Fast focus-scanning optical-resolution photoacoustic microscopy with extended depth of field

Photoacoustic imaging is a promising technique that combines optical contrast with ultrasonic detection to map the distribution of the absorbing pigments in biological tissues. Optical-resolution photoacoustic microscopy (OR-PAM) is an important branch of PAM that can achieve spatial resolutions at the submicron level. In the OR-PAM, the laser light is tightly focused into the sample to achieve sharp excitation. However, in OR-PAM, the depth of field (DoF) is quite limited, for it is determined by the optical focus. The short DoF hinders OR-PAM from high-quality three-dimensional volumetric imaging or acquiring dynamic information in depth direction. Here, we developed a fast focus-scanning optical-resolution photoacoustic microscopy with extended depth of field by using tunable acoustic gradient lens (TAG). The TAG lens was designed to continuously changing the focal plane of OR-PAM by modulating its refractive power with fast-changing ultrasonic standing wave. A function generator generates a sinusoidal signal to drive the TAG lens at an eigenmode. The focusing power of the TAG lens will exhibit a sinusoidal oscillation at the frequency of the driving signal. By using home-made synchronization circuit to synchronize the laser pulse with the three vibration states of TAG lens in turn, the focal plane shifts periodically with each step in a B scan. In our experiments, the axial scanning time is as low as 1 ms, which is at least an order faster than any other axial scanning method in OR-PAM, to the best of our knowledge. Faster focus scanning could be achieved as long as we have a laser with higher repetition. The performance was shown by imaging a tungsten wire. We achieved a DoF of about 750 μm, which is three times of that of OR-PAM with single fixed focus. The head of a zebrafish was also imaged to further demonstrate the feasibility of our method. Our system could be used for various applications, including the in vivo imaging of non-flat thick biological tissues and samples subject to breathing movements.