Effect of Molecular Dynamics and Internal Water Contact on the Photophysical Properties of Red pH-Sensitive Proteins.

The pH dependence of the absorption and (time-resolved) fluorescence of two red-shifted fluorescent proteins, mCardinal and mNeptune, was investigated. Decay-associated spectra were measured following fluorescence excitation at 470 nm in PBS buffer with a pH that ranged from 5.5 to 8.0. The fluorescence of both proteins shows two different decay components. mCardinal exhibits an increase in the long-lived fluorescence component with acidification from 1.34 ns at pH 8.0 to 1.62 ns at pH 5.5. An additional fast decay component with 0.64 ns at pH 8.0 up to 1.1 ns at pH 5.5 was found to be blue-shifted compared to the long-lived component. The fluorescence lifetime of mNeptune is insensitive to pH. DAS of mCardinal were simulated assuming a coupled two-level system to describe the 1S state of the chromophore within two different conformations of the protein. MD simulations were conducted to correlate the experimentally observed pH-induced change in the lifetime in mCardinal with its molecular properties. While the chromophores of both protein variants are stabilized by the same number of hydrogen bonds, it was found that the chromophore in mCardinal exhibits more water contacts compared to mNeptune. In mCardinal, interaction between the chromophore and Glu-145 is reduced as compared to mNeptune, but interaction with Thr-147 which is Ser-147 in mNeptune is stronger in mCardinal. Therefore, the dynamics of the excited-state proton transfer (ESPT) might be different in mCardinal and mNeptune. The pH dependency of ESPT is suggested as a key mechanism for pH sensitivity.

Evaluation of ultrasound sensors for transcranial photoacoustic sensing and imaging.

Photoacoustic imaging through skull bone causes strong attenuation and distortion of the acoustic wavefront, which diminishes image contrast and resolution. As a result, transcranial photoacoustic measurements in humans have been challenging to demonstrate. In this study, we investigated the acoustic transmission through the human skull to design an ultrasound sensor suitable for transcranial PA imaging and sensing. We measured the frequency dependent losses of human cranial bones ex vivo, compared the performance of a range of piezoelectric and optical ultrasound sensors, and imaged skull phantoms using a PA tomograph based on a planar Fabry-Perot sensor. All transcranial photoacoustic measurements show the typical effects of frequency and thickness dependent attenuation and aberration associated with acoustic propagation through bone. The performance of plano-concave optical resonator ultrasound sensors was found to be highly suitable for transcranial photoacoustic measurements.

Thermoresponsive swelling of photoacoustic single-chain nanoparticles.

NIR-fluorescent LCST-type single-chain nanoparticles (SCNPs) change their photophysical behaviour upon heating, caused by depletion of water from the swollen SCNP interiors. This thermoresponsive effect leads to a fluctuating photoacoustic (PA) signal which can be used as a contrast mechanism for PA imaging.

Tuning the Internal Compartmentation of Single-Chain Nanoparticles as Fluorescent Contrast Agents.

Controlling the internal structures of single-chain nanoparticles (SCNPs) is an important factor for their targeted chemical design and synthesis, especially in view of nanosized compartments presenting different local environments as a main feature to control functionality. We here design SCNPs bearing near-infrared fluorescent dyes embedded in hydrophobic compartments for use as contrast agents in pump-probe photoacoustic (PA) imaging, displaying improved properties by the location of the dye in the hydrophobic particle core. Compartment formation is controlled via single-chain collapse and subsequent crosslinking of an amphiphilic polymer using external crosslinkers in reaction media of adjustable polarity. Different SCNPs with hydrodynamic diameters of 6-12 nm bearing adjustable label densities are synthesized. It is found that the specific conditions for single-chain collapse have a major impact on the formation of the desired core-shell structure, in turn adjusting the internal nanocompartments together with the formation of excitonic dye couples, which in turn increase their fluorescence lifetime and PA signal generation. SCNPs with the dye molecules accumulate at the core also show a nonlinear PA response as a function of pulse energy-a property that can be exploited as a contrast mechanism in molecular PA tomography.

A backward-mode optical-resolution photoacoustic microscope for 3D imaging using a planar Fabry-Pérot sensor.

Optical-resolution photoacoustic microscopy (OR-PAM) combines high spatial resolution and strong absorption-based contrast in tissue, which has enabled structural and spectroscopic imaging of endogenous chromophores, primarily hemoglobin. Conventional piezoelectric ultrasound transducers are typically placed far away from the photoacoustic source due to their opacity, which reduces acoustic sensitivity. Optical ultrasound sensors are an alternative as their transparency allows them to be positioned close to the sample with minimal source-detector distances. In this work, a backward-mode OR-PAM system based on a planar Fabry-Pérot ultrasound sensor and coaxially aligned excitation and interrogation beams was developed. Two 3D imaging modes, using raster-scanning for enhanced image quality and continuous-scanning for fast imaging, were implemented and tested on a leaf skeleton phantom. In fast imaging mode, a scan-rate of 100,000 A-lines/s was achieved. 3D images of a zebrafish embryo were acquired in vivo in raster-scanning mode. The transparency of the FP sensor in the visible and near-infrared wavelength region makes it suitable for combined functional and molecular imaging applications using OR-PAM and multi-photon fluorescence microscopy.

Translational Photoacoustic Imaging for Disease Diagnosis, Monitoring, and Surgical Guidance: introduction to the feature issue.

This feature issue of Biomedical Optics Express covered all aspects of translational photoacoustic research. Application areas include screening and diagnosis of diseases, imaging of disease progression and therapeutic response, and image-guided treatment, such as surgery, drug delivery, and photothermal/photodynamic therapy. The feature issue also covers relevant developments in photoacoustic instrumentation, contrast agents, image processing and reconstruction algorithms.

Quantitative PA tomography of high resolution 3-D images: Experimental validation in a tissue phantom.

Quantitative photoacoustic tomography aims to recover the spatial distribution of absolute chromophore concentrations and their ratios from deep tissue, high-resolution images. In this study, a model-based inversion scheme based on a Monte-Carlo light transport model is experimentally validated on 3-D multispectral images of a tissue phantom acquired using an all-optical scanner with a planar detection geometry. A calibrated absorber allowed scaling of the measured data during the inversion, while an acoustic correction method was employed to compensate the effects of limited view detection. Chromophore- and fluence-dependent step sizes and Adam optimization were implemented to achieve rapid convergence. High resolution 3-D maps of absolute concentrations and their ratios were recovered with high accuracy. Potential applications of this method include quantitative functional and molecular photoacoustic tomography of deep tissue in preclinical and clinical studies.

Quenching of nonlinear photoacoustic signal generation in gold nanoparticles through coating.

The photoacoustic signal generated from specific gold nanoparticles increases nonlinearly with respect to fluence. We demonstrate experimentally that this nonlinear behavior can be quenched with a particle coating, and present a theoretical analysis to explain this behavior. This effect has the potential to be developed into a photoacoustic-based biochemical sensor.

NIR- and thermo-responsive semi-interpenetrated polypyrrole nanogels for imaging guided combinational photothermal and chemotherapy.

Versatile, multifunctional nanomaterials for theranostic approaches in cancer treatment are highly on demand in order to increase therapeutic outcomes. Here, we developed thermo-responsive nanogels equipped with the efficient near-infrared (NIR) transducing polymer polypyrrole (PPY) for combinational photothermal and chemotherapeutic therapy along with photoacoustic imaging ability. Long-term stability and water-dispersibility of PPY was achieved using semi-interpenetration method for in situ polymerization of PPY into hydrophilic thermo-responsive nanogels. The semi-interpenetrated nanogels of spherical shape and with hydrodynamic sizes of around 200 nm retained the temperature response behaviour and exhibit excellent photothermal transducing abilities in the NIR region. The PPY nanogels served as photoacoustic contrast agents, which allowed determination of biodistribution profiles ex vivo. In addition, we developed a new method for biodistribution determination based on the photothermal response of the nanogels with an accuracy down to 12.5 µg/mL. We examined the ability of the nanogels as photothermal agents and drug delivery systems in vitro and in vivo. We showed that they efficiently inhibit tumor growth with combinational effects of chemotherapeutics and photothermal treatment. Our work encourages further exploration of nanogels as functional stabilizing matrix for photothermal transducers and their application as drug delivery devices in combination with photothermal therapy and imaging.

Three-dimensional quantitative photoacoustic tomography using an adjoint radiance Monte Carlo model and gradient descent.

Quantitative photoacoustic tomography aims to recover maps of the local concentrations of tissue chromophores from multispectral images. While model-based inversion schemes are promising approaches, major challenges to their practical implementation include the unknown fluence distribution and the scale of the inverse problem. We describe an inversion scheme based on a radiance Monte Carlo model and an adjoint-assisted gradient optimization that incorporates fluence-dependent step sizes and adaptive moment estimation. The inversion is shown to recover absolute chromophore concentrations, blood oxygen saturation, and the Grüneisen parameter from in silico three-dimensional phantom images for different radiance approximations. The scattering coefficient is assumed to be homogeneous and known a priori.

Characterization and modeling of Fabry-Perot ultrasound sensors with hard dielectric mirrors for photoacoustic imaging.

A Fabry-Perot ultrasound sensor with nonhygroscopic dielectric mirrors made out of Ta2O5 and SiO2 for use in photoacoustic tomography is described. The sensor offers flat frequency response up to 36 MHz, low noise-equivalent pressure (70 Pa), and near-omnidirectional response up to 20 MHz as well as optical transparency for near-infrared illumination. A numerical model was developed to predict its frequency response, and the results were validated experimentally. An image of the human palm was acquired to demonstrate in vivo imaging capabilities.

Photoacoustic imaging using genetically encoded reporters: a review.

Genetically encoded contrast in photoacoustic imaging (PAI) is complementary to the intrinsic contrast provided by endogenous absorbing chromophores such as hemoglobin. The use of reporter genes expressing absorbing proteins opens the possibility of visualizing dynamic cellular and molecular processes. This is an enticing prospect but brings with it challenges and limitations associated with generating and detecting different types of reporters. The purpose of this review is to compare existing PAI reporters and signal detection strategies, thereby offering a practical guide, particularly for the nonbiologist, to choosing the most appropriate reporter for maximum sensitivity in the biological and technological system of interest.

Photoacoustic pump-probe tomography of fluorophores in vivo using interleaved image acquisition for motion suppression.

In fluorophores, the excited state lifetime can be modulated using pump-probe excitation. By generating photoacoustic (PA) signals using simultaneous and time-delayed pump and probe excitation pulses at fluences below the maximum permissible exposure, a modulation of the signal amplitude is observed in fluorophores but not in endogenous chromophores. This provides a highly specific contrast mechanism that can be used to recover the location of the fluorophore using difference imaging. The practical challenges in applying this method to in vivo PA tomography include the typically low concentrations of fluorescent contrast agents, and tissue motion. The former results in smaller PA signal amplitudes compared to those measured in blood, while the latter gives rise to difference image artefacts that compromise the unambiguous and potentially noise-limited detection of fluorescent contrast agents. To address this limitation, a method based on interleaved pump-probe image acquisition was developed. It relies on fast switching between simultaneous and time-delayed pump-probe excitation to acquire PA difference signals in quick succession, and to minimise the effects of tissue motion. The feasibility of this method is demonstrated in tissue phantoms and in initial experiments in vivo.

Photoacoustic imaging of fluorophores using pump-probe excitation.

A pump-probe technique for the detection of fluorophores in tomographic PA images is introduced. It is based on inducing stimulated emission in fluorescent molecules, which in turn modulates the amount of thermalized energy, and hence the PA signal amplitude. A theoretical model of the PA signal generation in fluorophores is presented and experimentally validated on cuvette measurements made in solutions of Rhodamine 6G, a fluorophore of known optical and molecular properties. The application of this technique to deep tissue tomographic PA imaging is demonstrated by determining the spatial distribution of a near-infrared fluorophore in a tissue phantom.

In vitro characterization of genetically expressed absorbing proteins using photoacoustic spectroscopy.

Genetically expressed fluorescent proteins have been shown to provide photoacoustic contrast. However, they can be limited by low photoacoustic generation efficiency and low optical absorption at red and near infrared wavelengths, thus limiting their usefulness in mammalian small animal models. In addition, many fluorescent proteins exhibit low photostability due to photobleaching and transient absorption effects. In this study, we explore these issues by synthesizing and characterizing a range of commonly used fluorescent proteins (dsRed, mCherry, mNeptune, mRaspberry, AQ143, E2 Crimson) and novel non-fluorescent chromoproteins (aeCP597 and cjBlue and a non-fluorescent mutant of E2 Crimson). The photoacoustic spectra, photoacoustic generation efficiency and photostability of each fluorescent protein and chromoprotein were measured. Compared to the fluorescent proteins, the chromoproteins were found to exhibit higher photoacoustic generation efficiency due to the absence of radiative relaxation and ground state depopulation, and significantly higher photostability. The feasibility of converting an existing fluorescent protein into a non-fluorescent chromoprotein via mutagenesis was also demonstrated. The chromoprotein mutant exhibited greater photoacoustic signal generation efficiency and better agreement between the photoacoustic and the specific extinction coefficient spectra than the original fluorescent protein. Lastly, the genetic expression of a chromoprotein in mammalian cells was demonstrated. This study suggests that chromoproteins may have potential for providing genetically encoded photoacoustic contrast.

Quantitative spectroscopic photoacoustic imaging: a review.

Obtaining absolute chromophore concentrations from photoacoustic images obtained at multiple wavelengths is a nontrivial aspect of photoacoustic imaging but is essential for accurate functional and molecular imaging. This topic, known as quantitative photoacoustic imaging, is reviewed here. The inverse problems involved are described, their nature (nonlinear and ill-posed) is discussed, proposed solution techniques and their limitations are explained, and the remaining unsolved challenges are introduced.

In vivo photoacoustic imaging of mouse embryos.

The ability to noninvasively image embryonic vascular anatomy in mouse models is an important requirement for characterizing the development of the normal cardiovascular system and malformations in the heart and vascular supply. Photoacoustic imaging, which can provide high resolution non invasive images of the vasculature based upon optical absorption by endogenous hemoglobin, is well suited to this application. In this study, photoacoustic images of mouse embryos were obtained ex vivo and in vivo. The images show intricate details of the embryonic vascular system to depths of up to 10 mm, which allowed whole embryos to be imaged in situ. To achieve this, an all-optical photoacoustic scanner and a novel time reversal image reconstruction algorithm, which provide deep tissue imaging capability while maintaining high spatial resolution and contrast were employed. This technology may find application as an imaging tool for preclinical embryo studies in developmental biology as well as more generally in preclinical and clinical medicine for studying pathologies characterized by changes in the vasculature.

In vivo preclinical photoacoustic imaging of tumor vasculature development and therapy.

The use of a novel all-optical photoacoustic scanner for imaging the development of tumor vasculature and its response to a therapeutic vascular disrupting agent is described. The scanner employs a Fabry-Perot polymer film ultrasound sensor for mapping the photoacoustic waves and an image reconstruction algorithm based upon attenuation-compensated acoustic time reversal. The system was used to noninvasively image human colorectal tumor xenografts implanted subcutaneously in mice. Label-free three-dimensional in vivo images of whole tumors to depths of almost 10 mm with sub-100-micron spatial resolution were acquired in a longitudinal manner. This enabled the development of tumor-related vascular features, such as vessel tortuosity, feeding vessel recruitment, and necrosis to be visualized over time. The system was also used to study the temporal evolution of the response of the tumor vasculature following the administration of a therapeutic vascular disrupting agent (OXi4503). This revealed the well-known destruction and recovery phases associated with this agent. These studies illustrate the broader potential of this technology as an imaging tool for the preclinical and clinical study of tumors and other pathologies characterized by changes in the vasculature.

Automatic sound speed selection in photoacoustic image reconstruction using an autofocus approach.

The reconstruction of images in photoacoustic tomography is reliant on specifying the speed of sound within the propagation medium. However, for in vivo imaging, this value is not normally accurately known. Here, an autofocus approach for automatically selecting the sound speed is proposed. This is based on maximizing the sharpness of the reconstructed image as quantified by a focus function. Several focus functions are investigated, and their performance is discussed. The method is demonstrated using phantom measurements made in a medium with a known sound speed and in vivo measurements of the vasculature in the flank of an adult mouse.

Multimodal photoacoustic and optical coherence tomography scanner using an all optical detection scheme for 3D morphological skin imaging.

A noninvasive, multimodal photoacoustic and optical coherence tomography (PAT/OCT) scanner for three-dimensional in vivo (3D) skin imaging is described. The system employs an integrated, all optical detection scheme for both modalities in backward mode utilizing a shared 2D optical scanner with a field-of-view of ~13 × 13 mm(2). The photoacoustic waves were detected using a Fabry Perot polymer film ultrasound sensor placed on the surface of the skin. The sensor is transparent in the spectral range 590-1200 nm. This permits the photoacoustic excitation beam (670-680 nm) and the OCT probe beam (1050 nm) to be transmitted through the sensor head and into the underlying tissue thus providing a backward mode imaging configuration. The respective OCT and PAT axial resolutions were 8 and 20 µm and the lateral resolutions were 18 and 50-100 µm. The system provides greater penetration depth than previous combined PA/OCT devices due to the longer wavelength of the OCT beam (1050 nm rather than 829-870 nm) and by operating in the tomographic rather than the optical resolution mode of photoacoustic imaging. Three-dimensional in vivo images of the vasculature and the surrounding tissue micro-morphology in murine and human skin were acquired. These studies demonstrated the complementary contrast and tissue information provided by each modality for high-resolution 3D imaging of vascular structures to depths of up to 5 mm. Potential applications include characterizing skin conditions such as tumors, vascular lesions, soft tissue damage such as burns and wounds, inflammatory conditions such as dermatitis and other superficial tissue abnormalities.