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 μm to 2.3 μm and, at the same time, slightly improved lateral resolution from 0.63 μm to 0.41 μm. In addition, the optical sectioning capability facilitates the measurement of the absolute absorption coefficient without fluence calibration

Subwavelength-resolution photoacoustic microscopy for label-free detection of optical absorption in vivo

Mainstream optical microscopy technologies normally detect fluorescence or scattering, which may require undesirable labeling, but cannot directly sense optical absorption, which provides essential biological functional information. Here we reported in vivo and label-free subwavelength-resolution photoacoustic microscopy (SW-PAM) by using a waterimmersion optical objective with a 1.23 NA. Capable of detecting nonfluorescent endogenous pigments, SW-PAM provides exquisitely high optical-absorption contrast. And, as a result of background-free detection, the sensitivity of SW-PAM to optical absorption reaches 100%. SW-PAM was demonstrated with wide-field optical microscopy by imaging gold nanospheres, ex vivo cells, and in vivo vasculature and melanoma. It was shown that SW-PAM has approached the ultimate diffraction-limited optical resolution-220 nm resolution at 532 nm wavelength. Subcellular organelles, such as melanosomes, can be resolved by SW-PAM. Vasculature and early-stage melanoma were imaged with 21:1 and 34:1 contrasts, respectively, without labeling. For all these applications, SW-PAM has contrasts orders of magnitude higher than wide-field optical microscopy. Therefore, SW-PAM is expected to join the mainstream microscopy technologies

Effects of light scattering on optical-resolution photoacoustic microscopy

The imaging depth of ballistic optical imaging technologies is limited by light scattering. To study the effects of scattering on optical-resolution photoacoustic microscopy (OR-PAM), the signals were divided into target and background signals. A method to simulate the point spread function (PSF) of the PAM system considering both optical illumination and acoustic detection was proposed, then the PSF was used to calculate the contribution of each class of signal at different depths of the focal plane (z_f). How image contrast is degraded when there is a uniformly absorbing background as well as when there are small targets densely packed in the acoustic resolution cell were studied. By using the hyperboloid-focusing-based Monte Carlo method, optical focusing into a scattering medium was simulated. It was found that the lateral resolution provided by optical focusing is degraded by only 14% when z_f=1.1 transport mean free path (l’_t), compared with the case of no scattering. When z_f =1.7 l′_t, the fluence at 50 μm radial distance away from the focal point is 93% of that at the focal point, which shows optical focusing is very weak at this depth. The method to simulate the PSF of PAM can be used in the future to optimize parameters so as to improve the system performance

Fast and Robust Deconvolution-Based Image Reconstruction for Photoacoustic Tomography in Circular Geometry: Experimental Validation

Photoacoustic tomography (PAT) is a fast-developing biomedical imaging technology suitable for in vivo imaging. PAT in spherical or circular geometry gives good image resolution yet is slow or expensive in signal acquisition and image formation. Reducing the number of detection angles can ameliorate such issues, usually at the expense of image quality. This paper introduces a deconvolution-based algorithm that models the imaging process as a linear and shift-invariant system. As demonstrated by the in vivo experiment, this algorithm not only runs much faster than the back-projection algorithm but also shows stronger robustness in that it provides better image quality when detection angles are sparse. Therefore, this algorithm promises to enable real-time PAT in circular geometry

Subwavelength-resolution label-free photoacoustic microscopy of optical absorption in vivo

Optical absorption provides essential biological functional information but cannot be sensed by mainstream optical microscopy technologies directly, which detect fluorescence or scattering and may require undesirable labeling. Here we developed in vivo subwavelength-resolution photoacoustic microscopy (SW-PAM) that provides exquisitely high optical-absorption contrast due to nonfluorescent, or fluorescent, endogenous pigments. Having approached the ultimate diffraction-limited optical resolution, SW-PAM can resolve subcellular organelles. Vasculature and early-stage melanoma were imaged with 12:1 and 17:1 contrasts, respectively, without labeling. SW-PAM along with the scaled-up macroscopy, as the only technology that measures the same contrast origin over such a wide length scale, can potentially accelerate translation from microscopic research to clinical practice

In vivophotoacoustic microscopy with 7.6-µm axial resolution using a commercial 125-MHz ultrasonic transducer

Photoacoustic microscopy has achieved submicron lateral resolution, but its axial resolution is much lower. Here an axial resolution of 7.6 μm, the highest axial resolution validated by experimental data, has been achieved by using a commercial 125 MHz ultrasonic transducer for signal detection followed by the Wiener deconvolution for signal processing. Limited by the working distance, the high-frequency ultrasonic transducer can penetrate 1.2 mm into biological tissue from the ultrasound detection side. At this depth, the signal-to-noise ratio decreases by 11 dB, and the axial resolution degrades by 36%. The new system was demonstrated in imaging melanoma cells ex vivo and mouse ears in vivo

Photoacoustic microscopy with an enhanced axial resolution of 5.8 μm

The axial resolution of photoacoustic microscopy (PAM) can be enhanced by reducing the speed of sound within the imaging region of interest. This principle was demonstrated on a previously-reported PAM system, which utilized a 125 MHz ultrasonic transducer for signal detection and the Wiener deconvolution for signal processing. With sound slowed by silicone oil immersion, we have achieved a finest axial resolution of 5.8 μm for PAM, as validated by phantom experiments. The axial resolution was also enhanced in vivo when mouse ears injected with silicone oil were imaged. After injection of silicone oil, the blood vessels were resolved more clearly. When tissue-compatible low-speed liquids become available, this approach may find applications in PAM as well as in other imaging modalities, such as photoacoustic computed tomography and ultrasound imaging

Slow-sound photoacoustic microscopy

We propose to enhance the axial resolution of photoacoustic microscopy (PAM) by reducing the speed of sound within the imaging region of interest. With silicone oil immersion, we have achieved a finest axial resolution of 5.8 μm for PAM, as validated by phantom experiments. The axial resolution was also enhanced in vivo when mouse ears injected with silicone oil were imaged. When tissue-compatible low-speed liquid becomes available, this approach may find broad applications in PAM as well as in other imaging modalities, such as photoacoustic computed tomography and ultrasound imaging

Photoacoustic microscopy with 7.6-μm axial resolution

The axial resolution of photoacoustic microscopy (PAM) is much lower than its lateral resolution, which resolves down to the submicron level. Here we achieved so far the highest axial resolution of 7.6 μm by using a commercial 125 MHz ultrasonic transducer for signal detection, followed by the Wiener deconvolution for signal processing. The axial resolution was validated by imaging two layers of red ink in a wedge shape. Melanoma cells were imaged ex vivo with high axial resolution. Compared with a PAM system with a 50 MHz ultrasonic transducer, our high-axial-resolution PAM system resolved the blood vessels in mouse ears in vivo much more clearly in the depth direction