Mechanical characterization of intraluminal tissue with phase-resolved photoacoustic viscoelasticity endoscopy

We developed a phase-sensitive side-scanning photoacoustic viscoelasticity endoscopy (PAVEE) for mechanical characterization of intraluminal tissues. In PAVEE, the PA phase can be extracted from the optical absorption induced ultrasonic waves and provides an index of viscoelasticity that is closely linked to tissue compositions. The transverse resolution of the PAVEE measured by carbon fiber was about 32 μm. The imaging capability of the PAVEE was verified using a vessel-mimicking phantom with different agar density. Moreover, PAVEE was investigated in processed lumen-shaped vascular tissues to evaluate the biomechanical features, which was highly consistent with the histology. The results demonstrated that the PAVEE can obtain viscoelastic properties of intraluminal tissues, which puts a new insight into the intravascular disease and holds great promise for plaque vulnerability detection. ©2015 Optical Society of America OCIS codes: (170.3880) Medical and biological imaging; (170.5120) Photoacoustic imaging; (170.2150) Endoscopic imaging; (120.5050) Phase measurement. References and links 1. A. V. Finn, M. Nakano, J. Narula, F. D. Kolodgie, and R. Virmani, “Concept of vulnerable/unstable plaque,” Arterioscler. Thromb. Vasc. Biol. 30(7), 1282–1292 (2010). 2. G. W. Stone, A. Maehara, A. J. Lansky, B. de Bruyne, E. Cristea, G. S. Mintz, R. Mehran, J. McPherson, N. Farhat, S. P. Marso, H. Parise, B. Templin, R. White, Z. Zhang, and P. W. Serruys; PROSPECT Investigators, “A prospective natural-history study of coronary atherosclerosis,” N. Engl. J. Med. 364(3), 226–235 (2011). 3. V. 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Introduction
Acute myocardial infarction is the leading cause of death worldwide, which makes the intravascular atherosclerosis detection become extremely important.Current clinical endoscopes are focused on detecting the anatomical structure of atherosclerotic plaque.However, autopsy studies reveal that morphologic feature is not a sufficient predictor of acute events and the altered mechanical property is also a vital consideration [1,2].These findings enforce the need for new endoscopic technique to obtain mechanical characterization of atherosclerosis to provide other new insights into its etiology.
In order to compensate this shortage, we have studied a novel imaging modality of PA effect with detection of the PA phase [21][22][23].Typically, nanosecond pulsed laser is used to illuminate the tissues.The optical absorption of chromophores in local region leads to a cyclical temperature variation and induces a thermal stress.Then a strain in the form of force-produced PA wave is generated based on the thermo-elastic expansion.Due to the viscoelasticity of tissues would introduce a damping effect, the strain response would produce a phase lag behind the stress.Thus, the PA signal has the same frequency but a phase delay with the laser excitation.In the rheological Kelvin-Voigt model, the PA phase δ of the strain response delay to the stress can be derived as arc tan , where η is the viscosity coefficient, ω is the modulation frequency, and E is the Young's modulus.According to the formula, image contrast  , where η and E usually change in opposite directions in many kinds of disease.So with a constant modulation frequency, PA phase reflects the viscosity-elasticity ratio η/E and is sensitive to the viscoelastic change.There is much supporting evidence that viscoelastic factors actively regulate and influence atherosclerosis progression [24,25].Thus, in this paper, we first proposed a photoacoustic viscoelasticity endoscopy (PAVEE) to measure the mechanics of intraluminal tissues.With its phase-sensitive image contrast, PAVEE has the potential to achieve early detection and in vivo intravascular mechanical characterization, which enables a meticulous understanding and diagnosis of atherosclerosis.

Methods and materials
The lock-in detector is widely used in the detection of weak signals.In the process of calculating the phase delay between laser and PA signal, the synchronized laser output is used as a reference signal, the PA signal E 1 sin(ωt + α) and the reference signal E 2 sin(ωt + β) are input into the multiplier within the lock-in detector, and then the output is which is fed into a low-pass filter within the lock-in detector to output the dc component E 1 E 2 cos(α-β)/2, where E 1 E 2 and α-β are the amplitude and phase delay of the PA signal respectively.
The experimental setup of the PAVEE was shown in Fig. 1.A quasi-continuous laser (DS20HE-1064D/R, PHOTONICS) with 22 ns of pulse width, 1064 nm of wavelength, 65 KHz of repetition frequency was used as the excitation source.The collimated laser was focused by a microscope objective and coupled into an optical fiber.The distal end of the optical fiber passed through a hollow motor and ultrasound transducer (65-KHz central frequency) to fire laser on a customized parabolic reflector, which could simultaneously reflect and focus the laser.The reflector was fixed on a rotating motor to realize a 360° cross-sectional scanning.The transducer and scanning mirror were held in a nickel tube (16-mm in out diameter), which had a side opened window for firing laser and detecting PA signal.All the components were installed on a motorized pullback stage to realize a longitudinal scanning.At each angular step (0.225°) of the scan, time-averaged laser intensity on the inner surface of the vessel sample was limited well within the American National Standard Institute's safety limit (100 mJ/cm 2 ) [26].During data acquisition, the distance between the laser focus and transducer was maintained strictly consistent.The generated PA signals were acoustically coupled with distilled water and detected by the ultrasound transducer, then delivered to a low-noise low-pass preamplifier (SR552, Stanford Research Systems), and calculated by a lock-in detector (SR830, Stanford Research Systems) to resolve the phase lag behind the laser.The phase data were collected and analyzed on a computer which controlled the motorized scanner with a Labview program simultaneously.

Transverse-resolution evaluation of the PAVEE
The imaging resolution of the PAVEE was firstly investigated with a ~20 μm diameter carbon fiber, which was inserted in a hollow agar phantom at a radial position of 9.5 mm.Each cross-sectional image was acquired with 6400 sampling points.The cross-sectional PAVEE image of the phantom was shown in Fig. 2(a), and the enlarged view of the dashed box in Fig. 2(a) was shown in Fig. 2(b).As is evident from the images, the carbon fiber revealed a lower viscoelasticity than the agar due to its higher elasticity.In Fig. 2(c), the transverse point spread function (PSF) of the carbon fiber at α in Fig. 2(b) was presented.The transverse resolution, defined as the full-width half-maximum (FWHM) of the PSF, was about 32 μm.

PAVEE of a vessel-mimicking phantom
The PAVEE was subsequently verified with a vessel-mimicking phantom with a luminal diameter of 19 mm, which was composed of two counterparts with the same optical absorption coefficient and different viscoelastic properties.The photograph of the phantom from an upper view was shown in Fig. 3(a) and the corresponding cross-sectional PAVEE image at z = 1.25 mm was illustrated in Fig. 3(b), which matched well with the former in morphology as well as viscoelasticity distribution.The PAVEE imaging of the vessel-mimicking phantom was depicted in Fig. 3(c), where the counterpart with 6% agar was harder than the counterpart with 3% agar, and revealed a lower viscoelasticity than the latter.Both sides exhibited a uniform viscoelasticity distribution due to the homogeneous sample.The demonstrated the capability of the PAVEE for intraluminal mechanical characterization.

PAVEE of vascular tissues
To further validate the feasibility of PAVEE for biomedical application, ex vivo experiments were conducted on vessels harvested from a 3-months high-fat/high-cholesterol diet feeding rabbit.The vessel-shaped sample with a 19-mm luminal diameter was prepared with a normal-looking and atherosclerotic segment fixed in agar, where the atherosclerotic segment was affected by large-area plaques, then the PAVEE imaging was performed in the lumen of the sample.3D PAVEE image and en-face viscoelasticity distribution were shown in Fig. 4(a).The longitudinal PAVEE image enabled an overall viscoelastic evaluation of the luminal sample.The atherosclerotic tissue revealed an inhomogeneity of PA viscoelasticity distribution, which was relatively uniform in the normal-looking tissue.This was mainly attributed to the different degree of lipid accumulation for the atherosclerosis.As expected, the lipid-rich regions with higher viscoelasticity lead to higher PA phase.The PAVEE result verified a well correlation with both the sample morphology and the corresponding en-face PA viscoelasticity image.After PA experiment, the specimen was cross-sectional sliced and stained with oil red.In Fig. 4(b), the cross-sectional PAVEE section and histological staining at Z = 0.8 mm demonstrated a good coherency between PAVEE and histology.The lipid-rich plaques in atherosclerotic segment suggested high viscoelasticity and dense oil red staining.The normal-looking segment had a mean phase value of 18.4° and ~2° of phase fluctuations.This phase instability was caused by the unapparent early atherosclerosis, since the normal-looking and the atherosclerotic segment were harvested from the same vessel.The atherosclerotic segment had a phase range from 17.25° to 23.43°.The experiment proved that the PAVEE can sensitively differentiate atherosclerosis, and has the feasibility for accurate medical evaluation.

Discussion and conclusions
We first propose a PAVEE to evaluate intraluminal mechanics, which is an average viscoelasticity within the light excitation depth, so the PAVEE results are circle-line and cylindrical-surface characterization in cross-sectional and three-dimensional (3D) PAVEE image respectively.PAVEE combines the elasticity and viscosity of tissues, which is more comprehensive to describe the biomechanical properties.Benefiting from the phase-sensitive image contrast, PAVEE has the potential to differentiate subtle changes in mechanical properties, which enables early detection of disease.Besides, PAVEE may be combined with other endoscopic modalities to allow simultaneous multiple-characteristic measurements.We will integrate the PAVEE with conventional PA endoscopy by using a proper transducer to obtain mechanical and structural features.Furthermore, since the distance between laser focus and transducer would affect the phase detection, an algorithm using a sound propagation model in conjunction with the in-depth structural information obtained from conventional PA endoscopy could be applied to correct the distance-dependence phase deviation with the integrated endoscope.Meanwhile, the rotating speed of the PAVEE is limited by the time constant (30 ms) of the lock-in detector and the current low-frequency piezoelectric transducer holds large diameter, which restrict its in vivo clinical applications.In the future, a small-diameter focused transducer will be employed to improve the detecting sensitivity and accelerate the speed for further in vivo intravascular detection, which indicates an attractive prospect in the vulnerability evaluation of plaque.
In summary, a phase-sensitive PAVEE for intraluminal mechanical characterization was developed.The method holds great promise for detecting the altered mechanics accompany many pathologies, particularly in cardiovascular disease.The development and ex vivo demonstration of the PAVEE opens the opportunity for future clinical studies to get valuable insights on vital mechanical metrics associated with plaque rupture.

Fig. 2 .
Fig. 2. Transverse-resolution evaluation of the system.(a) Cross-sectional PAVEE image of the carbon fiber embedded in hollow agar.(b) Enlarged view of the dashed box in (a).(c) Transverse point spread function (PSF) of the carbon fiber at α in (b).

Fig. 3 .
Fig. 3. (a) Photograph of vessel-mimicking phantom composed of two counterparts with same absorption coefficient and different viscoelasticity (upper view).(b) Cross-sectional PAVEE image at z = 1.25 mm.(c) 3D PAVEE image of the agar phantom.

Fig. 4 .
Fig. 4. (a) Longitudinal PAVEE image and en-face viscoelasticity image viewed from inside over a 360° field of the sample.In the flattened tissue photograph, region within the dashed frame is scanning area.(b) PAVEE section and corresponding histology and phase distribution at z = 0.8 mm.The section was stained with oil red (red) targeting lipid.