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.

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.

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.

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.

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.

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.

Quantitative determination of chromophore concentrations from 2D photoacoustic images using a nonlinear model-based inversion scheme.

A model-based inversion scheme was used to determine absolute chromophore concentrations from multiwavelength photoacoustic images. The inversion scheme incorporated a forward model, which predicted 2D images of the initial pressure distribution as a function of the spatial distribution of the chromophore concentrations. It comprised a multiwavelength diffusion based model of the light transport, a model of acoustic propagation and detection, and an image reconstruction algorithm. The model was inverted by fitting its output to measured photoacoustic images to determine the chromophore concentrations. The scheme was validated using images acquired in a tissue phantom at wavelengths between 590 nm and 980 nm. The phantom comprised a scattering emulsion in which up to four tubes, filled with absorbing solutions of copper and nickel chloride at different concentration ratios, were submerged. Photoacoustic signals were detected along a line perpendicular to the tubes from which images of the initial pressure distribution were reconstructed. By varying the excitation wavelength, sets of multiwavelength photoacoustic images were obtained. The majority of the determined chromophore concentrations were within +/-15% of the true value, while the concentration ratios were determined with an average accuracy of -1.2%.

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.

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.

Three-dimensional noninvasive imaging of the vasculature in the mouse brain using a high resolution photoacoustic scanner.

The application of a novel photoacoustic imaging instrument based on a Fabry-Perot polymer film sensing interferometer to imaging the small animal brain is described. This approach provides a convenient backward mode sensing configuration that offers the prospect of overcoming the limitations of existing piezoelectric based detection schemes for small animal brain imaging. Noninvasive images of the vasculature in the mouse brain were obtained at different wavelengths between 590 and 889 nm, showing that the cerebral vascular anatomy can be visualized with high contrast and spatial resolution to depths up to 3.7 mm. It is considered that the instrument has a role to play in characterizing small animal models of human disease and injury processes such as stroke, epilepsy, and traumatic brain injury.

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.

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.

Quantitative spatially resolved measurement of tissue chromophore concentrations using photoacoustic spectroscopy: application to the measurement of blood oxygenation and haemoglobin concentration.

A new approach based on pulsed photoacoustic spectroscopy for non-invasively quantifying tissue chromophore concentrations with high spatial resolution has been developed. The technique is applicable to the quantification of tissue chromophores such as oxyhaemoglobin (HbO(2)) and deoxyhaemoglobin (HHb) for the measurement of physiological parameters such as blood oxygen saturation (SO(2)) and total haemoglobin concentration. It can also be used to quantify the local accumulation of targeted contrast agents used in photoacoustic molecular imaging. The technique employs a model-based inversion scheme to recover the chromophore concentrations from photoacoustic measurements. This comprises a numerical forward model of the detected time-dependent photoacoustic signal that incorporates a multiwavelength diffusion-based finite element light propagation model to describe the light transport and a time-domain acoustic model to describe the generation, propagation and detection of the photoacoustic wave. The forward model is then inverted by iteratively fitting it to measurements of photoacoustic signals acquired at different wavelengths to recover the chromophore concentrations. To validate this approach, photoacoustic signals were generated in a tissue phantom using nanosecond laser pulses between 740 nm and 1040 nm. The tissue phantom comprised a suspension of intralipid, blood and a near-infrared dye in which three tubes were immersed. Blood at physiological haemoglobin concentrations and oxygen saturation levels ranging from 2% to 100% was circulated through the tubes. The signal amplitude from different temporal sections of the detected photoacoustic waveforms was plotted as a function of wavelength and the forward model fitted to these data to recover the concentrations of HbO(2) and HHb, total haemoglobin concentration and SO(2). The performance was found to compare favourably to that of a laboratory CO-oximeter with measurement resolutions of +/-3.8 g l(-1) (+/-58 microM) and +/-4.4 g l(-1) (+/-68 microM) for the HbO(2) and HHb concentrations respectively and +/-4% for SO(2) with an accuracy in the latter in the range -6%-+7%.

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.

In vitro measurements of absolute blood oxygen saturation using pulsed near-infrared photoacoustic spectroscopy: accuracy and resolution.

Pulsed photoacoustic spectroscopy was used to measure blood oxygen saturation in vitro. An optical parametric oscillator laser system provided nanosecond excitation pulses over the wavelength range 740-1040 nm which were used to generate photoacoustic signals in a cuvette through which a saline suspension of red blood cells was circulated. The signal amplitude and the effective attenuation coefficient were extracted from the photoacoustic signals as a function of wavelength to provide photoacoustic spectra of the blood. From these, the relative concentrations of oxy- and deoxyhaemoglobin, and therefore blood oxygen saturation (SO2), were determined using forward models of the absorbed energy distribution based on diffusion theory. A standard linear model of the dependence of absorbance on the concentration of chromophores was also used to calculate the blood oxygen saturation from the signal amplitude spectra. The diffusion approximation model was shown to produce the highest accuracy in blood SO2. The photoacoustically determined oxygen saturation was found to have an accuracy of +/-4% SO2 for signal amplitude data and +/-2.5% SO2 for effective attenuation spectra. The smallest change in oxygen saturation that can be measured using this technique was +/-1% SO2.

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.

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.