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.

Analysis of substrate specificity and kinetics of cyclic nucleotide phosphodiesterases with N'-methylanthraniloyl-substituted purine and pyrimidine 3',5'-cyclic nucleotides by fluorescence spectrometry.

As second messengers, the cyclic purine nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) play an essential role in intracellular signaling. Recent data suggest that the cyclic pyrimidine nucleotides cytidine 3',5'-cyclic monophosphate (cCMP) and uridine 3',5'-cyclic monophosphate (cUMP) also act as second messengers. Hydrolysis by phosphodiesterases (PDEs) is the most important degradation mechanism for cAMP and cGMP. Elimination of cUMP and cCMP is not completely understood, though. We have shown that human PDEs hydrolyze not only cAMP and cGMP but also cyclic pyrimidine nucleotides, indicating that these enzymes may be important for termination of cCMP- and cUMP effects as well. However, these findings were acquired using a rather expensive HPLC/mass spectrometry assay, the technical requirements of which are available only to few laboratories. N'-Methylanthraniloyl-(MANT-)labeled nucleotides are endogenously fluorescent and suitable tools to study diverse protein/nucleotide interactions. In the present study, we report the synthesis of new MANT-substituted cyclic purine- and pyrimidine nucleotides that are appropriate to analyze substrate specificity and kinetics of PDEs with more moderate technical requirements. MANT-labeled nucleoside 3',5'-cyclic monophosphates (MANT-cNMPs) are shown to be substrates of various human PDEs and to undergo a significant change in fluorescence upon cleavage, thus allowing direct, quantitative and continuous determination of hydrolysis via fluorescence detection. As substrates of several PDEs, MANT-cNMPs show similar kinetics to native nucleotides, with some exceptions. Finally, they are shown to be also appropriate tools for PDE inhibitor studies.

Human cyclic nucleotide phosphodiesterases possess a much broader substrate-specificity than previously appreciated.

Phosphodiesterases (PDEs) capable of degrading cAMP and cGMP are indispensable for the regulation of cyclic nucleotide-mediated signals. The existence of other cyclic nucleotides such as cCMP and cUMP has been discussed controversially in the literature. Despite publications on PDEs hydrolyzing cCMP or cUMP, the molecular identity of such enzymes remained elusive. Recently, we have provided evidence for a role of cCMP as second messenger in vascular relaxation and inhibition of platelet aggregation. Using an HPLC-MS based assay, here, we show that human PDEs belonging to various families hydrolyze not only cAMP and cGMP but also other cyclic nucleotides.

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.

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.

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.

Thermoacoustic computed tomography using a conventional linear transducer array.

We report on methodology for employing a conventional linear transducer array as a thermoacoustic detector in a thermoacoustic computed tomography (TCT) device, which has been designed for imaging small animals, e.g., athymic nude mice. We tested this concept using a 5 MHz, 128-element linear array (Acuson model L538). Thermoacoustic emissions were induced in a tissue-mimicking phantom using a Nd:YAg laser, operated at 1064 nm. Two-dimensional, axial "slice" images were formed using a filtered-backprojection algorithm. In-plane spatial resolution was measured as better than 200 microns with a slice thickness of 1.5 mm (full width at half maximum). The same detector, when operated as a conventional phased array, produced conventional ultrasound images in perfect registration with the TCT images.