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. Photoacoustic microscopy (PAM) is an effective in vivo functional and molecular imaging tool. In the PA phenomenon, light is absorbed by molecules and converted to heat. Subsequent thermoelastic expansion generates an acoustic wave, termed the PA wave. 1,2 Detection of PA waves sequentially excited at multiple optical wavelengths provides quantitative information about the concentrations of multiple chromophores such as oxygenated and deoxygenated hemoglobin molecules in red blood cells. Thus, the relative concentration and the oxygen saturation (sO 2 ) of hemoglobin can be extracted. [3][4][5] Generally, the amplitude of the PA signal is assumed to be linearly proportional to the excitation pulse fluence. However, as the excitation laser intensity increases, both the saturation of the optical absorption 6,7 and the temperature dependence of thermal expansion 8-10 result in a measurable nonlinear dependence of the PA signal on the excitation pulse fluence. PA nonlinearity has recently been used in several applications such as quantifying picosecond absorption relaxation times with a nanosecond laser, 6 differentiating optical absorbers, 10 measuring oxygen saturation in vivo, 7 and performing label-free PA nanoscopy of biological structures having undetectable fluorescence. 11 In the presence of nonlinearity, quantitative PA measurements require a detailed analysis of the intensity-dependence of the PA signal, particularly for hemoglobin, the major intrinsic absorber in tissue for PA imaging. Based on thermal nonlinearity, analysis of the nonlinear PA signal from a single point source has been previously discussed. 8 However, the wavelengthand concentration-dependent effects of both optical saturation and thermal nonlinearity on the PA signals for multiple absorbers have not been reported. Here, we investigate nonlinear PA effects in oxygenated and deoxygenated hemoglobin using a PA spectrometer with a flat-top beam illumination, which effectively reduces uncertainty in our measurements that would otherwise arise from inhomogeneous spatial distribution of the optical fluence. In ox...

Molecular Photoacoustic Contrast Agents: Design Principles & Applications

“…Although nonlinear RSA materials are anticipated to be the most promising dye category for generation of an enhanced nonlinear PA response it must be kept in mind that their application is very much limited to scenarios where the application of high laser fluences is feasible. While a number of studies addressing nonlinear PAI have emerged very few have involved in vivo studies , none of which have relied solely on a molecular‐based contrast agent. As such, all of the in vivo imaging applications of MPACs discussed in this review have been conducted under low laser fluence conditions where the dye either behaves as a LA or SA material.…”

“…PAI can also be considered noninvasive. For example, hemoglobin can be used as an endogenous contrast agent to obtain histological imaging and has even been investigated ex vivo for nonlinear PAI . However, the addition of exogenous PA contrast agents allows for microscopic imaging with far greater selectivity and contrast at a cellular and molecular level.…”

Lock-in detection of photoacoustic feedback signal for focusing through scattering media using wave-front shaping

“…The lock-in detection method is also designed for measuring nonlinear PA, motivated by the potential for 3D enhanced resolution. The source of PA nonlinearities [31][32][33][34] varies between different samples and excitations and can be classified in the following categories; 1saturation and damage effects, 2-thermal nonlinearities and 3-phase transformations in the sample or its surroundings. All mechanisms are characterized by a nonlinear relation of the PA amplitude to the laser fluence.…”

Electromagnetic–Acoustic Sensing for Biomedical Applications

“…The most commonly photo absorbers for PA imaging and sensing can be classified into two categories based on their primary absorbing wavelengths: (1) in the visible regions (400–700 nm), the primary absorbers for PA include hemoglobin [ 40 , 60 ], cytochrome [ 61 ], melanin [ 62 ], DNA/RNA [ 49 , 63 ], myoglobin [ 64 ]; (2) in the near-infrared region (700–1400 nm), lipid [ 65 , 66 ], glucose [ 45 , 67 ] are also strong absorbers for PA emission. Figure 1 summarizes the absorption spectrum of common endogenous contrasts [ 68 ].…”