Eno Hysi

Photoacoustic ultrasound spectroscopy for assessing red blood cell aggregation and oxygenation.

Abstract. Red blood cell (RBC) aggregation and oxygenation are important markers for a variety of blood disorders. No current technique is capable of simultaneously measuring aggregation/oxygenation levels noninvasively. We propose using photoacoustic ultrasound spectroscopy (PAUS) for assessing both phenomena. This technique relies on frequency-domain analysis of the PA signals by extracting parameters such as the ultrasound spectral slope and the midband fit. To investigate the effect of hematocrit, aggregation, and oxygenation levels on PAUS parameters, a Monte Carlo-based theoretical model and an experimental protocol using porcine RBCs were developed. The samples were illuminated at 750 and 1064 nm and changes in the PAUS parameters were compared to the oxygen-dependent optical absorption coefficients to assess the oxygenation level. Good agreement between the theoretical and experimental spectral parameters was obtained for the spectral slope of the nonaggregated spectra (∼0.3  dB/MHz). The experimental midband fit increased by ∼5  dB for the largest aggregate size. Based on the analysis of the PA signals, the oxygen saturation level of the most aggregated sample was >20% greater than the nonaggregated sample. The results provide a framework for using PA signals’ spectroscopic parameters for monitoring the aggregation and oxygenation levels of RBCs.

On the use of photoacoustics to detect red blood cell aggregation.

The feasibility of detecting red blood cell (RBC) aggregation with photoacoustics (PAs) was investigated theoretically and experimentally using human and porcine RBCs. The theoretical PA signals and spectra generated from such samples were examined for several hematocrit levels and aggregates sizes. The effect of a finite transducer bandwidth on the received PA signal was also examined. The simulation results suggest that the dominant frequency of the PA signals from non-aggregated RBCs decreases towards clinical frequency ranges as the aggregate size increases. The experimentally measured mean spectral power increased by ~6 dB for the largest aggregate compared to the non-aggregated samples. Such results confirm the theoretical predictions and illustrate the potential of using PA imaging for detecting RBC aggregation.

Validity of a theoretical model to examine blood oxygenation dependent optoacoustics

A theoretical model investigating the dependence of optoacoustic (OA) signal on blood oxygen saturation (SO(2)) is discussed. The derivations for the nonbandlimited and bandlimited OA signals from many red blood cells (RBCs) are presented. The OA field generated by many RBCs was obtained by summing the OA field emitted by each RBC approximated as a fluid sphere. A Monte Carlo technique was employed generating the spatial organizations of RBCs in two-dimensional. The RBCs were assumed to have the same SO(2) level in a simulated configuration. The fractional number of oxyhemoglobin molecules, confined in a cell, determined the cellular SO(2) and also defined the blood SO(2). For the nonbandlimited case, the OA signal amplitude decreased and increased linearly with blood SO(2) when illuminated by 700 and 1000 nm radiations, respectively. The power spectra exhibited similar trends over the entire frequency range (MHz to GHz). For the bandlimited case, three acoustic receivers with 2, 10, and 50 MHz as the center frequencies were considered. The linear variations of the OA amplitude with blood SO(2) were also observed for each receiver at those laser sources. The good agreement between simulated and published experimental results validates the model qualitatively.

Photoacoustic Imaging of Cancer Treatment Response: Early Detection of Therapeutic Effect from Thermosensitive Liposomes

Imaging methods capable of indicating the potential for success of an individualized treatment course, during or immediately following the treatment, could improve therapeutic outcomes. Temperature Sensitive Liposomes (TSLs) provide an effective way to deliver chemotherapeutics to a localized tumoral area heated to mild-hyperthermia (HT). The high drug levels reached in the tumor vasculature lead to increased tumor regression via the cascade of events during and immediately following treatment. For a TSL carrying doxorubicin (DOX) these include the rapid and intense exposure of endothelial cells to high drug concentrations, hemorrhage, blood coagulation and vascular shutdown. In this study, ultrasound-guided photoacoustic imaging was used to probe the changes to tumors following treatment with the TSL, HaT-DOX (Heat activated cytoToxic). Levels of oxygen saturation (sO2) were studied in a longitudinal manner, from 30 min pre-treatment to 7 days post-treatment. The efficacious treatments of HT-HaT-DOX were shown to induce a significant drop in sO2 (>10%) as early as 30 min post-treatment that led to tumor regression (in 90% of cases); HT-Saline and non-efficacious HT-HaT-DOX (10% of cases) treatments did not show any significant change in sO2 at these timepoints. The changes in sO2 were further corroborated with histological data, using the vascular and perfusion markers CD31 and FITC-lectin. These results allowed us to further surmise a plausible mechanism of the cellular events taking place in the TSL treated tumor regions over the first 24 hours post-treatment. The potential for using photoacoustic imaging to measure tumor sO2 as a surrogate prognostic marker for predicting therapeutic outcome with a TSL treatment is demonstrated.

Simultaneous assessment of red blood cell aggregation and oxygen saturation under pulsatile flow using high-frequency photoacoustics.

We investigate the feasibility of photoacoustic (PA) imaging for assessing the correlation between red blood cell (RBC) aggregation and the oxygen saturation (sO2) in a simulated pulsatile blood flow system. For the 750 and 850 nm illuminations, the PA amplitude (PAA) increased and decreased as the mean blood flow velocity decreased and increased, respectively, at all beat rates (60, 120 and 180 bpm). The sO2 also cyclically varied, in phase with the PAA for all beat rates. However, the linear correlation between the sO2 and the PAA at 850 nm was stronger than that at 750 nm. These results suggest that the sO2 can be correlated with RBC aggregation induced by decreased mean shear rate in pulsatile flow, and that the correlation is dependent on the optical wavelength. The hemodynamic properties of blood flow assessed by PA imaging may be used to provide a new biomarker for simultaneous monitoring blood viscosity related to RBC aggregation, oxygen delivery related to the sO2 and their clinical correlation.

Photoacoustic tissue characterization using envelope statistics and ultrasonic spectral parameters

Photoacoustic (PA) tissue characterization relies on the analysis of ultrasound (US) signals generated through the PA effect. The probability distributions of PA signal amplitude as well as the frequency content of the PA signals are typically not considered. We present a phantom study where we introduce the combined use of US/PA signal envelope statistics along with analysis of the frequency content of the US/PA signals for the purposes of monitoring physical changes in the absorbers. The phantoms were constructed using black polystyrene beads (radius 1.77 μm and 7.36 μm). Tissue microenvironment was emulated by homogenously mixing 10 beads/imaging-transducer-resolution-volume in order to accommodate large numbers of sub-resolved beads. The phantoms were imaged with the Vevo LAZR US/PA integrated imaging system (Fujifilm-VisualSonics) using a 40 MHz linear array probe and 680 nm illumination. US/PA signals from the same region of interest were analyzed by mapping the signal amplitudes distributions from 5 phantom locations, fitting the data to the Generalized Gamma (GG) distribution and extracting the fit parameters while computing the normalized power spectra to retrieve the spectral slope (SS) and midband fit (MBF) spectral parameters. The GG scale parameter increased by 500x for US images and 12x for PA equivalents as the size of the beads increased by ~4.5x. The SS decreased by 0.8x for US and 0.4x for PA. These changes can be attributed variations in size and spatial organization of the beads suggesting that combined US/PA statistical and spectral analysis can potentially monitor normal and abnormal tissue physiological changes.

Photoacoustic measurements of single red blood cells

A photoacoustic method to infer the size, shape and orientation of single RBCs is presented. Diseased or damaged RBCs frequently have abnormal shapes and sizes, which can result from a variety of diseases. When using frequencies between 100 and 500 MHz, the photoacoustic power spectrum from RBCs has periodically varying minima and maxima that depend on the size and shape of the RBC. The signals from RBCs in a vertical and horizontal orientation relative to the transducer were compared to numerical simulations using a finite element model. Three different shapes commonly used to model RBCs were examined (at constant volume): a biconcave disk, an oblate ellipsoid and a sphere. For a single biconcave shaped RBC, good agreement in the shape of the power spectrum was observed between measured and numerical simulations for the RBC in both the vertical and horizontal orientation relative to the transducer. When the RBC was placed in a hypotonic solution, it swelled to a spherical shape. In this case, good agreement was observed between the measured signal and the spherical model. In this paper, we show that the photoacoustic power spectrum can be used to infer the size, shape and orientation of single RBCs.

Photoacoustic signal characterization of cancer treatment response: Correlation with changes in tumor oxygenation

Frequency analysis of the photoacoustic radiofrequency signals and oxygen saturation estimates were used to monitor the in-vivo response of a novel, thermosensitive liposome treatment. The liposome encapsulated doxorubicin (HaT-DOX) releasing it rapidly (<20 s) when the tumor was exposed to mild hyperthermia (43 °C). Photoacoustic imaging (VevoLAZR, 750/850 nm, 40 MHz) of EMT-6 breast cancer tumors was performed 30 min pre- and post-treatment and up to 7 days post-treatment (at 2/5/24 h timepoints). HaT-DOX-treatment responders exhibited on average a 22% drop in oxygen saturation 2 h post-treatment and a decrease (45% at 750 nm and 73% at 850 nm) in the slope of the normalized PA frequency spectra. The spectral slope parameter correlated with treatment-induced hemorrhaging which increased the optical absorber effective size via interstitial red blood cell leakage. Combining frequency analysis and oxygen saturation estimates differentiated treatment responders from non-responders/control animals by probing the treatment-induced structural changes of blood vessel.

Quantitative photoacoustic assessment of red blood cell aggregation under pulsatile blood flow: experimental and theoretical approaches

In the present paper, the optical wavelength dependence on the photoacoustic (PA) assessment of the pulsatile blood flow was investigated by means of the experimental and theoretical approaches analyzing PA radiofrequency spectral parameters such as the spectral slope (SS) and mid-band fit (MBF). For the experimental approach, the pulsatile flow of human whole blood at 60 bpm was imaged using the VevoLAZR system (40-MHz-linear-array probe, 700-900 nm illuminations). For the theoretical approach, a Monte Carlo simulation for the light transmit into a layered tissue phantom and a Green’s function based method for the PA wave generation was implemented for illumination wavelengths of 700, 750, 800, 850 and 900 nm. The SS and MBF for the experimental results were compared to theoretical ones as a function of the illumination wavelength. The MBF increased with the optical wavelength in both theory and experiments. This was expected because the MBF is representative of the PA magnitude, and the PA signal from red blood cell (RBC) is dependent on the molar extinction coefficient of oxyhemoglobin. On the other hand, the SS decreased with the wavelength, even though the RBC size (absorber size which is related to the SS) cannot depend on the illumination wavelength. This conflicting result can be interpreted by means of the changes of the fluence pattern for different illumination wavelengths. The SS decrease with the increasing illumination wavelength should be further investigated.

Probing the in vivo changes in oxygen saturation with photoacoustic imaging as a non-invasive means of assessing treatment progression

In vivo photoacoustic estimations of tumor oxygenation were used to assess the therapeutic efficacy of a thermosensitive liposome treatment in a pre-clinical mouse model. The treated group (n = 12) was administered doxorubicin-loaded, heat sensitive liposomes and exposed to mild hyperthermia (43°C) in order to deliver doxorubicin locally within the tumor micro-vessels. Control groups received systemic doxorubicin (n = 7) or saline (n = 12). The changes in tumor blood vessels after treatment were probed by analyzing the frequency content of the photoacoustic radiofrequency signals. Tumor oxygenation dropped by 15-20% during the first 30 minutes post-treatment when the tumors were exposed to encapsulated (Heat-Activated cyToxic – HaT-DOX) or free doxorubicin (DOX). The early (30 minutes to 5 hours) decrease in oxygen saturation strongly correlated to the reduction in tumor size assessed by caliper measurements. Control animals did not exhibit significant changes in tumor oxygenation at the early time points. The oxygenation at 7 days increased significantly for all groups. Measurements of the spectral slope from the normalized power spectra of the photoacoustic signals could also be used to differentiate between responder and non-responder mice. The results of this study suggest that photoacoustic imaging of tumors undergoing vascular-targeted cancer therapy can be used to assess treatment response early (hours) post-treatment through a combined analysis of oxygen saturation and photoacoustic radiofrequency spectroscopy.