 7 clearly shows the spine of the mouse. The image formation in the spinal region could be from the network of small and unresolved blood vessels supplying the vertebra. While the spine is visible in most mouse volumes it was especially clear in this volume, because the mouse was located in the holder in a way that placed the mouse spine at the center of rotation where the sensitivity and resolution of the probe is the highest.

Fig. 7

Optoacoustic volume of a nude mouse illuminated at $755\mathrm{nm}$ with optical fluence of $1\mathrm{mJ}∕{\mathrm{cm}}^2$ clearly showing the spine (contrast of the spine is provided by spinal microvasculature), the descending inferior vena cava, and its bifurcation into external and internal femoral veins, and a view of the left and right kidneys.

Video 3

This video corresponds to Fig. 7, showing an optoacoustic volume of a nude mouse illuminated at $755\mathrm{nm}$ with optical fluence of $1\mathrm{mJ}∕{\mathrm{cm}}^2$ clearly showing the spine (contrast of the spine is provided by spinal microvasculature), the descending inferior vena cava, and its bifurcation into external and internal femoral veins, and a view of the left and right kidneys (QuickTime, 2 MEG). .

The optoacoustic volume from another mouse scan (Fig. 8 ) was performed using the Nd:YAG laser at $1064\mathrm{nm}$ (also see Videos 4 ). It shows the descending aorta, the abdominal aorta, and its bifurcation into the femoral veins. Additionally, the three veins in the tail were imaged. The left and right kidneys are fainter than comparable images performed with an illumination of $755\mathrm{nm}$ . Blood in the mouse vasculature was highly oxygenated due to the use of 50% oxygen along with anesthesia. Therefore, laser illumination at $1064\mathrm{nm}$ , which is strongly absorbed by oxygenated blood, revealed both arterial and venous vasculature, while emphasizing veins due to their larger diameters.

Fig. 8

Optoacoustic volume of a nude mouse illuminated at $1064\mathrm{nm}$ , at optical fluence of $1\mathrm{mJ}∕{\mathrm{cm}}^2$ , showing the mouse body vasculature including descending inferior vena cava and its bifurcation into the external and internal femoral veins and the three tail veins. Additionally, there is a partial view of the left and right kidney vasculature.

Video 4

This video corresponds to Fig. 8, showing an optoacoustic volume of a nude mouse illuminated at $1064\mathrm{nm}$ at optical fluence of $1\mathrm{mJ}∕{\mathrm{cm}}^2$ , which displays the mouse body vasculature including descending inferior vena cava and its bifurcation into the external and internal femoral veins and the three tail veins. Additionally, there is a partial view of the left and right kidney vasculature (QuickTime, 758 KB). .