Nonlinear photoacoustic spectroscopy of hemoglobin

Abstract

As light intensity increases in photoacoustic imaging, the saturation of optical absorption and the temperature dependence of the thermal expansion coefficient result in a measurable nonlinear dependence of the photoacoustic (PA) signal on the excitation pulse fluence. Here, under controlled conditions, we investigate the intensity-dependent photoacoustic signals from oxygenated and deoxygenated hemoglobin at varied optical wavelengths and molecular concentrations. The wavelength and concentration dependencies of the nonlinear PA spectrum are found to be significantly greater in oxygenated hemoglobin than in deoxygenated hemoglobin. These effects are further influenced by the hemoglobin concentration. These nonlinear phenomena provide insights into applications of photoacoustics, such as measurements of average inter-molecular distances on a nm scale or with a tuned selection of wavelengths, a more accurate quantitative PA tomography.

FIG. 1.

(a) Densely and loosely packed absorbers within the acoustic voxel. (b) A photoacoustic spectrometer with flat-top beam illumination. (c) 2D beam profile.

FIG. 2.

(a) and (b) PA amplitude versus laser wavelength in fully oxygenated lysed blood (0.07 mM) at (a) low incident fluence and (b) high incident fluence. The red dashed line is the absorption spectrum, which fits the theoretical absorption of 98% oxygenated hemoglobin, measured by a spectrophotometer (Cary-50, Agilent). (c) and (d) PA amplitude as a function of the incident fluence at (c) 578 nm and (d) 560 nm. Each red dashed line is the tangent of the nonlinear curve at the origin. Error bars represent the standard deviations of ten measurements. (e) and (f) A typical (e) linear coefficient, $c_1$, and (f) 2nd order coefficient, $c_2$, as a function of the laser wavelength. The red dashed line represents $c_2=0$.

FIG. 3.

(a) Photoacoustic amplitude versus laser wavelength in deoxygenated whole blood. (b) 2nd order coefficient, $c_2$, versus laser wavelength for deoxygenated whole blood. (c)–(e) $c_2/{\sigma }^2(\lambda )$ versus laser wavelength for (c) deoxygenated whole blood, (d) oxygenated whole blood, and (e) different concentrations of oxygenated lysed blood. The red dashed line represents $c_2=0$. (f) The concentration's power dependence of $c_2$ at 560 nm as a function of average distance between molecules. The red dashed line represents the thermal diffusion length (26 nm) in water for a 4.9 ns laser pulse width. Error bars in (a) represent the standard deviations of ten measurements. Error bars in (c)–(f) represent the standard errors of 3–4 experiments.