Pushing the Technology Envelope of Multi-Parametric Photoacoustic Microscopy

Photoacoustic microscopy (PAM) is a hybrid imaging modality that combines the deep-penetration advantage of ultrasonic imaging with the high-resolution and high-contrast advantage of optical imaging. It detects the short pulsed laser induced photoacoustic waves, whose amplitudes reflect the localized laser energy absorption, to image the internal optical absorption distributions. Mathematically, the photoacoustic wave pressure can be described as p_0=Γη_th μ_a F where p_0 is the pressure, Γ is Grueneisen parameter, η_th is percentage that the abosorbed optical energy is converted into heat, μ_a is the optical absorption coefficient and F is the optical fluence (J/cm2). Currently, conventional optical resolution PAM (OR-PAM) is facing two main challenges which limit its application in physiology studies, the highly anisotropic spatial resolution and slow imaging speed. This dissertation focuses on addressing these two problems and provides several potential solutions.
In OR-PAM, while the lateral resolution is determined by the optical focusing which can achieve micron level, the axial resolution determined by ultrasound detection bandwidth is usually at least one order of magnitude worse than the lateral resolution. Thus, compared to other optical imaging technique such as two photon microscopy (TPM) or optical coherence tomography (OCT), OR-PAM has poor performance in volumetric imaging with comparable axial and lateral resolution. This limitation strongly constraints the application of PAM in physiology and biology studies.
To address this issue, we present two potential methods. 1. OR-PAM with surface plasmon resonance (SPR) based ultrasound detector (SPR-PAM) that can achieve broad ultrasound detection bandwidth. Experimentally, an ultra-flat frequency response (±0.7 dB) from 680 kHz to 126 MHz has been examined. With the broad detection bandwidth, high spatial resolution (2.0 µm laterally and 8.4 µm axially) is achieved. 3D PA imaging of a melanoma cell with isotropic spatial resolution is also presented. 2. OR-PAM with multi-angle illumination (MAI-PAM). With multi-angle illumination, PAM images from different view angles can be simultaneously acquired for multi-view deconvolution. We experimentally examine the system performance both in phantom and in vivo. The measurement results reveals that MAI-PAM achieved a high axial resolution of 3.7 μm, which is 10-fold higher than that of conventional PAM and approached the lateral resolution of 2.7 μm.
Conventional PAM employs pure mechanical scan and commercial 559-nm Raman pulsed laser with low pulse repetition rate (PRR) to realize oxygen saturation (sO2) measurement. These two factors significantly limits the imaging speed of PAM. To address these problems, we realize a high speed multi-parametric PAM with A-line rate of 300-kHz by employing optical-mechanical hybrid scan mode and stimulated Raman scattering (SRS) based wavelength conversion method to generate 558-nm pulsed laser. Compared to conventional PAM, 20-fold imaging speed improvement is achieved. The system performance is examined both in vitro and in vivo. Employing two 600-kHz PRR pulsed lasers and a weakly focused ultrasound transducer with 250-μm focal zone diameter, we develop an ultra-high speed multi-parametric PAM with A-line rate of 1.2-MHz which further improves imaging speed by 6-fold over the 300-kHz high-speed PAM. The system is validated by performing side-by-side measurement comparison between our previously well-developed multi-parametric PAM and the ultra-high speed multi-parametric PAM.