Multiscale photoacoustic microscopy imaging with image improvement and quantification technique

Photoacoustic (PA) imaging is one of the fastest growing imaging technologies nowadays in both research and clinical applications, especially due to its unique capability to visualize blood vessels. The PA microscopy (PAM) is classified into two types: optical-resolution PAM (OR-PAM) and acoustic-resolution PAM (AR-PAM). OR-PAM image has a point spread function (PSF) much smaller than AR-PAM because it uses a tightly focused optical beam and the PSF is determined by the optical focus. In contrast, AR-PAM uses an unfocused optical illumination to excite a relatively large area and detects the PA signal from a small area determined by its acoustic focus. Because ultrasound is less scattered than light in biological tissue, AR-PAM can achieve deeper imaging depth than OR-PAM at the expense of image resolution. Due to the limited resolution and imaging depth scale of each PAM type, it is challenging to image vessels in various area of small animals. In this study, we demonstrated in vivo OR-/AR-PAM imaging of blood vessels in various areas such as eye, ear, and hind limb by using a single commercial PAM system. Additionally, we quantified micro-vessel density (MVD) of the mouse eye and ear images, and applied a synthetic aperture focusing technique (SAFT) to correct the distorted PA signal at the out-of-focus in AR-PAM image. As a result, we have demonstrated multiscale PAM imaging of small animal vasculature in various areas with vessel quantification and resolution enhancement, so we believe that this multiscale PAM imaging technique would be helpful in biology research such as ischemia and neovascularization.