Grueneisen Relaxation Photoacoustic Microscopy

The temperature-dependent property of the Grueneisen parameter has been employed in photoacoustic imaging mainly to measure tissue temperature. Here we explore this property using a different approach and develop Grueneisen relaxation photoacoustic microscopy (GR-PAM), a technique that images nonradiative absorption with confocal optical resolution. GR-PAM sequentially delivers two identical laser pulses with a microsecond-scale time delay. The first laser pulse generates a photoacoustic signal and thermally tags the in-focus absorbers. When the second laser pulse excites the tagged absorbers within the thermal relaxation time, a photoacoustic signal stronger than the first one is produced, owing to the temperature dependence of the Grueneisen parameter. GR-PAM detects the amplitude difference between the two colocated photoacoustic signals, confocally imaging the nonradiative absorption. We greatly improved axial resolution from $45\mu \mathrm{m}$ to $2.3\mu \mathrm{m}$ and, at the same time, slightly improved lateral resolution from $0.63\mu \mathrm{m}$ to $0.41\mu \mathrm{m}$. In addition, the optical sectioning capability facilitates the measurement of the absolute absorption coefficient without fluence calibration.

Figure 1

(a) Fluence distribution for laser pulse 1 ($L_1$). (b) Residual temperature rise from laser pulse 1 and fluence distribution of laser pulse 2 ($L_2$). (c) Differential pressure rise induced by the Grueneisen relaxation effect. (d) Schematic of a GR-PAM system. Amp, 40-dB broadband amplifier; DAQ, high-speed digitizer; $p_{01}$, initial pressure rise from the first laser pulse; $p_{02}$, initial pressure rise from the second laser pulse; $\mathrm{\Delta }{p}_0=p_{02}-p_{01}$; PC, computer; ${\text{PD}}_1$ and ${\text{PD}}_2$, photodiodes 1 and 2; PM, parabolic mirror; UT, ultrasonic transducer.

Figure 2

(a) Lateral profiles of a sharp ink edge measured by OR-PAM and GR-PAM. OR-PAM has a lateral resolution of $0.65\mu \mathrm{m}$, while GR-PAM has a lateral resolution of $0.41\mu \mathrm{m}$. ESFs, edge spread functions; LSFs, line spread functions. (b) Axial profiles of OR-PAM and GR-PAM. The FWHM of the OR-PAM profile is $45\mu \mathrm{m}$. The FWHM of the GR-PAM profile is $2.3\mu \mathrm{m}$.

Figure 3

(a)–(c) Optically sectioned GR-PAM images of red blood cells at three depths. (d)–(f) Optically sectioned OR-PAM images of the same sample as in (a)–(c). (g) A side-view GR-PAM image of red blood cells in the $x-z$ plane. (h) A side-view OR-PAM image of the same sample as in (g). (i) PA profiles along the dashed lines in (c) and (f). The contrasts of the doughnut-shaped features were measured in GR-PAM and OR-PAM images. Based on measurements from nine RBCs, the contrast of GR-PAM is $0.46\pm 0.04$, while the contrast of OR-PAM is $0.11\pm 0.03$ ($\text{mean}\pm \text{standard}\text{error}$). The average ratio between the contrasts is 4.2. (j) Volumetric GR-PAM image of red blood cells.

Figure 4

(a) Diagram of a thick blood sample. (b) An OR-PAM cross-sectional image. (c) A GR-PAM cross-sectional image. (d) Time-resolved PA signals from the two laser pulses and the differential PA signal. The focus was $60\mu \mathrm{m}$ below the blood surface. (e) Exponential decay of GR-PAM amplitude along depth. (f) Measured absolute absorption coefficient versus set absorption coefficient.