In vivo characterization of connective tissue remodeling using infrared photoacoustic spectra.

Premature cervical remodeling is a critical precursor of spontaneous preterm birth, and the remodeling process is characterized by an increase in tissue hydration. Nevertheless, current clinical measurements of cervical remodeling are subjective and detect only late events, such as cervical effacement and dilation. Here, we present a photoacoustic endoscope that can quantify tissue hydration by measuring near-infrared cervical spectra. We quantify the water contents of tissue-mimicking hydrogel phantoms as an analog of cervical connective tissue. Applying this method to pregnant women in vivo, we observed an increase in the water content of the cervix throughout pregnancy. The application of this technique in maternal healthcare may advance our understanding of cervical remodeling and provide a sensitive method for predicting preterm birth.

Dry coupling for whole-body small-animal photoacoustic computed tomography.

We have enhanced photoacoustic computed tomography with dry acoustic coupling that eliminates water immersion anxiety and wrinkling of the animal and facilitates incorporating complementary modalities and procedures. The dry acoustic coupler is made of a tubular elastic membrane enclosed by a closed transparent water tank. The tubular membrane ensures water-free contact with the animal, and the closed water tank allows pressurization for animal stabilization. The dry coupler was tested using a whole-body small-animal ring-shaped photoacoustic computed tomography system. Dry coupling was found to provide image quality comparable to that of conventional water coupling.

A forward-adjoint operator pair based on the elastic wave equation for use in transcranial photoacoustic computed tomography.

Photoacoustic computed tomography (PACT) is an emerging imaging modality that exploits optical contrast and ultrasonic detection principles to form images of the photoacoustically induced initial pressure distribution within tissue. The PACT reconstruction problem corresponds to an inverse source problem in which the initial pressure distribution is recovered from measurements of the radiated wavefield. A major challenge in transcranial PACT brain imaging is compensation for aberrations in the measured data due to the presence of the skull. Ultrasonic waves undergo absorption, scattering and longitudinal-to-shear wave mode conversion as they propagate through the skull. To properly account for these effects, a wave-equation-based inversion method should be employed that can model the heterogeneous elastic properties of the skull. In this work, a forward model based on a finite-difference time-domain discretization of the three-dimensional elastic wave equation is established and a procedure for computing the corresponding adjoint of the forward operator is presented. Massively parallel implementations of these operators employing multiple graphics processing units (GPUs) are also developed. The developed numerical framework is validated and investigated in computer19 simulation and experimental phantom studies whose designs are motivated by transcranial PACT applications.

Use of a single xenon flash lamp for photoacoustic computed tomography of multiple-centimeter-thick biological tissue ex vivo and a whole mouse body in vivo.

While lasers have been commonly used as illumination sources in photoacoustic (PA) imaging, their high purchase and maintenance costs, as well as their bulkiness, have hindered the rapid clinical dissemination of PA imaging. With this in mind, we explore an alternative illumination source for PA tomography­a xenon flash lamp with high pulse energy and a microsecond pulse width. We demonstrate that, by using a single xenon flash lamp, we can image both a black latex cord placed in chicken breast tissue at a depth of up to 3.5 cm ex vivo and an entire mouse body in vivo. Our findings indicate that the xenon flash lamp, producing optical illumination that is safe for humans, can be potentially applied to human tissue imaging.

Label-free photoacoustic tomography of whole mouse brain structures ex vivo.

Capitalizing on endogenous hemoglobin contrast, photoacoustic-computed tomography (PACT), a deep-tissue high-resolution imaging modality, has drawn increasing interest in neuroimaging. However, most existing studies are limited to functional imaging on the cortical surface and the deep brain structural imaging capability of PACT has never been demonstrated. Here, we explicitly studied the limiting factors of deep brain PACT imaging. We found that the skull distorted the acoustic signal and blood suppressed the structural contrast from other chromophores. When the two effects are mitigated, PACT can potentially provide high-resolution label-free imaging of structures in the entire mouse brain. With [Formula: see text] in-plane resolution, we can clearly identify major structures of the brain, which complements magnetic resonance microscopy for imaging small-animal brain structures. Spectral PACT studies indicate that structural contrasts mainly originate from cytochrome distribution and that the presence of lipid sharpens the image contrast; brain histology results provide further validation. The feasibility of imaging the structure of the brain in vivo is also discussed. Our results demonstrate that PACT is a promising modality for both structural and functional brain imaging.

Dual-Modality Photoacoustic and Ultrasound Imaging System for Noninvasive Sentinel Lymph Node Detection in Patients with Breast Cancer.

The detection of regional lymph node metastases is important in cancer staging as it guides the prognosis of the patient and the strategy for treatment. Sentinel lymph node biopsy (SLNB) is an accurate, less invasive alternative to axillary lymph node dissection. The sentinel lymph node hypothesis states that the pathological status of the axilla can be accurately predicted by determining the status of the first lymph nodes that drain from the primary tumor. Physicians use radio-labeled sulfur colloid and/or methylene blue dye to identify the SLN, which is most likely to contain metastatic cancer cells. However, the surgical procedure causes morbidity and associated expenses. To overcome these limitations, we developed a dual-modality photoacoustic and ultrasonic imaging system to noninvasively detect SLNs based on the accumulation of methylene blue dye. Ultimately, we aim to guide percutaneous needle biopsies and provide a minimally invasive method for axillary staging of breast cancer.

Label-free photoacoustic nanoscopy.

Super-resolution microscopy techniques - capable of overcoming the diffraction limit of light - have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents. Here, we report label-free photoacoustic (PA) nanoscopy, which is exquisitely sensitive to optical absorption, with an 88 nm resolution. At each scanning position, multiple PA signals are successively excited with increasing laser pulse energy. Because of optical saturation or nonlinear thermal expansion, the PA amplitude depends on the nonlinear incident optical fluence. The high-order dependence, quantified by polynomial fitting, provides super-resolution imaging with optical sectioning. PA nanoscopy is capable of super-resolution imaging of either fluorescent or nonfluorescent molecules.

Photobleaching imprinting microscopy: seeing clearer and deeper.

We present a generic sub-diffraction-limited imaging method - photobleaching imprinting microscopy (PIM) - for biological fluorescence imaging. A lateral resolution of 110 nm was measured, more than a twofold improvement over the optical diffraction limit. Unlike other super-resolution imaging techniques, PIM does not require complicated illumination modules or specific fluorescent dyes. PIM is expected to facilitate the conversion of super-resolution imaging into a routine lab tool, making it accessible to a much broader biological research community. Moreover, we show that PIM can increase the image contrast of biological tissue, effectively extending the fundamental depth limit of multi-photon fluorescence microscopy.

Photoacoustic recovery after photothermal bleaching in living cells.

We present an innovative method, photoacoustic recovery after photothermal bleaching (PRAP), for studying particle dynamics at micron scale via photoacoustic imaging. As an intuitive way to visualize and quantify dynamic processes, PRAP is demonstrated first in a simple phantom study and then in a more complex measurement involving live cells. Compared with the conventional fluorescence-based approach, PRAP provides high signal-to-noise ratio (SNR) imaging with minimal bleaching-induced artifacts during the recovery stage, ideal for monitoring the diffusive and kinetic processes inside a cell.

Photothermal bleaching in time-lapse photoacoustic microscopy.

We studied the phenomenon of photothermal bleaching - a gradual reduction of contrast agent particles during repeated scans in photoacoustic microscopy. The dependence of the photothermal bleaching rate on the excitation pulse energy, pulse duration, and the absorber's size was determined while the laser focal diameter was held constant. Our results showed that, the dependence of the photothermal bleaching rate on the excitation pulse energy differed before and after the absorbers were raised to their melting point by the deposited laser energy. Based on this finding, we suggested an optimal excitation pulse energy, which balances the photothermal bleaching rate and signal amplitude, for time-lapse imaging applications. (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

Video-rate functional photoacoustic microscopy at depths.

We report the development of functional photoacoustic microscopy capable of video-rate high-resolution in vivo imaging in deep tissue. A lightweight photoacoustic probe is made of a single-element broadband ultrasound transducer, a compact photoacoustic beam combiner, and a bright-field light delivery system. Focused broadband ultrasound detection provides a 44-µm lateral resolution and a 28-µm axial resolution based on the envelope (a 15-µm axial resolution based on the raw RF signal). Due to the efficient bright-field light delivery, the system can image as deep as 4.8 mm in vivo using low excitation pulse energy (28 µJ per pulse, 0.35 mJ/cm² on the skin surface). The photoacoustic probe is mounted on a fast-scanning voice-coil scanner to acquire 40 two-dimensional (2-D) B-scan images per second over a 9-mm range. High-resolution anatomical imaging is demonstrated in the mouse ear and brain. Via fast dual-wavelength switching, oxygen dynamics of mouse cardio-vasculature is imaged in realtime as well.

Photoacoustic tomography through a whole adult human skull with a photon recycler.

ABSTRACT. Photoacoustic tomography (PAT) of the human brain is challenging due to the fact that the skull strongly absorbs and scatters light, and attenuates and distorts ultrasound as well. For the first time, we demonstrated the feasibility of PAT through a whole adult human skull. A photon recycler (PR) was built to increase light transmittance through the skull. Both a graphite target and a canine brain were imaged through the skull. Use of the PR was found to improve the photoacoustic signal-to-noise ratio by a factor of 2.4. In addition, subtraction of photoacoustic signals that arise from light absorption within the skull significantly improved the contrast of the target. Our results indicate that PAT can potentially be applied to in vivo human brain imaging.

Quantitative photoacoustic microscopy of optical absorption coefficients from acoustic spectra in the optical diffusive regime.

Photoacoustic (PA) microscopy (PAM) can image optical absorption contrast with ultrasonic spatial resolution in the optical diffusive regime. Conventionally, accurate quantification in PAM requires knowledge of the optical fluence attenuation, acoustic pressure attenuation, and detection bandwidth. We circumvent this requirement by quantifying the optical absorption coefficients from the acoustic spectra of PA signals acquired at multiple optical wavelengths. With the acoustic spectral method, the absorption coefficients of an oxygenated bovine blood phantom at 560, 565, 570, and 575 nm were quantified with errors of <3%. We also quantified the total hemoglobin concentration and hemoglobin oxygen saturation in a live mouse. Compared with the conventional amplitude method, the acoustic spectral method provides greater quantification accuracy in the optical diffusive regime. The limitations of the acoustic spectral method was also discussed.

In vivo diagnosis of melanoma and nonmelanoma skin cancer using oblique incidence diffuse reflectance spectrometry.

Early detection and treatment of skin cancer can significantly improve patient outcome. However, present standards for diagnosis require biopsy and histopathologic examinations that are relatively invasive, expensive, and difficult for patients with many early-stage lesions. Here, we show an oblique incidence diffuse reflectance spectroscopic (OIDRS) system that can be used for rapid skin cancer detection in vivo. This system was tested under clinical conditions by obtaining spectra from pigmented and nonpigmented skin lesions, including melanomas, differently staged dysplastic nevi, and common nevi that were validated by standard pathohistologic criteria. For diagnosis of pigmented melanoma, the data obtained achieved 90% sensitivity and specificity for a blinded test set. In a second analysis, we showed that this spectroscopy system can also differentiate nonpigmented basal cell or squamous cell carcinomas from noncancerous skin abnormalities, such as actinic keratoses and seborrheic keratoses, achieving 92% sensitivity and specificity. Taken together, our findings establish how OIDRS can be used to more rapidly and easily diagnose skin cancer in an accurate and automated manner in the clinic.

In-vivo characterization of optical properties of pigmented skin lesions including melanoma using oblique incidence diffuse reflectance spectrometry.

In this letter, we report the first use of oblique incidence diffuse reflectance spectrometry to conduct in-vivo measurements of optical properties of three different types of pigmented skin lesions, including melanoma, dysplastic, and common nevi. Both absorption and reduced scattering coefficient spectra were estimated from the spatially resolved diffuse reflectance within the wavelength range of 455-765 nm for 144 pigmented skin lesions including 16 melanomas. The absorption and reduced scattering spectra were found to change with the malignancy of the skin lesions, which were generally higher for the malignant cases than the benign ones. Based on the measurement results, the physiological origin leading to the change of the absorption and scattering properties is also discussed.

High-efficiency and side-viewing micro fiber optic probe for in-vivo diffuse reflectance measurements of human epithelial tissues.

In this paper, we present a new micro fiber optic probe for in-vivo diffuse reflectance measurements of human epithelial tissues. The probe consists of seven, 120 x 120 microm collection fibers and one 200 microm incidence source fiber, arranged into a side-viewing configuration by using curved air-core waveguides with high optical transmittance. The outer dimension of the probe is 2 x 8mm(2). The extremely small size, high transmission efficiency and side-viewing capability of the new probe make it suitable for endoscopic spectroscopic applications inside the human body.

Skin cancer detection by spectroscopic oblique-incidence reflectometry: classification and physiological origins.

Data obtained from 102 skin lesions in vivo by spectroscopic oblique-incidence reflectometry were analyzed. The participating physicians initially divided the skin lesions into two visually distinguishable groups based on the lesions' melanocytic conditions. Group 1 consisted of the following two cancerous and benign subgroups: (1) basal cell carcinomas and squamous cell carcinomas and (2) benign actinic keratoses, seborrheic keratoses, and warts. Group 2 consisted of (1) dysplastic nevi and (2) benign common nevi. For each group, a bootstrap-based Bayes classifier was designed to separate the benign from the dysplastic or cancerous tissues. A genetic algorithm was then used to obtain the most effective combination of spatiospectral features for each classifier. The classifiers, tested with prospective blind studies, reached statistical accuracies of 100% and 95% for groups 1 and 2, respectively. Properties that related to cell-nuclear size, to the concentration of oxyhemoglobin, and to the concentration of deoxyhemoglobin as well as the derived concentration of total hemoglobin and oxygen saturation were defined to explain the origins of the classification outcomes.