Daniel R. Reinecke

Photoacoustic angiography of the breast

PURPOSE The authors report a noninvasive technique and instrumentation for visualizing vasculature in the breast in three dimensions without using either ionizing radiation or exogenous contrast agents, such as iodine or gadolinium. Vasculature is visualized by virtue of its high hemoglobin content compared to surrounding breast parenchyma. The technique is compatible with dynamic contrast-enhanced studies. METHODS Photoacoustic sonic waves were stimulated in the breast with a pulsed laser operating at 800 nm and a mean exposure of 20 mJ/pulse over an area of approximately 20 cm2. These waves were subsequently detected by a hemispherical array of piezoelectric transducers, the temporal signals from which were filtered and backprojected to form three-dimensional images with nearly uniform k-space sampling. RESULTS Three-dimensional vascular images of a human volunteer demonstrated a clear visualization of vascular anatomy with submillimeter spatial resolution to a maximum depth of 40 mm using a 24 s image acquisition protocol. Spatial resolution was nearly isotropic and approached 250 microm over a 64 x 64 x 50 mm field of view. CONCLUSIONS The authors have successfully visualized submillimeter breast vasculature to a depth of 40 mm using an illumination intensity that is 32 times less than the maximum permissible exposure according to the American National Standard for Safe Use of Lasers. Clearly, the authors can achieve greater penetration depth in the breast by increasing the intensity and the cross-sectional area of the illumination beam. Given the 24 s image acquisition time without contrast agent, dynamic, contrast-enhanced, photoacoustic breast imaging using optically absorbing contrast agents is conceivable in the future.

Thermoacoustic molecular imaging of small animals.

We have designed, constructed, and tested a thermoacoustic computed tomography (TCT) scanner for imaging optical absorption in small animals in three dimensions. The device utilizes pulsed laser irradiation (680-1064 nm) and a unique, 128-element transducer array. We quantified the isotropic spatial resolution of this scanner to be 0.35 mm. We describe a dual-wavelength subtraction technique for isolating optical dyes with TCT. Phantom experiments demonstrate that we can detect 5 fmol of a near-infrared dye (indocyanine green, ICG) in a 1-microL volume using dual-wavelength subtraction. Initial TCT imaging in phantoms and in two sacrificed mice suggests that three-dimensional, optical absorption patterns in small animals can be detected with an order of magnitude better spatial resolution and an order of magnitude better low-contrast detectability in small animals when compared to fluorescence imaging or diffusion optical tomography.

Dedicated 3D photoacoustic breast imaging

PURPOSE To report the design and imaging methodology of a photoacoustic scanner dedicated to imaging hemoglobin distribution throughout a human breast. METHODS The authors developed a dedicated breast photoacoustic mammography (PAM) system using a spherical detector aperture based on our previous photoacoustic tomography scanner. The system uses 512 detectors with rectilinear scanning. The scan shape is a spiral pattern whose radius varies from 24 to 96 mm, thereby allowing a field of view that accommodates a wide range of breast sizes. The authors measured the contrast-to-noise ratio (CNR) using a target comprised of 1-mm dots printed on clear plastic. Each dot absorption coefficient was approximately the same as a 1-mm thickness of whole blood at 756 nm, the output wavelength of the Alexandrite laser used by this imaging system. The target was immersed in varying depths of an 8% solution of stock Liposyn II-20%, which mimics the attenuation of breast tissue (1.1 cm(-1)). The spatial resolution was measured using a 6 μm-diameter carbon fiber embedded in agar. The breasts of four healthy female volunteers, spanning a range of breast size from a brassiere C cup to a DD cup, were imaged using a 96-mm spiral protocol. RESULTS The CNR target was clearly visualized to a depth of 53 mm. Spatial resolution, which was estimated from the full width at half-maximum of a profile across the PAM image of a carbon fiber, was 0.42 mm. In the four human volunteers, the vasculature was well visualized throughout the breast tissue, including to the chest wall. CONCLUSIONS CNR, lateral field-of-view and penetration depth of our dedicated PAM scanning system is sufficient to image breasts as large as 1335 mL, which should accommodate up to 90% of the women in the United States.

Thermoacoustic CT: imaging principles

We have developed instrumentation for measuring the tissue- absorption properties of radio waves in the human body using thermoacoustic interactions. The imaging principles upon which this instrumentation is based are applicable to other irradiation sources, such as visible and IR. We present the imaging reconstruction methodology that we have developed for mapping radiation absorption pattern sin 3D. Both simulated and experimental data are used to illustrate imaging principles.

Synthesis and Evaluation of Near-Infrared (NIR) Dye-Herceptin Conjugates as Photoacoustic Computed Tomography (PCT) Probes for HER2 Expression in Breast Cancer

We are evaluating PCT imaging in conjunction with NIR dye labeled Herceptin antibody for noninvasive assessment of HER2 expression in tumors. Herceptin was labeled with Alexa Fluor-750 amine reactive dye for characterization of photoacoustic and fluorescence signals. Measurements were performed in solution and after incubation in cultured cell lines that were positive or negative in expression of HER2. The dye to antibody ratio was controlled to achieve a broad range of degree of labeling (DOL = 2 to 15). Photoacoustic signal intensity of Herceptin-dye conjugates in solution increased with increases over the entire DOL range studied. In contrast, fluorescence exhibited significant quenching for higher DOL. In vitro PCT imaging of the labeled HER2 (+) and HER2 (-) cells revealed the targeting specificity of the NIR dye labeled Herceptin. In HER2 (+) cells lines, photoacoustic signal intensity gradually increased with increasing DOL and with increasing number of cells. These results demonstrate that PCT-based measurement of HER2 receptor binding using NIR dye labeled Herceptin is feasible. The absence of a quenching effect with increased DOL advantages this method over traditional methods based on fluorescence measurement.

Reconstruction of blood vessels from x-ray subtraction projections: limited angle geometry

Several algorithms have been investigated for reconstructing blood vessels from a limited number of x-ray subtraction projections, distributed over a limited range of angles. Both computer simulations and an in vivo animal study were carried out. The best reconstruction performance was achieved using an algorithm that folded in two pieces of a priori knowledge of the vascular density distributions: (1) the object is dilute, consisting mainly of a void; and (2) the density distribution in the reconstructions is most likely to be non-negative. Both the signal-to-noise ratio (SNR) and the signal to out-of-focus blur were quantitated. Compared to tomosynthetic reconstruction (backprojection), the amount of residual blur from out-of-focus planes was significantly reduced with only a small penalty in diminished SNR. The combined effect resulted in significant qualitative image improvement for real arterial distributions as demonstrated in a canine arterial imaging example.

Photoacoustic Spectroscopic Imaging of Intra-Tumor Heterogeneity and Molecular Identification

Purpose. To evaluate photoacoustic spectroscopy as a potential imaging modality capable of measuring intra-tumor heterogeneity and spectral features associated with hemoglobin and the molecular probe indocyanine green (ICG). Material and Methods. Immune deficient mice were injected with wildtype and VEGF enhanced MCF-7 breast cancer cells or SKOV3x ovarian cancer cells, which were allowed to grow to a size of 6-12 mm in diameter. Two mice were imaged alive and after euthanasia for (oxy/deoxy)-hemoglobin content. A 0.4 mL volume of 1 μg/mL concentration of ICG was injected into the tail veins of two mice prior to imaging using the photoacoustic computed tomography (PCT) spectrometer (Optosonics, Inc., Indianapolis, IN 46202) scanner. Mouse images were acquired for wavelengths spanning 700-920 nm, after which the major organs were excised, and similarly imaged. A histological study was performed by sectioning the organ and optically imaging the fluorescence distribution. Results. Calibration of PCT-spectroscopy with different samples of oxygenated blood reproduced a hemoglobin dissociation curve consistent with empirical formula with an average error of 5.6%. In vivo PCT determination of SaO2 levels within the tumor vascular was measurably tracked, and spatially correlated to the periphery of the tumor. Statistical and systematic errors associated with hypoxia were estimated to be 10 and 13%, respectively. Measured ICG concentrations determined by contrast-differential PCT images in excised organs (tumor, liver) were approximately 0.8 μg/mL, consistent with fluorescent histological results. Also, the difference in the ratio of ICG concentration in the gall bladder-to-vasculature between the mice was consistent with excretion times between the two mice. Conclusion. PCT spectroscopic imaging has shown to be a noninvasive modality capable of imaging intra-tumor heterogeneity of (oxy/deoxy)-hemoglobin and ICG in vivo, with an estimated error in SaO2 at 17% and in ICG at 0.8 μg/mL in excised tissue. Ongoing development of spectroscopic analysis techniques, probe development, and calibration techniques are being developed to improve sensitivity to both exogenous molecular probes and (oxy/deoxy)-hemoglobin fraction.

Application of thermoacoustic computed tomography to breast imaging

Acoustic pressure waves are induced in soft tissue whenever time-varying radiation is absorbed. By recording these time- dependent pressure waves over a sufficient number of angles surrounding the tissue being imaged, it is possible to reconstruct the pattern of radiation absorption within the tissue in three dimensions with spatial resolution that is independent of the carrier frequency of the irradiating energy. We recently constructed the worlds first thermoacoustic computed tomography (TACT) scanner, which exploits this physical interaction. Initial in vivo imaging of a human breast was performed using safe levels of 434 MHz radiation. Good soft tissue differentiation with 2 - 5 mm spatial resolution to a depth of 40 mm was achieved. The absorption properties of the breast and the irradiation pattern within the breast determined the TACT image contrast. The length of the RF pulse, the size of the transducers and their frequency response, the geometry of the detector array, and the reconstruction algorithm that was used determined the spatial resolution. We conclude that TACT imaging may have application to breast cancer detection.

Improving limited-view reconstruction in photoacoustic tomography by incorporating a priori boundary information

Photoacoustic tomography (PAT) is an emerging ultrasound-mediated biophotonic imaging modality that has great potential for many biomedical imaging applications. In many practical implementations of PAT, the photoacoustic signals are recorded over an aperture that does not enclose the object, which results in a limitedview tomographic reconstruction problem. When conventional reconstruction algorithms are applied to limitedview measurement data, the resulting images can contain severe image artifacts and distortions. To circumvent such artifacts, we exploit a priori information about the locations of boundaries within the object (optical absorption function) to improve the fidelity of the reconstructed images. Such boundary information can be inferred, for example, from a co-registered B-mode ultrasound image or other adjunct imaging study. We develop and implement an iterative reconstruction algorithm that exploits a priori object information in the form of support constraints. We demonstrate that the developed iterative reconstruction algorithm produces images with reduced artifact levels as compared to those produced by a conventional PAT reconstruction algorithm.

Co-registered photoacoustic, thermoacoustic and ultrasound mouse imaging

We have constructed and tested a prototype test bed that allows us to form 3D photoacoustic CT images using near-infrared (NIR) irradiation (700 - 900 nm), 3D thermoacoustic CT images using microwave irradiation (434 MHz), and 3D ultrasound images from a commercial ultrasound scanner. The device utilizes a vertically oriented, curved array to capture the photoacoustic and thermoacoustic data. In addition, an 8-MHz linear array fixed in a horizontal position provides the ultrasound data. The photoacoustic and thermoacoustic data sets are co-registered exactly because they use the same detector. The ultrasound data set requires only simple corrections to co-register its images. The photoacoustic, thermoacoustic, and ultrasound images of mouse anatomy reveal complementary anatomic information as they exploit different contrast mechanisms. The thermoacoustic images differentiate between muscle, fat and bone. The photoacoustic images reveal the hemoglobin distribution, which is localized predominantly in the vascular space. The ultrasound images provide detailed information about the bony structures. Superposition of all three images onto a co-registered hybrid image shows the potential of a trimodal photoacoustic-thermoacoustic-ultrasound small-animal imaging system.