Optical imaging is drawing broad attentions to visualize the multiscale samples, such as cells, tissue, and organs. Lots of advanced optical microscopy modalities have occurred in the past three decades, such as two photon excitation microscopy, optical coherence tomography, phase contrast microscopy and super-resolution microscopy, etc. Optical-resolution photoacoustic microscopy (OR-PAM) is a hybrid imaging technique involving optical contrast and resolution and improved penetration depth, opening a broad avenue to image the absorber in the deep tissue without labelling. However, the sensitivity of OR-PAM is always a problem especially for imaging the weak absorber and functional imaging. To address this problem, we develop two efficient methods, both of which could be able to significantly improve the sensitivity of OR-PAM.

The first method is to enlarge the numerical aperture of the spherical glass acoustic lens to its physical limit 0.74. We designed and customized a spherical acoustic lens and registered it into our previous developed OR-PAM system. As well, we improved the optics and acoustic alignment method to mitigate the effect introduced by the stress and the optical illumination approach to enhance the optical numerical aperture. Our high acoustic numerical aperture optical resolution photoacoustic microscopy (HNA-OR-PAM) improved the sensitivity as 160% as the state-of-art OR-PAM system. It can image the oxygen saturation in the mice’s ear with only 10-nJ pulse energy at wave-length 532 nm and 558 nm. In addition, due to the extended acoustic detection aperture, HNA-OR-PAM could visualize the tilted vessels clearer. Such high sensitivity could enable this technique for many potential applications, such as neural science and ophthalmology.

The second method is to develop a double-illumination strategy to improve the imaging signal-to-noise ratio (SNR) without increasing the pulse energy. We placed the optical thin sample upon a single first-surface silver mirror, in which the laser beam transmitted the sample and was reflected back to the bottom surface of the sample by the mirror. Such a simple and efficient illumination method could enhance the sensitivity by 37% to 100% to image the sample with thickness ranging from 0 to one transport mean free path. In addition, via statistical analysis of Monte Carlo method, we found that our double-illumination strategy could offer the more uniform optical illumination along the depth direction. We combined the double-illumination strategy and a home-built 1750 nm pulse laser to build the near-infrared double-illumination optical-resolution photoacoustic microscopy (DIOR-PAM). We used our near-infrared DIOR-PAM to image the thin lipid-rich slides. We demonstrated improved imaging sensitivity 50% to 100% on both nonbiological and biological slides. We believed the uniform optical illumination and improved sensitivity could facilitate this technique toward the high spatial resolution and extended volume photoacoustic imaging of thin lipid-rich samples.

In conclusion, both the HNA-OR-PAM and near-infrared DIOR-PAM would significantly improve the sensitivity of OR-PAM. Both works facilitated the photoacoustic imaging to image deeper and weaker absorbers.