Simplified method for ultra high-resolution photoacoustic microscopy via transient absorption

Photoacoustic microscopy (PAM) is a hybrid imaging modality that combines optical illumination with ultrasonic detection to achieve absorption contrast imaging of endogenous and exogenous chromophores. Optical resolution PAM achieves high lateral-resolution by tightly focusing the excitation light; however the axial resolution is still dependent upon the bandwidth of the ultrasonic transducer. As a result, PAM images have highly asymmetric voxels with submicron lateral resolution and axial resolution typically limited to tens of microns. We have previously reported on a resonant multiphoton approach to PAM called transient absorption ultrasonic microscopy (TAUM), which enables high axial resolution by frequency encoding the photoacoustic signal at the overlap of a pump and a probe beam. This approach enables photoacoustic imaging with subcellular resolution on par with other multiphoton microscopy techniques. Here, we report on an innovation that enables TAUM imaging with a much less sophisticated optical system than previously reported. If we allow the time delay between the pump and probe to collapse to zero, the pump and probe optical paths can be combined. An amplitude modulator in the single beam path is sufficient to encode the TAUM signal at the second harmonic of the modulation frequency. The resulting system is essentially a standard optical resolution PAM system that incorporates an amplitude modulator and utilizes a Fourier post processing algorithm to improve the axial resolution by approximately an order of magnitude. A prototype system based on this approach has been assembled and tested on fixed bovine erythrocytes.