2.1.

Phantoms and Ex Vivo Tissue Samples

The experiments were first performed using tissue-mimicking phantoms with models of acute and chronic DVT. A $6.37\text{-}\mathrm{mm}$ -diam tube (WLS25275-BC, Sargent-Welch, Incorporated, Buffalo, New York) was used to mimic a vessel wall. This artificial vein was attached to a fixture and then embedded into $100\text{-}\times 50\text{-}\times 45\text{-}\mathrm{mm}$ $(\mathrm{L}\times \mathrm{W}\times \mathrm{H})$ rectangular block made out of 10% weight concentration of gelatin (type A, Sigma-Aldrich, Saint Louis, Missouri). To produce a background material with similar optical and ultrasonic properties of soft tissues, a 20% volume concentration of fat-free milk was added to elevate optical scattering,^{24, 25} and 0.8% weight concentration of $40\text{-}\mu \mathrm{m}$ silica particles²⁶ (Sigma-Aldrich) was added for ultrasonic backscattering. The light absorption and reduced scattering coefficients of this gelatin were estimated to be $0.09\pm 0.01{\mathrm{cm}}^{-1}$ and $5.82\pm 0.04{\mathrm{cm}}^{-1}$ , respectively ( $\text{mean}\pm \text{standard}$ deviation).

Venous blood was modeled by a solution of heparinized human RBCs obtained from a local blood and tissue center. The RBCs were 50% diluted in saline to match the normal concentration of RBC in the whole human blood. The light extinction coefficient, measured in a thin layer of this solution, was $293\pm 9{\mathrm{cm}}^{-1}$ .

The $13\text{-}\text{to}20\text{-}\mathrm{mm}$ -long and $6.4\text{-}\mathrm{mm}$ -diam artificial clots were constructed using 7% weight concentration of PVA (Celvol 165SF, Celanese, Dallas, Texas). A 2% weight concentration of $40\text{-}\mu \mathrm{m}$ silica particles was added in all artificial clots for ultrasound scattering, while PVA by itself is already a light scattering material. The light absorption and reduced light scattering coefficients were estimated to be $0.4\pm 0.3{\mathrm{cm}}^{-1}$ and $6.8\pm 0.3{\mathrm{cm}}^{-1}$ , respectively. These values are similar to those reported in the literature.²⁷ To position the clots reliably in the vein and to avoid the penetration of the RBC solution between the clot and the vessel wall, the diameter of the clot was slightly larger than the diameter of the dialysis tubing. To model acute clots, a base RBC solution, was added in PVA, and pure saline with no RBCs was used in artificial chronic clots. The light absorption coefficient of an acute clot was estimated to be 9% lower than that of RBC solution, since both PVA and silica particles have negligible optical absorption coefficients in comparison with RBC solution. The clot-containing vein was embedded in a gelatin phantom, as shown in Fig. 1 . The remaining part of the vessel was filled with the 50% RBC solution representing venous blood.

Fig. 1

Diagram of a phantom and an experimental setup used in ultrasound and photoacoustic imaging of DVT. The dashed rectangle schematically represents the imaging area, and the vertical lines indicate the positions of the photoacoustic signals used for quantitative comparison of different clots.

The combined ultrasound and photoacoustic imaging were also explored to image acute and chronic thrombi obtained from a well established small animal model of stasis-induced venous thrombosis,²³ which closely reproduces thrombus development in clinical disease. Sprague-Dawley rats underwent surgery to initiate thrombus formation in the inferior vena cava, and $3\text{-}\text{day}$ -old (acute thrombus) and $9\text{-}\text{day}$ -old (chronic thrombus) clots were surgically removed from the animals. The thrombi were then positioned in a gelatin phantom at $15\mathrm{mm}$ depth and $13\mathrm{mm}$ apart from each other and imaged ex vivo. The diameters of the acute and chronic thrombi in the imaging plane were approximately 2 and $3.5\mathrm{mm}$ , respectively.

2.2.

Imaging System

The combined ultrasound and photoacoustic imaging system is schematically shown in Fig. 1. During the experiments, the phantoms were placed in a water cuvette with an optical window at the bottom to allow laser light to enter from underneath (Fig. 1). The phantoms were irradiated with $5\text{-}\mathrm{ns}$ pulses using a Q-switched Nd:YAG laser (Polaris II, New Wave Research, Freemont, California) operating at $532\text{-}\mathrm{nm}$ wavelength. A focused, single element ultrasound transducer (Panametrics-NDT, Waltham, Massachusetts) with a center frequency of $7.5\mathrm{MHz}$ , a fractional bandwidth of 60%, an aperture of $12.7\mathrm{mm}$ , and an f/number of 4 was positioned on the opposite side of the phantom. Assuming the speed of sound of $1500\mathrm{m}∕\mathrm{s}$ , the ultrasound images obtained using this transducer should have $170\text{-}\mu \mathrm{m}$ axial (i.e., along the ultrasound beam) and $800\text{-}\mu \mathrm{m}$ lateral (i.e., across the beam) resolutions, respectively. During the imaging, both the ultrasound transducer and the laser beam were simultaneously moved by $200\mu \mathrm{m}$ in the horizontal direction using a computer controlled, high-precision positioning system (model MN10-0050-M02-21, Velmex, Bloomfield, New York). Using the same transducer, both photoacoustic and ultrasound signals were acquired by the $12\text{-}\text{bit}$ , $50\text{-}\mathrm{MHz}$ bandwidth, $100\text{-}\mathrm{MS}∕\mathrm{s}$ digitizer (CompuScope 12100, GaGe Applied, Lockport, Illinois). Overall, all components of the imaging system including the pulsed laser, an ultrasound pulser/receiver (model 5910PR, Panametrics-NDT), the motion controller (nuDrive, National Instruments, Austin, Texas) and the digitizer were interfaced and controlled using a developed software interface written in LabVIEW.

2.3.

Data Acquisition and Signal/Image Processing

The dependence of a photoacoustic signal magnitude on a concentration of the RBC solution was measured using the phantom with an embedded modeled vein filled with RBC solution. For each concentration, the photoacoustic measurements were performed with laser pulses of different $\left(9\text{to}23\mathrm{mJ}\right)$ energy levels, and then normalized. The baseline signal was estimated using a vein filled with saline.

The imaging of the tissue-mimicking phantoms with modeled thrombi was performed along the coronal cross section of the vessel, as shown in Fig. 1. To qualitatively and quantitatively compare the acute and chronic thrombi obtained from the rat model of DVT, the transverse cross sections were imaged within one scan. In all imaging experiments, an average of more than ten laser pulses was applied at each lateral position to reduce the pulse-to-pulse laser energy variations. The corresponding photoacoustic signals, followed by ultrasound signals, were acquired and stored for off-line processing. Due to a weak light absorption in chronic clots, these clots were imaged with high $\left(23\mathrm{mJ}\right)$ laser energy, while the phantom with an artificial acute clot was imaged using low $\left(9\mathrm{mJ}\right)$ laser pulses.

The captured photoacoustic and ultrasound rf signals were averaged, demodulated, and scan converted to cover the desired region of interest, pictorially shown in Fig. 1. In addition, the magnitude of the photoacoustic signal was corrected for the exponential (Beer’s law) losses of optical fluence with depth. Both ultrasound and photoacoustic images were formed using the magnitude of the basebanded signal. No sophisticated reconstruction techniques were applied to produce photoacoustic images. For quantitative comparison, the photoacoustic signals from clots and blood-filled veins were analyzed (the positions of the rf lines are shown in Fig. 1). Finally, the maximum of each photoacoustic rf signal within the region of interest was estimated and plotted as a function of the lateral position. No compensation for the optical fluence was applied to calculate these photoacoustic profiles or to quantitatively compare photoacoustic rf signals.

3.1.

Phantom Studies

The dependence of the magnitude of the photoacoustic signal on the concentration of the RBC solution is shown in Fig. 2 . As expected, the dependence is monotonic and a higher concentration of RBC solution produces a higher photoacoustic response. Therefore, an acute clot with a higher concentration of RBC should produce a greater photoacoustic response compared to a chronic clot with lower concentration of RBC.

Fig. 2

Dependence of normalized magnitude (mean value ± standard deviation) of the photoacoustic pressure transient on volume concentration of RBC solution in saline. RBC concentration of 50% corresponds to a concentration of RBC solution in normal human blood.

The ultrasound and photoacoustic images of the phantoms with the artificial clots are presented in Fig. 3 . Specifically, images of the acute and chronic clots in the vein filled with blood are presented in Figs. 3a and 3b, and Figs. 3c and 3d, correspondingly. The chronic clot inside the vein filled with saline solution is presented in Figs. 3e and 3f.

Fig. 3

(a), (c), and (e) The ultrasound and (b), (d), and (f) photoacoustic images depict the coronal views of the phantoms with artificial clots inside of the modeled veins and background tissue. All images are $30.4\times 18.0\mathrm{mm}$ . The acute and chronic clots in the vein filled with blood are presented in (a) and (b), and (c) and (d), correspondingly. The chronic clot inside the vein filled with saline solution is presented in (e) and (f). The compensation for exponential decay of optical fluence with depth was applied to all photoacoustic images.

In ultrasound images [Figs. 3a, 3c, 3e], the structural features of all phantoms such as background tissue, vessel walls and blood clot are clearly depicted. These B-scans are slightly different due to the differences between the phantoms and the imaging planes. As expected, acute clots cannot be differentiated from chronic clots in ultrasound images. The acute clot in Fig. 3a appears darker than the chronic clots in Figs. 3c and 3e and does not produce an ultrasound shadow. However, these differences are more likely attributed to a slight off-center position of the imaging plane where the variations of the phantom material along the vein result in a different refraction of the ultrasound waves from the boundaries. Indeed, the acute clot and the background tissue in Fig. 3a appear to be similar, although no RBCs were added in the background material.

The photoacoustic image of the phantom with an acute clot is shown in Fig. 3b. Using the band-limited ultrasound transducer, the photoacoustic response from an object with relatively uniform distribution of optical absorption coefficient will be represented by two signals corresponding to the boundaries of the vessel or the blood clot.^{28, 29} Furthermore, the lower boundary exposed to a greater light intensity (or fluence) will exhibit a larger photoacoustic response generated in the acute clot and the lumen of the vessel. However, since the concentrations of RBC in acute clot and venous blood are nearly the same, the photoacoustic imaging cannot differentiate between acute clot and luminal blood.

The photoacoustic image of the phantom with a chronic clot is shown in Fig. 3d. As expected, the photoacoustic response from the luminal blood is relatively high. However, the magnitude of the photoacoustic signal from the chronic clot is reduced. This is due to almost no optical absorption in the chronic clot. This effect cannot be explained by additional ultrasound attenuation, which produces ultrasound shadow in Figs. 3c and 3e. Brightness of the chronic clots near upper and lower tissue-clot interfaces is similar, thus indicating low attenuation of ultrasound. The upper clot-tissue interface in the area of thrombus is no longer visible, although it was exposed to the higher laser energy again indicating the weak optical absorption of the clot compared with RBC solution. Overall, a chronic clot produces relatively small photoacoustic response in comparison with acute thrombi or luminal blood.

Compared to RBCs, the other materials of the phantom such as PVA, gelatin, and dialysis tubing do not significantly absorb light at this wavelength, and therefore do not generate photoacoustic transients—it is demonstrated by photoacoustic imaging of a phantom with a chronic clot and without the RBC solution in the lumen of the vessel [Fig. 3f]. Indeed, the photoacoustic image is nearly featureless, indicating the modest optical absorption coefficient in the phantom at the given wavelength.

The photoacoustic rf signals generated in the acute and chronic clots and in RBC/saline solution are quantitatively contrasted in Fig. 4 . The transducer is located closer to the origin of the plot, while the laser irradiation is directed from an opposite side (see also Fig. 1).

Fig. 4

The photoacoustic rf signals from the phantoms with (a) and (b) the artificial acute and (c) and (d) chronic thrombi and RBC solution in the lumen, and the phantom with chronic clot and saline in the vein [(e) and (f)]. The photoacoustic rf signals taken along the vertical line passing through the clots are presented in (a), (c), and (d). The rf signals recorded from the vessel with no clots are presented in (b), (d), and (f).

The photoacoustic rf signals from the acute clot [Fig. 4a] and from the vein with RBC solution [Figs. 4b and 4d] consist of two peaks corresponding to the boundaries of optical absorbers. Due to the strong optical absorption of the acute clot and venous blood, and given the orientation of laser irradiation, the magnitude of the photoacoustic signals from the upper tissue-vein interface (located at $5.5\mathrm{mm}$ ) is significantly lower than the magnitude of peaks corresponding to the rf signal from lower tissue-vein interface (located at $12\mathrm{mm}$ ). The magnitudes of photoacoustic rf signals from the chronic clots [Figs. 4c and 4e] and from the vein with saline [Fig. 4f] are significantly lower in comparison with both the acute clot [Fig. 4a] and the RBC solution [Figs. 4b and 4d]. The signal from the upper tissue-vein interface in the area of the chronic clot is not noticeable due to insignificant light absorption.

To further underline the differences in the photoacoustic signals from the acute and chronic clots, the photoacoustic profiles obtained from the images in Figs. 3b, 3d, 3f are plotted in Fig. 5 . The photoacoustic profile for the chronic clot in the vein filled with saline has low magnitude and does not vary with lateral position, i.e., the chronic clot cannot be distinguished from the background. The phantom with the acute clot also exhibits a nearly uniform photoacoustic profile, but the magnitude is high. The photoacoustic signal in the area of the acute clot itself is slightly lower because approximately 9% of RBCs were replaced by PVA and silica particles, thus reducing the optical absorption in acute clot in comparison with venous blood. On the contrary, the magnitude of the photoacoustic profile of the phantom with chronic clot is low in the region of the clot itself, while the photoacoustic signal from the vein filled by the RBC solution is considerably higher.

Fig. 5

The photoacoustic profiles estimated in the phantoms with the acute (solid line) and chronic (dot-dashed line) clots and blood-filled vein, and the phantom with the chronic clot and saline solution in the lumen (dashed line).

3.2.

Ex Vivo Studies

The photoacoustic and ultrasound imaging of DVT was further evaluated using thrombi obtained from a rat model of DVT. The ultrasound and photoacoustic images of chronic ( $9\text{-}\text{day}$ -old) and acute ( $3\text{-}\text{day}$ -old) thrombi are shown in Fig. 6 . The boundary between two layers of the gelatin is clearly visible in the ultrasound image [Fig. 6a] due to a slight mismatch in acoustic impedances. This boundary, however, is not visible on the photoacoustic image [Fig. 6b], since there are no differences in optical absorption between these two layers. This fact again demonstrates that the contrast mechanisms in ultrasound and photoacoustic images are not related. The differences in ultrasound appearance of both clots may be attributed to the difference of both the thrombi sizes and content.³⁰ The photoacoustic image of the acute thrombus consists of two transient pressure waves from the boundaries of the clot. The lower clot-tissue interface generates a higher signal than the upper interface. These features of the photoacoustic response are very similar to those of the artificial acute clot shown in Fig. 3b. In contrast, the chronic clot appears darker and is represented by the several transient pressure waves.

Fig. 6

The (a) ultrasound and (b) photoacoustic images representing the cross sectional view of the chronic, $9\text{-}\text{day}$ -old (left) and acute $3\text{-}\text{day}$ -old (right) clots embedded in the gelatin phantom. The thrombi were obtained from a rat model of stasis-induced DVT. Both images are $22\times 13\mathrm{mm}$ . The locations and sizes of both clots in the ultrasound image are denoted by dashed circles.

The photoacoustic rf signals generated in the chronic and acute thrombi are shown in Fig. 7 . The rf signal from the acute clot [Fig. 7a] closely resembles the rf signal from the artificial clot [Fig. 4a]. Again, the magnitude of the transient pressure from the lower clot-tissue interface is higher than the magnitude of the wave from the upper interface. However, both waves are clearly noticeable because higher laser energy reached the upper boundary of the smaller ( $\sim 2\mathrm{mm}$ diam) acute clot compared with the artificial $6.4\text{-}\mathrm{mm}$ -diam acute clot. The overall magnitude of the rf signal from the chronic clot is lower compared with the acute clot, indicating lower optical absorption of chronic DVT. However, the noticeable transient pressures generated within the chronic clot suggest a heterogeneous structure of the clot, and therefore a variation of light absorption coefficient inside the clot.

Fig. 7

The photoacoustic rf signals from the phantom with (a) acute and (b) chronic animal thrombi taken along the vertical lines indicated by arrows in Fig. 6b.

The differences between chronic and acute thrombi are further demonstrated in the photoacoustic profile shown in Fig. 8 . As expected, the magnitude of the photoacoustic response from the acute clot is greater than from the chronic clot. The photoacoustic signal from the chronic clot is slightly greater than the background level of the photoacoustic response. It is clear that the older clot is characterized by a weaker photoacoustic signal.

Fig. 8

The profile of the photoacoustic signals for the chronic ( $9\text{-}\text{day}$ -old) and acute ( $3\text{-}\text{day}$ -old) clots.