Photoacoustic tomography: in vivo imaging from organelles to organs.

It is well documented that photoacoustic imaging has the capability to differentiate tissue based on the spectral characteristics of tissue in the optical regime. The imaging depth in tissue exceeds standard optical imaging techniques, and systems can be designed to achieve excellent spatial resolution. A natural extension of imaging the intrinsic optical contrast of tissue is to demonstrate the ability of photoacoustic imaging to detect contrast agents based on optically absorbing dyes that exhibit well defined absorption peaks in the infrared. The ultimate goal of this project is to implement molecular imaging, in which Herceptin^TM, a monoclonal antibody that is used as a therapeutic agent in breast cancer patients that over express the HER2 gene, is labeled with an IR absorbing dye, and the resulting in vivo bio-distribution is mapped using multi-spectral, infrared stimulation and subsequent photoacoustic detection. To lay the groundwork for this goal and establish system sensitivity, images were collected in tissue mimicking phantoms to determine maximum detection depth and minimum detectable concentration of Indocyanine Green (ICG), a common IR absorbing dye, for a single angle photoacoustic acquisition. A breast mimicking phantom was constructed and spectra were also collected for hemoglobin and methanol. An imaging schema was developed that made it possible to separate the ICG from the other tissue mimicking components in a multiple component phantom. We present the results of these experiments and define the path forward for the detection of dye labeled Herceptin^TM in cell cultures and mice models.

Owing to its unique ability to capture volumetric tomographic information with a single light flash, optoacoustic (OA) tomography has recently demonstrated ultrafast imaging speeds ultimately limited by the ultrasound time-of-flight. The method’s scalability and the achievable spatial resolution are yet limited by the narrow bandwidth of piezo-composite arrays currently employed for OA signal detection. Here we report on the first implementation of high-density spherical array technology based on flexible polyvinylidene difluoride films featuring ultrawideband (0.3–40 MHz) sub mm2 area elements, thus enabling real-time multi-scale volumetric imaging with 22–35 µm spatial resolution, superior image fidelity and over an order of magnitude signal-to-noise enhancement compared to piezo-composite equivalents. We further demonstrate five-dimensional (spectroscopic, time-resolved, volumetric) imaging capabilities by visualizing fast stimulus-evoked cerebral oxygenation changes in mice and performing real-time functional angiography of deep human micro-vasculature. The new technology thus leverages the true potential of OA for quantitative high-resolution visualization of rapid bio-dynamics across scales.

Photoacoustic imaging is a recent and rapidly developping technique that can provide images of optical absorption at depth in soft tissue. In this work, photoacoustic imaging is applied to detect tissue lesion induced by high-intensity focused-ultrasound (HIFU).Tissue changes during HIFU therapy include changes in optical tissue properties that may be detected using photoacoustics. However, although it has been demonstrated that HIFU lession could indeed be detected using photoacoustics (Chitnis et al., JBO 15(2), 2010, Cui et al., JBO 15(2), 2010), the exact origin of the observed contrast remains unclear. In this study, lesions induced in different types of biological tissue are imaged in vitro using a photoacoustic microscope. Lesions are induced in the tissue samples with therapeutic ultrasound in the MHz frequency range, and a photoacoustic microscope with imaging frequencies from the MHz to the tens of MHz range, are used to obtain acoustic-resolution photoacoustic images of lesions located at different depths in tissue. Several optical wavelengths, from 532 nm to the therapeutic window in the near-infrared are used to perform a spectroscopic study of the observed contrast.

Visualizing and characterizing vascular structures is important for many areas of health care, from accessing difficult veins and arteries for laboratory testing, to diagnosis and treatment of cardiovascular disease. Photoacoustic (PA) imaging, one of the fastest growing fields of biomedical imaging, is well suited for this task. PA imaging is based on the photoacoustic effect, which starts with a pulsed laser source incident on biological tissue. If the wavelength of the source matches an absorption wavelength of a chromophore within the tissue, a portion of the pulse energy is absorbed by the chromophore and converted into heat. A subsequent increase in temperature, followed by an increase in pressure occurs. Acoustic waves are emitted when this pressure relaxes, which can be detected at the surface of the tissue. PA imaging is considered absorption based, therefore spectroscopic information can be extracted. Yet, unlike purely optical imaging techniques, multiple centimeters of depth can be imaged. Vascular structures, in particular, can be viewed with high contrast using PA imaging, because the absorption coefficient of blood is up to six orders of magnitude higher than surrounding tissues [1]. Additional chromophores, such as lipids in atherosclerotic plaque, are beginning to be imaged using PA techniques in vitro [2]. Contacting piezoelectric transducers are often used for detecting acoustic waves in PA imaging. In many clinical situations, however, these transducers are unfavorable due to environmental constraints, or frequency response and spatial resolution needs [3]. The ability to image without contacting the patient, and without the need for personnel to manually control a transducer, creates the opportunity for this technique to be useful in both clinical and surgical procedures. A non-contact system has the potential to be used by practitioners who require access to the vascular system such as surgeons, nurses, and phlebotomists. An additional application of this device is as an aid for surgical procedures, such as catheter interventions. Interferometry is beginning to be explored as a method of non-contact PA imaging [4]. In this study, an experiment toward non-contact photoacoustic imaging was accomplished. A broadband interferometer was used, which measured particle velocity remotely. Absorbing structures in tissue mimicking phantoms were detected at various depths with the use of an all-optical, computer-controlled photoacoustic system.

Photoacoustic imaging has attracted interest for its capacity to capture functional spectral information with high spatial and temporal resolution in biological tissues. Several photoacoustic imaging systems have been commercialized recently, but they are variously limited by non-clinically relevant designs, immobility, single anatomical utility (e.g., breast only), or non-programmable interfaces. Here, we present a real-time clinical photoacoustic and ultrasound imaging system which consists of an FDA-approved clinical ultrasound system integrated with a portable laser. The system is completely programmable, has an intuitive user interface, and can be adapted for different applications by switching handheld imaging probes with various transducer types. The customizable photoacoustic and ultrasound imaging system is intended to meet the diverse needs of medical researchers performing both clinical and preclinical photoacoustic studies.

Photoacoustic (PA) imaging (PAI), or optoacoustic imaging, is a hybrid imaging modality that combines optical absorption contrast and ultrasound image formation. In PAI, the target is excited by a short laser pulse and subsequently absorbs the photon energy, leading to a transient local temperature rise. The temperature rise induces a local pressure rise that propagates as acoustic waves. As acoustic waves generally undergo less scattering and attenuation in tissue compared with light, PAI can provide high-resolution images in both the optical (quasi)ballistic and (quasi)diffusive regimes (1,2). Based on the image formation methods, PAI can be classified into two categories: photoacoustic microscopy (PAM) and photoacoustic computed tomography (PACT). PAM uses a focused excitation light beam and/or a focused single-element ultrasonic transducer for direct image formation through position scanning (1,2). PAM has a maximum imaging depth ranging from a few hundred micrometers to a few millimeters with spatial resolution ranging from sub-micrometer to sub-millimeter (2,3). PAM can be further classified into optical-resolution PAM (OR-PAM) and acoustic-resolution PAM (AR-PAM). For both OR-PAM and AR-PAM, the axial resolution is determined by the bandwidth of the ultrasonic transducer (4). OR-PAM works in the optical (quasi)ballistic regime, whereas the light is tightly focused that it can penetrate about one optical transport mean free path (~1 mm in soft tissue). Therefore, the lateral resolution of OR-PAM is mainly determined by the optical focal spot size (4-6). The optical focusing is diffraction-limited as λ/2NA, where λ is the light wavelength, and NA is the numerical aperture of objective lens. On the contrary, in AR-PAM, the laser is loosely focused to fulfill the entire acoustic focal spot, thereby penetrating a few optical transport mean free paths, i.e., in the quasi-diffusive regime. The lateral resolution of AR-PAM is thus determined by the size of acoustic focus (4,7,8), limited by acoustic diffraction. In PACT, the object is illuminated with a wide-field laser beam in the diffusive regime, and the generated acoustic waves are detected at multiple locations or by using a multi-element transducer array. The image formed by PACT is reconstructed by an inverse algorithm. The spatial resolution of PACT is fundamentally limited by acoustic diffraction, and additionally affected by the directionality and spacing of the detector elements (9). Recently, several studies have shown that sub-diffraction imaging of biological samples can be achieved through PAI by breaking optical-diffraction limit in the (quasi)ballistic regime or acoustic-diffraction limit in the (quasi)diffusive regime, which have opened new possibilities for fundamental biological studies. Yao et al. developed a photoimprint PAM using the intensity-dependent photobleaching effect and acquired a melanoma cell PA image with a lateral resolution of 90 nm (10). Danielli et al. reported a label-free PA nanoscopy based on the optical-absorption saturation effect and acquired a mitochondria PA image with a lateral resolution of 88 nm (11). Chaigne et al. exploited the sample-dynamics-induced inherent temporal fluctuation in the PA signals and achieved a resolution enhancement of about 1.4 over conventional PACT (12). Murray et al. broke the acoustic diffraction limit by implementing a blind speckle illumination and block-FISTA reconstruction algorithm and achieved a resolution close to the acoustic speckle size (13). Dean-Ben et al. also overcame the acoustic diffraction limit by incorporating rapid sequential acquisition of 3D PA images of flowing absorbing particles and further enhanced the visibility of structures under limited-view tomographic conditions (14). Conkey et al. optimized wavefront shaping with photoacoustic feedback and achieved up to ten times improvement in signal-to-noise ratio and five to six times sub-acoustic-diffraction resolution (15). In this concise review, we summarize and analyze the recent development in super-resolution (SR) PAI (SR-PAI) in both the optical (quasi)ballistic and (quasi)diffusive regime, as well as their representative applications. We also discuss the current challenges in SR-PAI and envision the potential breakthroughs.

Current systems designed for deep photoacoustic (PA) imaging typically use a low repetition rate, high power pulsed laser to provide a ns-scale pulse illuminating a large tissue volume. Acoustic signals recorded on each laser firing can be used to reconstruct a complete 2-D (3-D) image of sources of heat release within that region. Using broad-beam excitation, the maximum frame rate of the imaging system is restricted by the pulse repetition rate of the laser. An alternate illumination approach is proposed based on fast scanning by a low energy (~ 1 mJ) high repetition rate (up to a few kHz) narrow laser beam (~1 mm) along the tissue surface over a region of interest. A final PA image is produced from the summation of individual PA images reconstructed at each laser beam position. This concept can take advantage of high repetition rate fiber lasers to create PA images with much higher frame rates than current systems, enabling true real-time integration of photoacoustics with ultrasound imaging. As an initial proof of concept, we compare conventional broad beam illumination to a scanned beam approach in a simple model system. Two transparent teflon tubes with diameters of 1.6 mm and 0.8 mm were filled with ink having an absorption coefficient of 5 cm -1 . These tubes were buried inside chicken breast tissue acting as an optical scattering medium. They were separated by 3 mm or 10 mm to test spatial and contrast resolution for the two scan formats. The excitation wavelength was 700 nm. The excitation source is a traditional OPO pumped by a Q-switched Nd:YAG laser with doubler. Photoacoustic images were reconstructed using signals from a small, scanned PVDF transducer acting as an acoustic array. Two different illumination schemes were compared: one was 15 mm x 10 mm in cross section and acted as the broad beam; the other was 5 mm x 2 mm in cross section (15 times smaller than the broad beam case) and was scanned over an area equivalent to broad beam illumination. Multiple images obtained during narrow beam scanning were added together to form one PA image equivalent to the single-shot broad beam one. Results of the phantom study indicate that PA images formed by narrow beam scanning excitation can be equivalent to one shot broad beam illumination in signal to noise ratio and spatial resolution. Future studies will focus on high repetition-rate laser sources and scan formats appropriate for real-time, integrated deep photoacoustic/ultrasonic imaging.

Numerical investigation on improving photoacoustic imaging contrast based on temperature difference effect in biological tissues

Chapter 13 - Photoacoustic Molecular Imaging: Principles and Practice

Recent progress in photoacoustic molecular imaging

Real-time photoacoustic and ultrasonic dual-modality imaging system for early gastric cancer: Phantom and ex vivo studies

Photoacoustic (PA) imaging using contrast agents with strong near-infrared-II (NIR-II, 1000-1700 nm) absorption enables deep penetration into biological tissue. Besides, biocompatibility and biodegradability are essential for clinical translation. Herein, we developed biocompatible and biodegradable germanium nanoparticles (GeNPs) with high photothermal stability as well as strong and broad absorption for NIR-II PA imaging. We first demonstrate the excellent biocompatibility of the GeNPs through experiments, including the zebrafish embryo survival rates, nude mouse body weight curves, and histological images of the major organs. Then, comprehensive PA imaging demonstrations are presented to showcase the versatile imaging capabilities and excellent biodegradability, including in vitro PA imaging which can bypass blood absorption, in vivo dual-wavelength PA imaging which can clearly distinguish the injected GeNPs from the background blood vessels, in vivo and ex vivo PA imaging with deep penetration, in vivo time-lapse PA imaging of a mouse ear for observing biodegradation, ex vivo time-lapse PA imaging of the major organs of a mouse model for observing the biodistribution after intravenous injection, and notably in vivo dual-modality fluorescence and PA imaging of osteosarcoma tumors. The in vivo biodegradation of GeNPs is observed not only in the normal tissue but also in the tumor, making the GeNPs a promising candidate for clinical NIR-II PA imaging applications.

High-resolution volumetric optical imaging modalities, such as confocal microscopy, two-photon microscopy, and optical coherence tomography, have become increasing important in biomedical imaging fields. However, due to strong light scattering, the penetration depths of these imaging modalities are limited to the optical transport mean free path (~1 mm) in biological tissues. Photoacoustic imaging, an emerging hybrid modality that can provide strong endogenous and exogenous optical absorption contrasts with high ultrasonic spatial resolution, has overcome the fundamental depth limitation while keeping the spatial resolution. The image resolution, as well as the maximum imaging depth, is scalable with ultrasonic frequency within the reach of diffuse photons. In biological tissues the imaging depth can be up to a few centimeters deep. In this presentation, the following topics of photoacoustic imaging will be discussed; (1) multi-scale photoacoustic imaging systems, (2) morphological, functional, and molecular photoacoustic imaging, (3) potential clinical applications, and (4) contrast agents for photoacoustic imaging.

Photoacoustic imaging is becoming a very promising tool for the research of living organisms. It combines the high contrast of optical imaging and the high resolution of acoustic imaging to realize the imaging of absorption clusters in biological tissues. Since the scattering of ultrasound signals in biological tissues is 2-3 orders of magnitude weaker than the scattering of light in biological tissues, the endogenous absorption difference of tissues is directly used in the imaging process, so photoacoustic imaging has the advantages of deep imaging depth and non-destructive. As an important branch of photoacoustic imaging, photoacoustic microscopy can provide micron-level or even sub-micron-level imaging resolution, which is of great significance for biological research such as blood vessel detection. Since the lateral resolution of the photoacoustic microscopy imaging system depends on the focus of the laser, a higher resolution can be obtained by increasing the numerical aperture of the condenser objective. However, a large numerical aperture usually means a shorter working distance and makes the entire imaging system very sensitive to small optical defects. Therefore, the improvement of resolution through this method will be limited in practical applications. This paper implements a method of using iterative deconvolution to obtain a high-resolution photoacoustic image of the brain. The focal spot of the photoacoustic microscopy is measured to obtain the lateral PSF (point spread function) of the system. Making the measured PSF as the initial system PSF to perform Lucy- Richardson (LR) deconvolution. The image resolution of cerebral vasculature obtained by this method is higher. The full width at half maximum (FWHM) of width of the same cerebral capillaries before and after deconvolution are 7 μm and 3.6 μm, respectively, and the image definition is increased by about 1.9 times. Experiments show that this method can further improve the clarity of photoacoustic images of cerebral capillaries, which lays the foundation for further research on brain imaging.

Photoacoustic imaging is becoming a very promising tool for the research of living organisms. It combines the high contrast of optical imaging and the high resolution of acoustic imaging to realize the imaging of absorption clusters in biological tissues. Since the scattering of ultrasound signals in biological tissues is 2-3 orders of magnitude weaker than the scattering of light in biological tissues, the endogenous absorption difference of tissues is directly used in the imaging process, so photoacoustic imaging has the advantages of deep imaging depth and non-destructive. As an important branch of photoacoustic imaging, photoacoustic microscopy can provide micron-level or even sub-micron-level imaging resolution, which is of great significance for biological research such as blood vessel detection. Since the lateral resolution of the photoacoustic microscopy imaging system depends on the focus of the laser, a higher resolution can be obtained by increasing the numerical aperture of the condenser objective. However, a large numerical aperture usually means a shorter working distance and makes the entire imaging system very sensitive to small optical defects. Therefore, the improvement of resolution through this method will be limited in practical applications. This paper implements a method of using iterative deconvolution to obtain a high-resolution photoacoustic image of the brain. The focal spot of the photoacoustic microscopy is measured to obtain the lateral PSF (point spread function) of the system. Making the measured PSF as the initial system PSF to perform Lucy- Richardson (LR) deconvolution. The image resolution of cerebral vasculature obtained by this method is higher. The full width at half maximum (FWHM) of width of the same cerebral capillaries before and after deconvolution are 7 μm and 3.6 μm, respectively, and the image definition is increased by about 1.9 times. Experiments show that this method can further improve the clarity of photoacoustic images of cerebral capillaries, which lays the foundation for further research on brain imaging.