Volumetric Lissajous scanning OR-PAM

Zebrafish is one of the most important animal models currently used in preclinical research. Optical resolution photoacoustic microscopy (ORPAM) provides high resolution and molecular contrast in imaging in-vivo zebrafish larvae. One of the significant disadvantages of ORPAM is shallow plane of focus, which hinders visualization of microvasculature across whole larva. 2D Lissajous scanning pattern has been implemented in multiple scanning microscopy fields, improving the utilization of the actuation mechanisms. We present system extending Lissajous pattern into 3D, which functionally extends the focal plane. This extension allows for microvasculature imaging across whole larva without the need for sample repositioning. Combined with large field of view of the system it allows for easy batch imaging of the zebrafish larvae, previously requiring micrometer-level precision of vertical sample placement. Capabilities of the system are demonstrated on in-vivo and synthetic samples.