Study of Tissue Phantoms, Tissues, and Contrast Agent with the Biophotoacoustic Radar and Comparison to Ultrasound Imaging for Deep Subsurface Imaging

In this study, the imaging capability of our wide-spectrum frequency-domain photoacoustic (FD-PA) imaging alias ??photoacoustic radar?? methodology for imaging of soft tissues is explored. A practical application of the mathematical correlation processing method with relatively long (1 ms) frequency-modulated optical excitation is demonstrated for reconstruction of the spatial location of the PA sources. Image comparison with ultrasound (US) modality was investigated to see the complementarity between the two techniques. The obtained results with a phased array probe on tissue phantoms and their comparison to US images demonstrated that the FD-PA technique has strong potential for deep subsurface imaging with excellent contrast and high signal-to-noise ratio. FD-PA images of blood vessels in a human wrist and an in vivo subcutaneous tumor in a rat model are presented. As in other imaging modalities, the employment of contrast agents is desirable to improve the capability of medical diagnostics. Therefore, this study also evaluated and characterized the use of Food and Drug Administration (FDA)-approved superparamagnetic iron oxide nanoparticles (SPION) as PA contrast agents.     

Performance benchmarks of an array-based hand-held photoacoustic probe adapted from a clinical ultrasound system for non-invasive sentinel lymph node imaging

Clinical translation of photoacoustic (PA) imaging can be facilitated by integration with commercial ultrasound (US) scanners to enable dual-modality imaging. An array-based US scanner was modified for hand-held PA imaging. The performance was benchmarked in terms of signal-to-noise ratio (SNR), axial spatial resolution and sensitivity. PA images of a tube, filled with methylene blue (MB; approx. 30?mM) and placed at various depths in chicken tissue, were acquired. A 5?cm penetration depth was achieved with an 18.6?dB SNR using a laser fluence of 3?mJ?cm(-2), only one-seventh of the safety limit (20?mJ?cm(-2)). An axial resolution of approximately 400?μm was maintained at all imaging depths. The PA sensitivity to MB placed 2.3?cm deep in chicken tissue was less than 100?μM. Further, after intradermal injection of MB (approx. 30?mM), a rat sentinel lymph node was clearly identified in vivo, beneath a 3.8?cm thick layer of chicken breast. The accumulated concentration of MB in the node was estimated to be approximately 7?mM. The noise-equivalent sensitivities (approx. 2?cm depth) were 17 and 85?μM, ex vivo and in vivo, respectively. These results support the use of this PA system for non-invasive mapping and image-guided needle biopsy of sentinel nodes in breast cancer patients.     

In Vivo Volumetric Photoacoustic Molecular Angiography and Therapeutic Monitoring with Targeted Plasmonic Nanostars

Photoacoustic (PA) imaging promises deeper tissue penetration while maintaining rich optical contrast as compared to other high resolution optical imaging techniques. In this report, a near‐infrared pulse laser serves as the excitation source, and 128 ultrasonic transducers are spirally distributed on a hemispherical surface to receive PA signals for three‐dimensional (3D) image reconstruction. With these attributes, the unique modality produces an isotropic and homogeneous spatial resolution (～200 μm) with penetration depth of centimeters. Cyclic Arg‐Gly‐Asp (RGD) peptides conjugated plasmonic gold nanostars (RGD‐GNS) are designed to specifically target over‐expressed integrin α_vβ₃ on tumor neovasculature, enabling highly sensitive angiography and photothermal therapy (PTT). After the administration of RGD‐GNS, tumor angiogenesis is clearly imaged with enhanced contrast, and the growth of tumor is effectively inhibited by PTT after laser irradiation. This study suggest that the PA angiography with plasmonic RGD‐GNS can be applied as a triple functional platform for tumor diagnosis, PTT, and treatment monitoring. This PA technique offers deeper imaging depth with homogeneous resolution over existing optical imaging techniques for early diagnosis of tumor angiogenesis as well as on‐the‐spot nanotherapeutic evaluation.     

Minimum Variance Beamforming Combined with Adaptive Coherence Weighting Applied to Medical Ultrasound Imaging

Currently, the nonadaptive delay-and-sum (DAS) beamformer is used in medical ultrasound imaging. However, due to its data-independent nature, DAS leads to images with limited resolution and contrast. In this paper, an adaptive minimum variance (MV)-based beamformer that combines the MV and coherence factor (CF) weighting is introduced and adapted to medical ultrasound imaging. MV-adaptive beamformers can improve the image quality in terms of resolution and sidelobes by suppressing off-axis signals, while keeping onaxis ones. In addition, CF weighting can improve contrast and sidelobes by emphasizing the in-phase signals and reducing the out-of-phase ones. Combining MV and CF weighting results in simultaneous improvement of imaging resolution and contrast, outperforming both DAS and MV beamformers. In addition, because of the power of CF in reducing the focusing errors, the proposed method presents satisfactory robustness against sound velocity inhomogeneities, outperforming the regularized MV beamformer. The excellent performance of the proposed beamforming approach is demonstrated by several simulated examples.     

Photoacoustic Imaging: Contrast Agents and Their Biomedical Applications

Photoacoustic (PA) imaging as a fast‐developing imaging technique has great potential in biomedical and clinical applications. It is a noninvasive imaging modality that depends on the light‐absorption coefficient of the imaged tissue and the injected PA‐imaging contrast agents. Furthermore, PA imaging provides superb contrast, super spatial resolution, and high penetrability and sensitivity to tissue functional characteristics by detecting the acoustic wave to construct PA images. In recent years, a series of PA‐imaging contrast agents are developed to improve the PA‐imaging performance in biomedical applications. Here, recent progress of PA contrast agents and their biomedical applications are outlined. PA contrast agents are classified according to their components and function, and gold nanocrystals, gold‐nanocrystal assembly, transition‐metal chalcogenides/MXene‐based nanomaterials, carbon‐based nanomaterials, other inorganic imaging agents, small organic molecules, semiconducting polymer nanoparticles, and nonlinear PA‐imaging contrast agents are discussed. The applications of PA contrast agents as biosensors (in the sensing of metal ions, pH, enzymes, temperature, hypoxia, reactive oxygen species, and reactive nitrogen species) and in bioimaging (lymph nodes, vasculature, tumors, and brain tissue) are discussed in detail. Finally, an outlook on the future research and investigation of PA‐imaging contrast agents and their significance in biomedical research is presented.     

Light‐Responsive Biodegradable Nanorattles for Cancer Theranostics

Cancer nanotheranostics, integrating both diagnostic and therapeutic functions into nanoscale agents, are advanced solutions for cancer management. Herein, a light‐responsive biodegradable nanorattle‐based perfluoropentane‐(PFP)‐filled mesoporous‐silica‐film‐coated gold nanorod (GNR@SiO₂‐PFP) is strategically designed and prepared for enhanced ultrasound (US)/photoacoustic (PA) dual‐modality imaging guided photothermal therapy of melanoma. The as‐prepared nanorattles are composed of a thin mesoporous silica film as the shell, which endows the nanoplatform with flexible morphology and excellent biodegradability, as well as large cavity for PFP filling. Upon 808 nm laser irradiation, the loaded PFP will undergo a liquid–gas phase transition due to the heat generation from GNRs, thus generating nanobubbles followed by the coalescence into microbubbles. The conversion of nanobubbles to microbubbles can improve the intratumoral permeation and retention in nonmicrovascular tissue, as well as enhance the tumor‐targeted US imaging signals. This nanotheranostic platform exhibits excellent biocompatibility and biodegradability, distinct gas bubbling phenomenon, good US/PA imaging contrast, and remarkable photothermal efficiency. The results demonstrate that the GNR@SiO₂‐PFP nanorattles hold great potential for cancer nanotheranostics.     

Encapsulated Conjugated Oligomer Nanoparticles for Real‐Time Photoacoustic Sentinel Lymph Node Imaging and Targeted Photothermal Therapy

Noninvasive and nonionizing imaging of sentinel lymph nodes (SLN) is highly desirable for the detection of breast cancer metastasis through sentinel lymph node biopsy. Photoacoustic (PA) imaging is an emerging imaging technique that can serve as a suitable approach for SLN imaging. Herein, novel conjugated oligomer based nanoparticles (NPs) with strong NIR absorption, good biocompatibility, excellent PA contrast, and good photothermal conversion efficiency are reported. Real‐time PA imaging of SLN reveals high resolution of the NPs via injection from the left forepaw pad. In addition, the surface functionalized NPs can target breast cancer cells and kill them efficiently and specifically through photothermal therapy upon 808 nm laser irradiation. This work shows great potential of the nanoparticle PA contrast agent to serve as a multifunctional probe for photothermal therapy at SLNs to achieve the inhibition of cancer cell metastasis in the near future.     

Post-beamforming second-order Volterra filter for pulse-echo ultrasonic imaging

We present a new algorithm for deriving a second-order Volterra filter (SVF) capable of separating linear and quadratic components from echo signals. Images based on the quadratic components are shown to provide contrast enhancement between tissue and ultrasound contrast agents (UCAs) without loss in spatial resolution. It is also shown that the quadratic images preserve the low scattering regions due to their high dynamic range when compared with standard B-mode or harmonic images. A robust algorithm for deriving the filter has been developed and tested on real-time imaging data from contrast and tissue-mimicking media. Illustrative examples from image targets containing contrast agent and tissue-mimicking media are presented and discussed. Quantitative assessment of the contrast enhancement is performed on both the RF data and the envelope-detected log-compressed image data. It is shown that the quadratic images offer levels of enhancement comparable or exceeding those from harmonic filters while maintaining the visibility of low scattering regions of the image.     

Enhanced Performance of a Molecular Photoacoustic Imaging Agent by Encapsulation in Mesoporous Silicon Nanoparticles

Photoacoustic (PA) imaging allows visualization of the physiology and pathology of tissues with good spatial resolution and relatively deep tissue penetration. The method converts near‐infrared (NIR) laser excitation into thermal expansion, generating pressure transients that are detected with an acoustic transducer. Here, we find that the response of the PA contrast agent indocyanine green (ICG) can be enhanced 17‐fold when it is sealed within a rigid nanoparticle. ICG encapsulated in particles composed of porous silicon (pSiNP), porous silica, or calcium silicate all show greater PA contrast relative to equivalent quantities of free ICG, with the pSiNPs showing the strongest enhancement. A liposomal formulation of ICG performs similar to free ICG, suggesting that a rigid host nanostructure is necessary to enhance ICG performance. The improved response of the nanoparticle formulations is attributed to the low thermal conductivity of the porous inorganic hosts and their ability to protect the ICG payload from photolytic and/or thermal degradation. The translational potential of ICG‐loaded pSiNPs as photoacoustic probes is demonstrated via imaging of a whole mouse brain.     

Ultrasonic backscatter imaging by shear-wave-induced echo phase encoding of target locations

We present a novel method for ultrasound backscatter image formation wherein lateral resolution of the target is obtained by using traveling shear waves to encode the lateral position of targets in the phase of the received echo. We demonstrate that the phase modulation as a function of shear wavenumber can be expressed in terms of a Fourier transform of the lateral component of the target echogenicity. The inverse transform, obtained by measurements of the phase modulation over a range of shear wave spatial frequencies, yields the lateral scatterer distribution. Range data are recovered from time of flight as in conventional ultrasound, yielding a B-mode-like image. In contrast to conventional ultrasound imaging, where mechanical or electronic focusing is used and lateral resolution is determined by aperture size and wavelength, we demonstrate that lateral resolution using the proposed method is independent of the properties of the aperture. Lateral resolution of the target is achieved using a stationary, unfocused, single-element transducer. We present simulated images of targets of uniform and non-uniform shear modulus. Compounding for speckle reduction is demonstrated. Finally, we demonstrate image formation with an unfocused transducer in gelatin phantoms of uniform shear modulus.     

Fluorescence lifetime imaging with near-field scanning optical microscopy

Near-field scanning optical microscopy (NSOM) is a high-resolution scanning probe technique capable of obtaining simultaneous optical and topographic images with spatial resolution of tens of nanometers. We have integrated time-correlated single-photon counting and NSOM to obtain images of fluorescence lifetimes with high spatial resolution. The technique can be used to measure either full fluorescence lifetime decays at individual spots with a spatial resolution of <100 nm or NSOM fluorescence images using fluorescence lifetime as a contrast mechanism. For imaging, a pulsed Ti:sapphire laser was used for sample excitation and fluorescent photons were time correlated and sorted into two time delay bins. The intensity in these bins can be used to estimate the fluorescence lifetime at each pixel in the image. The technique is demonstrated on thin films of poly(9,9'-dioctylfluorene) (PDOF). The fluorescence of PDOF is the results of both inter- and intrapolymer emitting species that can be easily distinguished in the time domain. Fluorescence lifetime imaging with near-field scanning optical microscopy demonstrates how photochemical degradation of the polymer leads to a quenching of short-delay intrachain emission and an increase in the long-delay photons associated with interpolymer emitting species. The images also show how intra- and interpolymer species are uniformly distributed in the films.     

Photoacoustic Probes for Molecular Detection: Recent Advances and Perspectives

Photoacoustic (PA) imaging (PAI) is a noninvasive and nonionizing biomedical imaging modality that combines the advantages of optical imaging and ultrasound imaging. Based on PAI, photoacoustic detection (PAD) is an emerging approach that is involved with the interaction between PA probes and analytes resulting in the changes of photoacoustic signals for molecular detection with rich contrast, high resolution, and deep tissue penetration. This Review focuses on the recent development of PA probes in PAD. The following contents will be discussed in detail: 1) the construction of PA probes; 2) the applications and mechanisms of PAD to different types of analytes, including microenvironments, small biomolecules, or metal ions; 3) the challenges and perspectives of PA probes in PAD.     

Light profile microscopy based on Raman and wavelength resolved luminescence contrast

Light profile microscopy based on contrast from wavelength resolved Raman and luminescence measurements is demonstrated experimentally for the first time. A Raman/multispectral light profile microscope (RMSLPM) has been constructed based on a line profiling geometry in which the sample is irradiated with a tightly focused laser beam (of ten micrometers radius or less) behind a polished view surface and the resulting line image is dispersed over the wavelength using an imaging spectrograph. The instrumentation developed in this laboratory has a spectral resolution approaching 10 cm(-1) and an (actual) depth independent spatial resolution of 6-8 times the Rayleigh diffraction limit, limited at present by optical aberrations and alignment. The technique has the potential to image at approximately twice the Rayleigh diffraction limit. The spectral signatures reconstructed from a variety of common industrial polymers show excellent agreement with reference spectra from the literature, and may be used to identify individual layers in depth images of unknown materials. RMS-LPM image data based on luminescence contrast have also been used to provide concentration depth profiles of additives and degradation products in injection molded samples of high-density poly(ethylene) (HDPE).     

Optimal apodization design for medical ultrasound using constrained least squares part I: theory

Aperture weighting functions are critical design parameters in the development of ultrasound systems because beam characteristics affect the contrast and point resolution of the final output image. In previous work by our group, we developed a metric that quantifies a broadband imaging system's contrast resolution performance. We now use this metric to formulate a novel general ultrasound beamformer design method. In our algorithm, we use constrained least squares (CLS) techniques and a linear algebra formulation to describe the system point spread function (PSF) as a function of the aperture weightings. In one approach, we minimize the energy of the PSF outside a certain boundary and impose a linear constraint on the aperture weights. In a second approach, we minimize the energy of the PSF outside a certain boundary while imposing a quadratic constraint on the energy of the PSF inside the boundary. We present detailed analysis for an arbitrary ultrasound imaging system and discuss several possible applications of the CLS techniques, such as designing aperture weightings to maximize contrast resolution and improve the system depth of field.     

Evaluation of Wheat and Maize Seeds by Photoacoustic Microscopy

In this study photoacoustic microscopy (PAM) was used to evaluate the deterioration of wheat and maize seeds. The analyzed seeds were obtained from different productive cycles. In the PAM experimental setup a modulated focused laser beam impinges on the sample. Due to the periodic heating it is possible to obtain a photoacoustic signal (PA) by using a microphone sensor; from this sensor an electrical signal is obtained due to the temperature variation in the sample. Since there are local differences in seed structure, the optical absorption of the laser beam and thermal properties are inhomogeneous, which produces a different PA signal in each point of the sample. By taking the set of the obtained PA signals as a function of their positions, it is possible to obtain a PA image. The image resolution was 50 μm for each seed. Therefore, the main objective of the present study was to obtain PA images of wheat and maize seeds, from different productive cycles (i.e., from different annual cycles). The analysis has shown that PAM is an alternative technique to evaluate seed deterioration. Also, the optical absorption spectra of these seeds were obtained by PA spectroscopy.     

Eigenspace-based minimum variance beamforming applied to medical ultrasound imaging

Recently, adaptive beamforming methods have been successfully applied to medical ultrasound imaging, resulting in significant improvement in image quality compared with non-adaptive delay-and-sum (DAS) beamformers. Most of the adaptive beamformers presented in the ultrasound imaging literature are based on the minimum variance (MV) beamformer which can significantly improve the imaging resolution, although their success in enhancing the contrast has not yet been satisfactory. It is desirable for the beamformer to improve the resolution and contrast at the same time. To this end, in this paper, we have applied the eigenspace-based MV (EIBMV) beamformer to medical ultrasound imaging and have shown a simultaneous improvement in imaging resolution and contrast. EIBMV beamformer utilizes the eigenstructure of the covariance matrix to enhance the performance of the MV beamformer. The weight vector of the EIBMV is found by projecting the MV weight vector onto a vector subspace constructed from the eigenstructure of the covariance matrix. Using EIBMV weights instead of the MV ones leads to reduced sidelobes and improved contrast, without compromising the high resolution of the MV beamformer. In addition, the proposed EIBMV beamformer presents a satisfactory robustness against data misalignment resulting from steering vector errors, outperforming the regularized MV beamformer.     

Quantum dots as multimodal photoacoustic and photothermal contrast agents

Quantum dots (QDs) have primarily been developed as fluorescent probes with unique optical properties. We herein demonstrate an extension of these QD utilities to photoacoustic (PA) and photothermal (PT) microscopy, using a nanosecond pulse laser excitation (420-900 nm, 8 ns, 10(-3)-10 J/cm(2)). The laser-induced PA, PT and accompanying bubble formation phenomena were studied with an advanced multifunctional microscope, which integrates fluorescence, PA, PT imaging, and PT thermolens modules. It was demonstrated that QDs, in addition to being excellent fluorescent probes, can be used as PA and PT contrast agents and sensitizers, thereby providing an opportunity for multimodal high resolution (300 nm) PA-PT-fluorescent imaging as well as PT therapy. Further improvements for this technology are suggested by increasing the conversion of laser energy in PT, PA, and bubble phenomena in hybrid multilayer QDs that have optimized absorption, thermal, and acoustic properties.     

Raman microspectroscopy: a comparison of point, line, and wide-field imaging methodologies

Three different Raman microspectroscopic imaging methodologies using a single experimental configuration are compared; namely, point and line mapping, as representatives of serial imaging approaches, and direct or wide-field Raman imaging employing liquid-crystalline tunable filters are surveyed. Raman imaging data acquired with equivalent low-power 514.5-nm laser excitation and a cooled CCD camera are analyzed with respect to acquisition times, image quality, spatial resolution, intensity profiles along spatial coordinates, and spectral signal-to-noise ratios (SNRs). Point and line mapping techniques provide similar SNRs and reconstructed Raman images at spatial resolutions of approximately 1.1 microm. In contrast, higher spatial resolution is obtained by direct, global imaging (approximately 313 nm), allowing subtle morphological features on test samples to be resolved.     

High-resolution ab initio three-dimensional x-ray diffraction microscopy

Coherent x-ray diffraction microscopy is a method of imaging nonperiodic isolated objects at resolutions limited, in principle, by only the wavelength and largest scattering angles recorded. We demonstrate x-ray diffraction imaging with high resolution in all three dimensions, as determined by a quantitative analysis of the reconstructed volume images. These images are retrieved from the three-dimensional diffraction data using no a priori knowledge about the shape or composition of the object, which has never before been demonstrated on a nonperiodic object. We also construct two-dimensional images of thick objects with greatly increased depth of focus (without loss of transverse spatial resolution). These methods can be used to image biological and materials science samples at high resolution with x-ray undulator radiation and establishes the techniques to be used in atomic-resolution ultrafast imaging at x-ray free-electron laser sources.     

High-resolution coregistered intravascular imaging with integrated ultrasound and optical coherence tomography probe

We report an integrated ultrasound (US) and optical coherence tomography (OCT) probe and system for intravascular imaging. The dual-function probe is based on a 50 MHz focused ring US transducer, with a centric hole for mounting OCT probe. The coaxial US and light beams are steered by a 45° mirror to enable coregistered US∕OCT imaging simultaneously. Lateral resolution of US is improved due to focused ultrasonic beam. Mirror effects on US were investigated and invitro imaging of a rabbit aorta has been carried out. The combined US-OCT system demonstrated high resolution in visualizing superficial arterial structures while retaining deep penetration of ultrasonic imaging.