Pavel Subochev

Simultaneous photoacoustic and optically mediated ultrasound microscopy: phantom study.

An experimental setup for combined photoacoustic (PA) and optically mediated ultrasound (US) microscopy is presented. A spherically focused 35 MHz polyvinylidene fluoride (PVDF) ultrasonic detector with a numerical aperture of 0.28, a focal distance of 9 mm, and a bandwidth (-6 dB level) of 24 MHz was used to obtain PA and US data with a 3 mm imaging depth. A fiber-optic system was employed to deliver laser excitation pulses from a tunable laser to the studied medium. A single optical pulse was used to form both PA and US A-scans. The probing US pulses were generated thermoelastically due to absorption of backscattered laser radiation by the metalized surface of a PVDF film.

Simultaneous photoacoustic and optically mediated ultrasound microscopy: an in vivo study.

We propose the use of thermoelastic (TE) excitation of an ultrasonic (US) detector by backscattered laser radiation as a means of upgrading a single-modality photoacoustic (PA) microscope to dual-modality PA/US imaging at minimal cost. The upgraded scanning head of our dual-modality microscope consists of a fiber bundle with 14 output arms and a 32MHz polyvinylidene difluoride (PVDF) detector with a 34 MHz bandwidth (-6 dB level), 12.7 mm focal length, and a 0.25 numerical aperture. A single optical pulse delivered through the fiber bundle to the biotissue being investigated, in combination with a metalized surface on the PVDF detector allows us to obtain both PA and US A-scans. To demonstrate the in vivo capabilities of the proposed method we present the results of bimodal imaging of the brain of a newborn rat, a mouse tail and a mouse tumor.

Cost-effective imaging of optoacoustic pressure, ultrasonic scattering, and optical diffuse reflectance with improved resolution and speed

The idea of a method of cost-effective upgrades from an acoustic resolution photoacoustic microscope to a triple-modality imaging system is validated using phantoms. The newly developed experimental setup is based on a diode pumped solid state laser coupled to a fiber bundle with a spherically focused polyvinylidene fluoride detector integrated into the center of a ring shaped optical illuminator. Each laser pulse illuminating the sample performs two functions. While the photons absorbed by the sample provide a measurable optoacoustic (OA) signal, the photons absorbed by the detector provide the measurable diffuse reflectometry (DR) signal from the sample and the probing ultrasonic (US) pulse. At a 3 mm imaging depth, the axial resolution of the OA/US modalities is 38 μm/26 μm, while the lateral resolution of the DR/OA/US modalities is 3.5 mm/50 μm/35 μm. The maximum acquisition rate of the trimodal DR/OA/US A-scans is 2 kHz.

Photobleaching and phototoxicity of KillerRed in tumor spheroids induced by continuous wave and pulsed laser illumination

The purpose of this study was to evaluate photobleaching of the genetically encoded photosensitizer KillerRed in tumor spheroids upon pulsed and continuous wave (CW) laser irradiation and to analyze the mechanisms of cancer cell death after the treatment. We observed the light-dose dependent mechanism of KillerRed photobleaching over a wide range of fluence rates. Loss of fluorescence was limited to 80% at light doses of 150 J/cm(2) and more. Based on the bleaching curves, six PDT regimes were applied for irradiation using CW and pulsed regimes at a power density of 160 mW/cm(2) and light doses of 140 J/cm(2) , 170 J/cm(2) and 200 J/cm(2). Irradiation of KillerRed-expressing spheroids in the pulsed mode (pulse duration 15 ns, pulse repetition rate 10 Hz) induced predominantly apoptotic cell death, while in the case of CW mode the cancer cells underwent necrosis. In general, these results improve our understanding of photobleaching mechanisms in GFP-like proteins and show the importance of appropriate selection of treatment mode for PDT with KillerRed. Representative fluorescence image of two KillerRed-expressing spheroids before and immediately after CW irradiation.

Optimal wavelengths for optoacoustic measurements of blood oxygen saturation in biological tissues.

The non-invasive measurement of blood oxygen saturation in blood vessels is a promising clinical application of optoacoustic imaging. Nevertheless, precise optoacoustic measurements of blood oxygen saturation are limited because of the complexities of calculating the spatial distribution of the optical fluence. In the paper error in the determination of blood oxygen saturation, associated with the use of approximate methods of optical fluence evaluation within the blood vessel, was investigated for optoacoustic measurements at two wavelengths. The method takes into account both acoustic pressure noise and the error in determined values of the optical scattering and absorption coefficients used for the calculation of the fluence. It is shown that, in conditions of an unknown (or partially known) spatial distribution of fluence at depths of 2 to 8 mm, minimal error in the determination of blood oxygen saturation is achieved at wavelengths of 658 ± 40 nm and 1069 ± 40 nm.

Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model.

The strong phototoxicity of the red fluorescent protein KillerRed allows it to be considered as a potential genetically encoded photosensitizer for the photodynamic therapy (PDT) of cancer. The advantages of KillerRed over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to any desired cell compartment. The ability of KillerRed to affect cell division and to induce cell death has already been demonstrated in cancer cell lines in vitro and HeLa tumor xenografts in vivo. However, the further development of this approach for PDT requires optimization of the method of treatment. In this study we tested the continuous wave (593 nm) and pulsed laser (584 nm, 10 Hz, 18 ns) modes to achieve an antitumor effect. The research was implemented on CT26 subcutaneous mouse tumors expressing KillerRed in fusion with histone H2B. The results showed that the pulsed mode provided a higher rate of photobleaching of KillerRed without any temperature increase on the tumor surface. PDT with the continuous wave laser was ineffective against CT26 tumors in mice, whereas the pulsed laser induced pronounced histopathological changes and inhibition of tumor growth. Therefore, we selected an effective regimen for PDT when using the genetically encoded photosensitizer KillerRed and pulsed laser irradiation.

Method of bimodal photoacoustic and ultrasound microscopy for simultaneous structural and functional diagnostics of biotissues

The excitation of external electrode of focused photoacoustic (PA) detector by the backscattered laser radiation can ensure a cost-effective upgrade from single-modality PA microscopy to dual-modality PA/US imaging. In this work we approbated thermoelastic generation of probing ultrasonic pulses for bimodal photoacoustic (PA) and optically mediated Ultrasound (US) microscopy in vivo, using single optical pulse delivered from tunable laser to form both PA and US A-scans. The presented results of bimodal in vivo visualization of rat brain provide with the experimental evidence, that the proposed approach can be used for both functional and structural bioimaging simultaneously.

Simultaneous in vivo imaging of diffuse optical reflectance, optoacoustic pressure and ultrasonic scattering.

We present reflection-mode bioimaging system providing complementary optical, photoacsoutic and acoustic measurements by acoustic detector after each laser pulse. While the photons absorbed within the sample provide optoacoustic (OA) signals, the photons absorbed by the external electrode of a detector provide the measurable diffuse reflectance (DR) from the sample and the probing ultrasonic (US) pulse. To demonstrate the in vivo capabilities of the system we present the results of complementary DR/OA/US imaging of a mouse tumor, head of a newborn rat, and the back of a newborn rat with 3.5mm/50μm/35μm lateral resolution. Trimodal approach allows visualization of mechanical structures in healthy and pathological tissues along with peculiarities of blood supply. The system may be used for diagnostics of diseases accompanied by the defects of vascularization as well as for assessing the mechanisms of vascular changes when monitoring response to therapy.

Passive estimation of internal temperatures making use of broadband ultrasound radiated by the body

The internal temperatures of plasticine models and the human forearm in vivo were determined, based on remote measurements of their intrinsic ultrasonic radiation. For passive detection of the thermal ultrasonic radiation an acoustic radiometer was developed, based on a broadband 0.8-3.3 MHz disk-shaped ultrasonic detector with an 8 mm aperture. To reconstruct temperature profiles using the experimentally measured spectra of thermal acoustic radiation a priori information was used regarding the temperature distribution within the objects being investigated. The temperature distribution for heated plasticine was considered to be a monotonic function. The distribution for the human forearm was considered to fit a heat equation incorporating blood flow parameters. Using sampling durations of 45 s the accuracy of temperature measurement inside a plasticine model was 0.5 K. The measured internal temperature of the forearm in vivo, at 36.3 °C, corresponded to existing physiological data. The results obtained verify the applicability of this passive method of wideband ultrasonic thermometry to medical applications that involve local internal heating of biological tissue.

Fluence compensation in raster-scan optoacoustic angiography

Modern optical imaging techniques demonstrate significant potential for high resolution in vivo angiography. Optoacoustic angiography benefits from higher imaging depth as compared to pure optical modalities. However, strong attenuation of optoacoustic signal with depth provides serious challenges for adequate 3D vessel net mapping, and proper compensation for fluence distribution within biotissues is required. We report on the novel approach allowing to estimate effective in-depth fluence profiles for optoacoustic systems. Calculations are based on Monte Carlo simulation of light transport and account for complex illumination geometry and acoustic detection parameters. The developed fluence compensation algorithm was tested in in vivo angiography of human palm and allowed to overcome significant in-depth attenuation of probing radiation and enhance the contrast of lower dermis plexus while preserving high resolution of upper plexus imaging.