To quantitatively evaluate the optical absorption property of tissue based on the photoacoustic response, wavelength dependent optical properties of tissues need to be taken into account [17
]. In this study, a narrow wavelength range of 1200–1230 nm was used for IVPA imaging to minimize the effect of wavelength-dependent optical properties on the spectroscopic IVPA data analysis. The narrow imaging wavelength range benefited the spectroscopic analysis in two ways. First, the limited range of IVPA imaging wavelength guaranteed the nearly linear decline of the photoacoustic signal from lipid-rich regions within 1200–1230 nm. Second, the effect of wavelength-dependent laser fluence distribution was minimal and thus did not affect significantly the relative changes of photoacoustic signal amplitude.
The photoacoustic response of the suspected lipid-rich region in the diseased aorta followed a monotonic decrease in the 1200–1230 nm range (region 1 in ). However, other researchers have found that fatty acid, pork lard and human fat have an absorption peak at around 1210 nm wavelength [13
]. The discrepancy between multi-wavelength photoacoustic signal amplitude and the optical absorption spectrum of lipid may be due to several factors including the wavelength dependent fluence distribution in the tissue, the spectral line width of the laser source, the mixture of lipid and other tissue types in the intima of the diseased aorta, the small inaccuracy in the reported wavelength of our OPO laser, and the differences in absorption spectrum between fatty acid and lipid in plaques. To capture the peak absorption of the lipid, the artery should be imaged within, for example, 1170–1230 nm range to include anticipated peak absorption – our pulsed laser system was operating at the spectral edge of its idler mode (1200 nm to 2400 nm) and did not allow such measurements. However, if the measurements are performed around the peak optical absorption of tissue, other correlation based methods, e.g. intraclass correlation analysis [19
], may provide better sensitivity than the slope-based method. Nevertheless, the slope-based method is computationally efficient and is less sensitive to the shift or shape change of the measured photoacoustic signal peak caused by wavelength dependent optical properties. The slope-based algorithm only requires imaging at several wavelengths thus shortening the imaging time and reducing the complexity of image processing. In comparison, the correlation-based methods may require measurements covering a broader range of wavelengths and resulting in larger data sets.
The parameters of the slope-based spectroscopic analysis were selected based on the optical absorption spectrum of fatty acid. After the peak absorption at 1210 nm wavelength, the absorption coefficient of fatty acid drops 60% as the wavelength increases to 1230 nm. Considering the smoothing effect of spectral line width of the laser source, we selected the slopes ranging between –0.02/nm to –0.007/nm to represent lipid-rich areas. Given the wavelength dependent fluence distribution and laser pulse energy variation, we used a 20% error per wavelength to tolerate the change in the photoacoustic signal amplitude.
The false positive signals in the normal aorta and the media-adventitial layer of the diseased aorta may be due to inaccuracies in tissue displacement estimation due to out-of-plane motion during mechanical rotation of the tissue sample. The normal aorta was more likely subject to this type of artifact because it was more flexible compared to the diseased aorta characterized by a thick vessel wall. However, compensation for tissue motion may become even more critical in spectroscopic IVPA imaging in-vivo because cardiac motion will introduce tissue displacement between multi-wavelength IVPA scans.
The sensitivity and specificity of our measurements were affected by the laser system and the limited knowledge of optical properties of arterial tissue. Because the OPO laser was operating at the spectral edge of its idler mode, the line width of the laser pulse was around 10 nm. Such broad line width may reduce the sensitivity for imaging lipid which has a sharp absorption peak. Using a laser with narrower spectral line width may increase the sensitivity and reduce the required laser energy to detect lipid-rich regions in the vessels. Because of limited available measurements of optical spectra of different tissue types in the wavelength around 1200 nm, we generally classify tissues into two categories: lipid-rich tissues or water-based tissues. Knowledge of the absorption spectra of lipid and other tissue will improve the specificity of identifying lipid-rich regions.
Using integrated IVUS/IVPA catheter, high sensitivity and high resolution spectroscopic IVPA imaging in-vivo is possible [20
]. Indeed, the low optical scattering of blood and arterial tissue around 1200 nm wavelength ensures sufficient penetration depth in IVPA imaging [21
]. The higher threshold of the maximum permissible exposure at this wavelength range reduces the concern of laser thermal damage. Moreover, IVPA imaging with high frequency IVUS transducer can achieve axial resolution of tens of micrometers. Since modest low-pass filtering was applied in spectroscopic IVPA imaging, the axial resolution of the spectroscopic IVPA image is decreased by 2-3 times compared to the axial resolution of the IVPA image alone.
In the future, quantitative studies will be performed to examine the agreement between lipid-rich regions identified from spectroscopic IVPA and histological stain. Furthermore, human atherosclerotic lesions tend to have more complex plaques. Therefore, the performance of spectroscopic IVPA to differentiate various tissue types including lipid needs to be studied.
In conclusion, a method to differentiate lipid-rich regions in atherosclerotic vessels using spectroscopic IVPA imaging together with IVUS imaging was introduced. Ex-vivo tissue studies demonstrated that the spectroscopic IVPA imaging in the 1200-1230 nm wavelength range can successfully identify lipid-rich regions in the atherosclerotic rabbit aorta. Generally, spectroscopic IVPA imaging has the potential to identify tissue composition based on intrinsic optical absorption contrast between various types of tissues.