Photoacoustic imaging of living subjects offers higher spatial resolution and allows deeper tissues to be imaged compared with most optical imaging techniques 1-7 . As many diseases do not exhibit a natural photoacoustic contrast, especially in their early stages, it is necessary to administer a photoacoustic contrast agent. A number of contrast agents for photoacoustic imaging have been suggested previously 8-15 , but most were not shown to target a diseased site in living subjects. Here we show that single-walled carbon nanotubes conjugated with cyclic Arg-Gly-Asp (RGD) peptides can be used as a contrast agent for photoacoustic imaging of tumours. Intravenous administration of these targeted nanotubes to mice bearing tumours showed eight times greater photoacoustic signal in the tumour than mice injected with non-targeted nanotubes. These results were verified ex vivo using Raman microscopy. Photoacoustic imaging of targeted single-walled carbon nanotubes may contribute to non-invasive cancer imaging and monitoring of nanotherapeutics in living subjects 16 .Recently, we reported on the conjugation of cyclic RGD containing peptides to single-walled carbon nanotubes 17 (SWNT-RGD) that is stable in serum. The single-walled carbon nanotubes, which were 1-2 nm in diameter and 50-300 nm in length were coupled to the RGD peptides through polyethylene glycol-5000 grafted phospholipid (PL-PEG 5000 ). These SWNT-RGD conjugates bind with high affinity to α v β 3 integrin, which is overexpressed in tumour neovasculature, and to other integrins expressed by tumours but with lower *e-mail: sgambhir@stanford.edu. Author contributions A.D. built the photoacoustic instrument, designed and performed the experiments and wrote the paper. C.Z. designed, performed and analysed the Raman experiments. S.K. built the photoacoustic instrument and designed the experiments. S.V. designed and built the photoacoustic instrument. S.B. performed the experiments and helped write the paper. Z.L. synthesized the single-walled carbon nanotube conjugates. J.L. performed the cell uptake studies. B.R.S. helped write the paper. T.M. and O.O. helped design the photoacoustic instrument. Z.C. helped perform the comparison to fluorescence imaging. X.C. provided the RGD peptides, performed the fluorescence imaging of QD-RGD conjugates and helped write the manuscript. H.D. was responsible for single-walled carbon nanotube conjugation synthesis. B.T.K. was responsible for building the photoacoustic instrument. S.S.G. was responsible for experimental design and wrote the paper.Author information Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to S.S.G. (Fig. 1a). Our photoacoustic instrument 20 used a single-element focused transducer to raster scan the object under study, which was illuminated through a fibre head (see Methods and Supplementary Information, Fig. S1). In a phantom study we measured the photoacoustic signal of pla...

The current state of novel technology, surface microfabricated ultrasonic transducers, is reported. Experiments demonstrating both air and water transmission are presented. Air-coupled longitudinal wave transmission through aluminum is demonstrated, implying a 110 dB dynamic range for transducers at 2.3 MHz in air. Water transmission experiments from 1 to 20 MHz are performed, with a measured 60 dB SNR at 3 MHz. A theoretical model is proposed that agrees well with observed transducer behavior. Most significantly, the model is used to demonstrate that microfabricated ultrasonic transducers constitute an attractive alternative to piezoelectric transducers in many applications.

Photoacoustic imaging is an emerging modality that overcomes to a great extent the resolution and depth limitations of optical imaging while maintaining relatively high-contrast. However, since many diseases will not manifest an endogenous photoacoustic contrast, it is essential to develop exogenous photoacoustic contrast agents that can target diseased tissue(s). Here we present a novel photoacoustic contrast agent, Indocyanine Green dye-enhanced single walled carbon nanotube (SWNT-ICG). We conjugated this contrast agent with cyclic Arg-Gly-Asp (RGD) peptides to molecularly target the α v β 3 integrins, which are associated with tumor angiogenesis. Intravenous administration of this tumor-targeted contrast agent to tumor-bearing mice showed significantly higher photoacoustic signal in the tumor than in mice injected with the untargeted contrast agent. The new contrast agent gave a markedly 300-times higher photoacoustic contrast in living tissues than previously reported SWNTs, leading to sub-nanomolar sensitivities. Finally, we show that the new contrast agent can detect ~20-times fewer cancer cells than previously reported SWNTs.Photoacoustic imaging is an emerging modality based on the photoacoustic effect where light is converted into ultrasound waves that are detected outside the subject of interest 1 . Photoacoustic imaging has been used in numerous applications where intrinsic contrast is available such as visualizing blood vessels structure 2 , thermal burns 3 and melanoma 4 . However, most diseases will not show photoacoustic contrast, thereby requiring the use of an exogenous contrast agent which will target the diseased tissue. The main challenge in designing such contrast agent remains creating an agent that produces sufficient photoacoustic signal in order to be detected in low concentration, while being able to target the diseased tissue(s). In * sgambhir@stanford.edu; hdai1@stanford.edu. † These authors contributed equally to this work Supporting Information Description of particles synthesis, photoacoustic imaging instrument, experimental procedures, statistical methods, and in-vitro characterization of the particles including serum-stability, photobleaching and cell-uptake study.

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Photoacoustic tomography is a rapidly growing imaging modality that can provide images of high spatial resolution and high contrast at depths up to 5 cm. We report here the design, synthesis and evaluation of an activatable probe that shows great promise in enabling detection of the cleaved probe in the presence of the high levels of non-activated, un-cleaved probe, a difficult task to attain in absorbance-based modality. Before the cleavage by its target, proteolytic enzyme MMP-2, the probe, an activatable cell penetrating peptide, Ceeee[Ahx]PLGLAGrrrrrK, labeled with two chromophores, BHQ3 and Alexa750, shows photoacoustic signal of similar intensity at the two wavelengths corresponding to the absorption maxima of the chromophores, 675 and 750 nm. Subtraction of the images taken at these two wavelengths makes the probe effectively photoacoustically silent as the signals at these two wavelengths essentially cancel out. After the cleavage, the dye associated with the cell penetrating part of the probe(CPP), BHQ3, accumulates in the cells, while the other dye diffuses away, resulting in photoacoustic signal seen only at one of the wavelengths, 675 nm. The subtraction of the photoacoustic images at two wavelengths reveals the location of the cleaved (activated) probe. In the search for the chromophores that are best suited for photoacoustic imaging we have investigated photoacoustic signal of five chromophores absorbing in the NIR region. We have found that the photoacoustic signal did not correlate with the absorbance and fluorescence of the molecules, as the highest photoacoustic signal arose from the least absorbing quenchers BHQ3 and QXL 680.

Photoacoustic imaging is a unique modality that overcomes to a great extent the resolution and depth limitations of optical imaging while maintaining relatively high-contrast. However, since many diseases will not manifest an endogenous photoacoustic contrast, it is essential to develop exogenous photoacoustic contrast agents that can target diseased tissue(s). Here we present a family of novel photoacoustic contrast agents that are based on the binding of small optical dyes to single walled carbon nanotubes (SWNT-dye). We synthesized five different SWNT-dye contrast agents using different optical dyes, creating five “flavors” of SWNT-dye nanoparticles. In particular, SWNT that were coated with either QSY21 (SWNT-QSY) or Indocyanine Green (SWNT-ICG) exhibited over 100-times higher photoacoustic contrast in living animals compared to plain SWNTs, leading to subnanomolar sensitivities. We then conjugated the SWNT-dye conjugates with cyclic Arg-Gly-Asp (RGD) peptides to molecularly target the αvβ3 integrin, which is associated with tumor angiogenesis. Intravenous administration of these tumor-targeted imaging agents to tumor-bearing mice showed significantly higher photoacoustic signal in the tumor than in mice injected with the untargeted contrast agent. Finally, we were able to spectrally separate the photoacoustic signals of SWNT-QSY and SWNT-ICG in living animals injected subcutaneously with both particles in the same location, opening the possibility for multiplexing in vivo studies.

For three-dimensional (3D) ultrasound imaging, connecting elements of a two-dimensional (2D) transducer array to the imaging system's front-end electronics is a challenge because of the large number of array elements and the small element size. To compactly connect the transducer array with electronics, we flip-chip bond a 2D 16 x 16-element capacitive micromachined ultrasonic transducer (CMUT) array to a custom-designed integrated circuit (IC). Through-wafer interconnects are used to connect the CMUT elements on the top side of the array with flip-chip bond pads on the back side. The IC provides a 25-V pulser and a transimpedance preamplifier to each element of the array. For each of three characterized devices, the element yield is excellent (99 to 100% of the elements are functional). Center frequencies range from 2.6 MHz to 5.1 MHz. For pulse echo operation, the average - 6-dB fractional bandwidth is as high as 125%. Transmit pressures normalized to the face of the transducer are as high as 339 kPa and input-referred receiver noise is typically 1.2 to 2.1 mPa/pHz. The flip-chip bonded devices were used to acquire 3D synthetic aperture images of a wire-target phantom. Combining the transducer array and IC, as shown in this paper, allows for better utilization of large arrays, improves receive sensitivity, and may lead to new imaging techniques that depend on transducer arrays that are closely coupled to IC electronics.

We report the use of focused acoustic beams to eject discrete droplets of controlled diameter and velocity from a free-liquid surface. No nozzles are involved. Droplet formation has been experimentally demonstrated over the frequency range of 5–300 MHz, with corresponding droplet diameters from 300 to 5 μm. The physics of droplet formation is essentially unchanged over this frequency range. For acoustic focusing elements having similar geometries, droplet diameter has been found to scale inversely with the acoustic frequency. A simple model is used to obtain analytical expressions for the key parameters of droplet formation and their scaling with acoustic frequency. Also reported is a more detailed theory which includes the linear propagation of the focused acoustic wave, the coupling of the acoustic fields to the initial surface velocity potential, and the subsequent dynamics of droplet formation. This latter phase is modeled numerically as an incompressible, irrotational process using a boundary integral vortex method. For simulations at 5 MHz, this numerical model is very successful in predicting the key features of droplet formation.