Label-free photoacoustic microscopy: super speed and super contrast

We have developed fast functional photoacoustic microscopy (ffPAM) for three-dimensional high-resolution, high-speed imaging of the mouse brain. Using endogenous contrast, ffPAM can perform high-speed, high-resolution imaging of the vascular morphology, blood oxygenation, blood flow dynamics and oxygen metabolism of the mouse brain. In particular, ffPAM has achieved a 1D time-resolved imaging rate of 500 kHz for morphological imaging and 100 kHz for blood oxygenation imaging.

We recently proposed a novel approach, called ultraviolet-localized mid-infrared (MIR) photoacoustic microscopy (ULM-PAM), to achieve high-resolution and nearly water-background-free MIR imaging of fresh biological samples. In our approach, a pulsed mid-MIR laser thermally excites the sample at a focal spot, and a confocal pulsed ultraviolet laser beam photoacoustically detects the resulting transient temperature rise, thereby reporting the magnitude of the MIR absorption by the sample. This detection scheme is based on the fact that a temperature rise in a sample enhances photoacoustic signals, a phenomenon called the Grüneisen relaxation effect. While our imaging method reveals MIR absorption contrast, its lateral resolution is determined by the ultraviolet wavelength, which is one order of magnitude shorter than the MIR wavelength. In addition, ultraviolet light in the range of 200–230 nm is highly absorbed by most biomolecules, such as lipids, proteins and nucleic acids, but it is totally transmissive in water. Thus, the strong water background of MIR absorption is suppressed in our method. For cultured cells, our method achieves water-background suppressed MIR imaging of lipids and proteins at ultraviolet resolution. Label-free histology using this method is also demonstrated in thick brain slices. Our approach provides convenient high-resolution and high-contrast MIR imaging, which can benefit the diagnosis of fresh biological samples.