Radioactive (90Y) upconversion nanoparticles conjugated with recombinant targeted toxin for synergistic nanotheranostics of cancer.

We report combined therapy using upconversion nanoparticles (UCNP) coupled to two therapeutic agents: beta-emitting radionuclide yttrium-90 (90Y) fractionally substituting yttrium in UCNP, and a fragment of the exotoxin A derived from Pseudomonas aeruginosa genetically fused with a targeting designed ankyrin repeat protein (DARPin) specific to HER2 receptors. The resultant hybrid complex UCNP-R-T was tested using human breast adenocarcinoma cells SK-BR-3 overexpressing HER2 receptors and immunodeficient mice, bearing HER2-positive xenograft tumors. The photophysical properties of UCNPs enabled background-free imaging of the UCNP-R-T distribution in cells and animals. Specific binding and uptake of UCNP complexes in SK-BR-3 cells was observed, with separate 90Y- and PE40-induced cytotoxic effects characterized by IC50 140 µg/mL (UCNP-R) and 5.2 µg/mL (UCNP-T), respectively. When both therapeutic agents were combined into UCNP-R-T, the synergetic effect increased markedly, ∼2200-fold, resulting in IC50 = 0.0024 µg/mL. The combined therapy with UCNP-R-T was demonstrated in vivo.

Spatiotemporal image reconstruction to enable high-frame-rate dynamic photoacoustic tomography with rotating-gantry volumetric imagers.

Significance: Dynamic photoacoustic computed tomography (PACT) is a valuable imaging technique for monitoring physiological processes. However, current dynamic PACT imaging techniques are often limited to two-dimensional spatial imaging. Although volumetric PACT imagers are commercially available, these systems typically employ a rotating measurement gantry in which the tomographic data are sequentially acquired as opposed to being acquired simultaneously at all views. Because the dynamic object varies during the data-acquisition process, the sequential data-acquisition process poses substantial challenges to image reconstruction associated with data incompleteness. The proposed image reconstruction method is highly significant in that it will address these challenges and enable volumetric dynamic PACT imaging with existing preclinical imagers. Aim: The aim of this study is to develop a spatiotemporal image reconstruction (STIR) method for dynamic PACT that can be applied to commercially available volumetric PACT imagers that employ a sequential scanning strategy. The proposed reconstruction method aims to overcome the challenges caused by the limited number of tomographic measurements acquired per frame. Approach: A low-rank matrix estimation-based STIR (LRME-STIR) method is proposed to enable dynamic volumetric PACT. The LRME-STIR method leverages the spatiotemporal redundancies in the dynamic object to accurately reconstruct a four-dimensional (4D) spatiotemporal image. Results: The conducted numerical studies substantiate the LRME-STIR method's efficacy in reconstructing 4D dynamic images from tomographic measurements acquired with a rotating measurement gantry. The experimental study demonstrates the method's ability to faithfully recover the flow of a contrast agent with a frame rate of 10 frames per second, even when only a single tomographic measurement per frame is available. Conclusions: The proposed LRME-STIR method offers a promising solution to the challenges faced by enabling 4D dynamic imaging using commercially available volumetric PACT imagers. By enabling accurate STIRs, this method has the potential to significantly advance preclinical research and facilitate the monitoring of critical physiological biomarkers.

Characterizing a photoacoustic and fluorescence imaging platform for preclinical murine longitudinal studies.

Significance: To effectively study preclinical animal models, medical imaging technology must be developed with a high enough resolution and sensitivity to perform anatomical, functional, and molecular assessments. Photoacoustic (PA) tomography provides high resolution and specificity, and fluorescence (FL) molecular tomography provides high sensitivity; the combination of these imaging modes will enable a wide range of research applications to be studied in small animals. Aim: We introduce and characterize a dual-modality PA and FL imaging platform using in vivo and phantom experiments. Approach: The imaging platform's detection limits were characterized through phantom studies that determined the PA spatial resolution, PA sensitivity, optical spatial resolution, and FL sensitivity. Results: The system characterization yielded a PA spatial resolution of 173 ± 17 µ m in the transverse plane and 640 ± 120 µ m in the longitudinal axis, a PA sensitivity detection limit not less than that of a sample with absorption coefficient µ a = 0.258 cm - 1 , an optical spatial resolution of 70 µ m in the vertical axis and 112 µ m in the horizontal axis, and a FL sensitivity detection limit not < 0.9 µ M concentration of IR-800. The scanned animals displayed in three-dimensional renders showed high-resolution anatomical detail of organs. Conclusions: The combined PA and FL imaging system has been characterized and has demonstrated its ability to image mice in vivo, proving its suitability for biomedical imaging research applications.

Indocyanine green dye based bimodal contrast agent tested by photoacoustic/fluorescence tomography setup.

Multimodal imaging systems are in high demand for preclinical research, experimental medicine, and clinical practice. Combinations of photoacoustic technology with other modalities including fluorescence, ultrasound, MRI, OCT have been already applied in feasibility studies. Nevertheless, only the combination of photoacoustics with ultrasound in a single setup is commercially available now. A combination of photoacoustics and fluorescence is another compelling approach because those two modalities naturally complement each other. Here, we presented a bimodal contrast agent based on the indocyanine green dye (ICG) as a single signalling compound embedded in the biocompatible and biodegradable polymer shell. We demonstrate its remarkable characteristics by imaging using a commercial photoacoustic/fluorescence tomography system (TriTom, PhotoSound Technologies). It was shown that photoacoustic signal of the particles depends on the amount of dye loaded into the shell, while fluorescence signal depends on the total amount of dye per particle. For the first time to our knowledge, a commercial bimodal photoacoustic/fluorescence setup was used for characterization of ICG doped polymer particles. Additionally, we conducted cell toxicity studies for these particles as well as studied biodistribution over time in vivo and ex vivo using fluorescent imaging. The obtained results suggest a potential for the application of biocompatible and biodegradable bimodal contrast agents as well as the integrated photoacoustic/fluorescence imaging system for preclinical and clinical studies.

Laser optoacoustic imaging system for detection of breast cancer.

We designed, fabricated and tested the laser optoacoustic imaging system for breast cancer detection (LOIS-64), which fuses optical and acoustic imaging techniques in one modality by utilizing pulsed optical illumination and ultrawide-band ultrasonic detection of resulting optoacoustic (OA) signals. The system was designed to image a single breast slice in craniocaudal or mediolateral projection with an arc-shaped array of 64 ultrawide-band acoustic transducers. The system resolution on breast phantoms was at least 0.5 mm. The single-channel sensitivity of 1.66 mVPa was estimated to be sufficient for single-pulse imaging of 6 to 11 mm tumors through the whole imaging slice of the breast. The implemented signal processing using the wavelet transform allowed significant reduction of the low-frequency (LF) acoustic noise, allowed localization of the optoacoustic signals from tumors, and enhanced the contrast and sharpened the boundaries of the optoacoustic images of the tumors. During the preliminary clinical studies on 27 patients, the LOIS-64 was able to visualize 18 out of 20 malignant lesions suspected from mammography and ultrasound images and confirmed by the biopsy performed after the optoacoustic tomography (OAT) procedure.

Preclinical Study of Biofunctional Polymer-Coated Upconversion Nanoparticles.

Upconversion nanoparticles (UCNPs) are new-generation photoluminescent nanomaterials gaining considerable recognition in the life sciences due to their unique optical properties that allow high-contrast imaging in cells and tissues. Upconversion nanoparticle applications in optical diagnosis, bioassays, therapeutics, photodynamic therapy, drug delivery, and light-controlled release of drugs are promising, demanding a comprehensive systematic study of their pharmacological properties. We report on production of biofunctional UCNP-based nanocomplexes suitable for optical microscopy and imaging of HER2-positive cells and tumors, as well as on the comprehensive evaluation of their pharmacokinetics, pharmacodynamics, and toxicological properties using cells and laboratory animals. The nanocomplexes represent a UCNP core/shell structure of the NaYF4:Yb, Er, Tm/NaYF4 composition coated with an amphiphilic alternating copolymer of maleic anhydride with 1-octadecene (PMAO) and conjugated to the Designed Ankyrin Repeat Protein (DARPin 9_29) with high affinity to the HER2 receptor. We demonstrated the specific binding of UCNP-PMAO-DARPin to HER2-positive cancer cells in cultures and xenograft animal models allowing the tumor visualization for at least 24 h. An exhaustive study of the general and specific toxicity of UCNP-PMAO-DARPin including the evaluation of their allergenic, immunotoxic, and reprotoxic properties was carried out. The obtained experimental body of evidence leads to a conclusion that UCNP-PMAO and UCNP-PMAO-DARPin are functional, noncytotoxic, biocompatible, and safe for imaging applications in cells, small animals, and prospective clinical applications of image-guided surgery.

Studies of plasma membrane mechanics and plasma membrane-cytoskeleton interactions using optical tweezers and fluorescence imaging.

We use optical tweezers in conjunction with an optical position-sensing system, which spectrally filters signals generated by a trapped fluorescent microsphere to study plasma membrane (PM) mechanics and its interactions with cytoskeleton. We dynamically measure the PM tethering force on human embryonic kidney cells that are a standard cultured cell line. Recorded tethering force vs. PM displacement profiles, revealed the tether formation process, initiated with linear deformation of the PM, followed by a nonlinear regime and terminated with the local separation of PM. Tethering force vs. displacement profiles were used to estimate tether formation force and stiffness parameter of the PM. Integration of the force-displacement profiles yielded the work of tether formation, including linear and nonlinear components. Our results demonstrate that spectral filtering of the optically trapped fluorescent microsphere image formed on the position-sensing system overcomes the artifacts introduced by the transillumination imaging and allows accurate measures of PM mechanics before and during the initial stages of tether formation.

Temperature-dependent optoacoustic response and transient through zero Grüneisen parameter in optically contrasted media.

Non-invasive optoacoustic mapping of temperature in tissues with low blood content can be enabled by administering external contrast agents. Some important clinical applications of such approach include temperature mapping during thermal therapies in a prostate or a mammary gland. However, the technique would require a calibration that establishes functional relationship between the measured normalized optoacoustic response and local tissue temperature. In this work, we investigate how a key calibration parameter - the temperature of zero optoacoustic response (T0 ) - behaves in different environments simulating biological tissues augmented with either dissolved or particulate (nanoparticles) contrast agents. The observed behavior of T0 in ionic and molecular solutions suggests that in-vivo temperature mapping is feasible for contrast agents of this type, but requires knowledge of local concentrations. Oppositely, particulate contrast agents (plasmonic or carbon nanoparticles) demonstrated concentration-independent thermal behavior of optoacoustic response with T0 defined by the thermoelastic properties of the local environment.

Red blood cell as a universal optoacoustic sensor for non-invasive temperature monitoring.

Optoacoustic (photoacoustic) temperature imaging could provide improved spatial resolution and temperature sensitivity as compared to other techniques of non-invasive thermometry used during thermal therapies for safe and efficient treatment of lesions. However, accuracy of the reported optoacoustic methods is compromised by biological variability and heterogeneous composition of tissues. We report our findings on the universal character of the normalized temperature dependent optoacoustic response (ThOR) in blood, which is invariant with respect to hematocrit at the isosbestic point of hemoglobin. The phenomenon is caused by the unique homeostatic compartmentalization of blood hemoglobin exclusively inside erythrocytes. On the contrary, the normalized ThOR in aqueous solutions of hemoglobin showed linear variation with respect to its concentration and was identical to that of blood when extrapolated to the hemoglobin concentration inside erythrocytes. To substantiate the conclusions, we analyzed optoacoustic images acquired from the samples of whole and diluted blood as well as hemoglobin solutions during gradual cooling from +37 to -15 °C. Our experimental methodology allowed direct observation and accurate measurement of the temperature of zero optoacoustic response, manifested as the sample's image faded into background and then reappeared in the reversed (negative) contrast. These findings provide a framework necessary for accurate correlation of measured normalized optoacoustic image intensity and local temperature in vascularized tissues independent of tissue composition.

Enabling in vivo measurements of nanoparticle concentrations with three-dimensional optoacoustic tomography.

In this report, we demonstrate the feasibility of using optoacoustic tomography (OAT) to evaluate biodistributions of nanoparticles in animal models. The redistribution of single-walled carbon nanotubes (SWCNTs) was visualized in living mice. Nanoparticle concentrations in harvested organs were measured spectroscopically using the intrinsic optical absorption and fluorescence of SWCNTs. Observed increases in optoacoustic signal brightness in tissues were compared with increases in optical absorption coefficients caused by SWCNT accumulation. The methodology presented in this report can further be extended to calibrate the sensitivity of an optoacoustic imaging system for a range of changes in optical absorption coefficient values at specific locations or organs in a mouse body to enable noninvasive measurements of nanoparticle concentrations in vivo. Additionally, qualitative information provided by OAT and quantitative information obtained ex vivo may provide valuable feedback for advancing methods of quantitative analysis with OAT.

Three-dimensional optoacoustic imaging as a new noninvasive technique to study long-term biodistribution of optical contrast agents in small animal models.

We used a 3-D optoacoustic (OA) tomography system to create maps of optical absorbance of mice tissues contrasted with gold nanorods (GNRs). Nude mice were scanned before and after injection of GNRs at time periods varying from 1 to 192 h. Synthesized GNRs were purified from hexadecyltrimethylammonium bromide and coated with polyethylene glycol (PEG) to obtain GNR-PEG complexes suitable for in vivo applications. Intravenous administration of purified GNR-PEG complexes resulted in enhanced OA contrast of internal organs and blood vessels compared to the same mouse before injection of the contrast agent. Maximum enhancement of the OA images was observed 24 to 48 h postinjection, followed by a slow clearance trend for the remaining part of the studied period (eight days). We demonstrate that OA imaging with two laser wavelengths can be used for noninvasive, long-term studies of biological distribution of contrast agents.

Highly purified biocompatible gold nanorods for contrasted optoacoustic imaging of small animal models.

We developed a methodology for high yield synthesis of gold nanorods (GNR) with narrow band optical absorption centered at 760 nm. GNR were purified from hexadecyltrimethylammonium bromide (CTAB) and coated with polyethylene glycol (PEG). The molar ratio between GNR and PEG (1÷50000) was optimized to make the conjugate a biocompatible PEG-GNR contrast agent for optoacoustic (OA) imaging. In vitro toxicity studies showed no significant change in survival rates of cultured normal (IEC-6, MDCK) and cancer (SKBR3 and HEPG2) cells after they were incubated with 0.125 to 1.25 nM PEG-GNR solutions. In vivo toxicity studies in nude mice showed no pathological changes in liver after the IV injection of GNR. Significant enhancements of OA contrast in comparison to images of untreated mice were observed 1 hour after the GNR injection in a dose of 20 mg gold per kg of body mass.

An imaging model incorporating ultrasonic transducer properties for three-dimensional optoacoustic tomography.

Optoacoustic tomography (OAT) is a hybrid imaging modality that combines the advantages of optical and ultrasound imaging. Most existing reconstruction algorithms for OAT assume that the ultrasound transducers employed to record the measurement data are point-like. When transducers with large detecting areas and/or compact measurement geometries are utilized, this assumption can result in conspicuous image blurring and distortions in the reconstructed images. In this work, a new OAT imaging model that incorporates the spatial and temporal responses of an ultrasound transducer is introduced. A discrete form of the imaging model is implemented and its numerical properties are investigated. We demonstrate that use of the imaging model in an iterative reconstruction method can improve the spatial resolution of the optoacoustic images as compared to those reconstructed assuming point-like ultrasound transducers.

Real-time optoacoustic monitoring and three-dimensional mapping of a human arm vasculature.

We present our findings from a real-time laser optoacoustic imaging system (LOIS). The system utilizes a Q-switched Nd:YAG laser; a standard 128-channel ultrasonic linear array probe; custom electronics and custom software to collect, process, and display optoacoustic (OA) images at 10 Hz. We propose that this system be used during preoperative mapping of forearm vessels for hemodialysis treatment. To demonstrate the real-time imaging capabilities of the system, we show OA images of forearm vessels in a volunteer and compare our results to ultrasound images of the same region. Our OA images show blood vessels in high contrast. Manipulations with the probe enable us to locate and track arteries and veins of a forearm in real time. We also demonstrate the ability to combine a series of OA image slices into a volume for spatial representation of the vascular network. Finally, we use frame-by-frame analysis of the recorded OA video to measure dynamic changes of the crossection of the ulnar artery.

Optoacoustic imaging of the prostate: development toward image-guided biopsy.

Optoacoustic (OA) tomography has demonstrated utility in identifying blood-rich malignancies in breast tissue. We describe the development and characterization of a laser OA imaging system for the prostate (LOIS-P). The system consists of a fiber-coupled Q-switched laser operating at 757 nm, a commercial 128-channel ultrasonic probe, a digital signal processor, and software that uses the filtered radial back-projection algorithm for image reconstruction. The system is used to reconstruct OA images of a blood-rich lesion induced in vivo in a canine prostate. OA images obtained in vivo are compared to images acquired using ultrasound, the current gold standard for guiding biopsy of the prostate. Although key structural features such as the urethra could be identified with both imaging techniques, a bloody lesion representing a highly vascularized tumor could only be clearly identified in OA images. The advantages and limitations of both forward and backward illumination modes are also evaluated by collecting OA images of phantoms simulating blood vessels within tissue. System resolution is estimated to be 0.2 mm in the radial direction of the acoustic array. The minimum detectable pressure signal is 1.83 Pa. Our results encourage further development toward a dual-modality OA/ultrasonic system for prostate imaging and image-guided biopsy.

Whole-body three-dimensional optoacoustic tomography system for small animals.

We develop a system for three-dimensional whole-body optoacoustic tomography of small animals for applications in preclinical research. The tomographic images are obtained while the objects of study (phantoms or mice) are rotated within a sphere outlined by a concave arc-shaped array of 64 piezocomposite transducers. Two pulsed lasers operating in the near-IR spectral range (755 and 1064 nm) with an average pulsed energy of about 100 mJ, a repetition rate of 10 Hz, and a pulse duration of 15 to 75 ns are used as optical illumination sources. During the scan, the mouse is illuminated orthogonally to the array with two wide beams of light from a bifurcated fiber bundle. The system is capable of generating images of individual organs and blood vessels through the entire body of a mouse with spatial resolution of approximately 0.5 mm.

Measurement of the spectral directivity of optoacoustic and ultrasonic transducers with a laser ultrasonic source.

Comprehensive characterization of wideband ultrasonic transducers and specifically optoacoustic detectors is achieved through the analysis of their frequency response as a function of the incident angle. The tests are performed under well-defined, repeatable operating conditions. Backillumination of a blackened, acoustically matched planar surface with a short laser pulse creates an acoustic impulse which is used as a wideband ultrasonic source. Upon illumination with a short laser pulse, the bandwidth of our source shows a -6 dB point of 12 MHz and a low-frequency roll-off around 300 kHz. Using proprietary software, we examine thoroughly the planarity of the emitted wave front within a specified amplitude cutoff and phase incoherence. Analysis of the angular dependence of the frequency response yields invaluable directivity information about the detector under study: a necessary component toward accurate optoacoustic image reconstruction and quantitative tomography. The laser ultrasonic source we developed is the main feature of our directivity measurement setup. Due to its simplicity, it can easily be adapted to various calibration devices. This paper focuses on the development and characterization of the flatness and the bandwidth of our wideband ultrasonic source.

Effects of salicylate on plasma membrane mechanics.

High concentrations of the amphipathic drug salicylate (Sal) block outer hair cell (OHC) electromotility resulting in reversible hearing loss. We used optical tweezers to study the effects of Sal on the mechanics of the cell plasma membrane. Membrane tethers were formed from guinea pig OHCs and cultured human embryonic kidney (HEK) cells as controls. HEK cells are commonly used in functional expression studies of electromotility. Effective tether viscosity (eta(eff)), steady-state tethering force extrapolated to zero pulling rate F(ss0), and time constant for tether growth (tau(tg)) were estimated from the measurements of the instantaneous tethering force at different tether pulling rates. Average values of eta(eff), F(ss0), and tau(tg) for the OHC lateral wall plasma membrane and control cell plasma membrane remained the same after Sal perfusion, which is consistent with the hypothesis that Sal-induced reversible hearing loss appears to be more the result of its competition with essential anions and less the result of a change in plasma membrane mechanics.

Effects of chlorpromazine on mechanical properties of the outer hair cell plasma membrane.

An optical tweezers system was used to characterize the effects of chlorpromazine (CPZ) on the mechanical properties of the mammalian outer hair cell (OHC) through the formation of plasma membrane tethers. Such tethers exhibited force relaxation when held at a constant length for several minutes. We used a second-order generalized Kelvin body to model tether-force behavior from which several mechanical parameters were then calculated including stiffness, viscosity-associated measures, and force relaxation time constants. The results of the analysis portray a two-part relaxation process characterized by significantly different rates of force decay, which we propose is due to the local reorganization of lipids within the tether and the flow of external lipid into the tether. We found that CPZ's effect was limited to the latter phenomenon since only the second phase of relaxation was significantly affected by the drug. This finding coupled with an observed large reduction in overall tether forces implies a common basis for the drug's effects, the plasma membrane-cytoskeleton interaction. The CPZ-induced changes in tether viscoelastic behavior suggest that alterations in the mechanical properties of the OHC lateral wall could play a role in the modulation of OHC electromotility by CPZ.