When stable, BphP1is at the On state. Upon 780 nm laser pulse train illumination, BphP1 molecules in the on state gradually switch to the off state. As a result, the amplitude of the generated PA signals decrease. The decay rate is proportional to the local excitation intensity. Thus, PA signal in the center of the excitation beam will decay faster compared to the surrounding. Using the difference image between the two states gives a high contrast image of the molecules (Figure 4), and sub-diffraction limited lateral and axial resolutions will be achieved. In the lateral direction, the decay dependence on the local excitation intensity results in a smaller FWHM of the decay PSF. The higher the order of the nonlinear decay, the higher the resolution enhancement will be. The effective lateral PSF for this imaging technique was shown to be formula_4 where b is the power dependence of the switching-off rate on the excitation intensity (for BphP1, b=1), and m is the order of the polynomial fitting. This technique achieves finer resolutions with a factor of formula_5 compared to conventional PAM. In the axial direction, the nonlinear optical effect will determine the resolution (since only in focus molecules contribute in the signal decay), thus, making it independent from the acoustic detection. This allows this technology to have optical sectioning capability. For point targets, the achievable axial resolution is formula_6 whereas for large targets, it is formula_7  Figure 4: PA images of BphP1 expressing U87 cells and HbO2 in scattering media. The differential image effectively removes the background signal, increasing the contrast of the cell area. As a demonstration (figure 5) this technique showed much finer lateral and axial resolutions compared to conventional PAM. The lateral and axial resolutions were quantified to be ∼141 nm and ~400 nm, respectively, which were about 2 and 75 times better than that of conventional PAM. Figure 5: Comparison of lateral and axial resolution of conventional PAM and RS-PAM in imaging. Non-radiative two-photon absorption can be utilized to achieve high 3D resolution in in vivo experiments. In conventional PAM temporal or spatial filtering is needed to eliminate background signal and achieve high axial resolution. However, in 2PAM, only the area within the focal spot generates nonlinear photoacoustic signals (figure 6). Based on the two-photon absorption PSF, the lateral (formula_8) and axial resolution (formula_9) of the 2PAM system are determined to be...