Multicolor Photoacoustic Volumetric Imaging of Subcellular Structures

Photoacoustic imaging (PAI) has been widely used in multiscale and multicontrast imaging of biological structures and functions. Optical resolution photoacoustic microscopy (OR-PAM), an emerging submodality of PAI, features high lateral resolution and rich optical contrast, indicating great potential in visualizing cellular and subcellular structures. However, three-dimensional (3D) imaging of subcellular structures using OR-PAM has remained a challenge due to the limited axial resolution. In this study, we propose a multicolor 3D photoacoustic microscopy with high lateral/axial resolutions of 0.42/2 and 0.5/2.5 μm at 532 and 780 nm excitation, respectively. Owing to the significantly increased axial resolution, we could visualize the volumetric subcellular structures of melanoma cells using intrinsic contrast. In addition, we carried out multicolor imaging of labeled microtubules/clathrin-coated pits (CCP) and microtubules/mitochondria, respectively, with one scanning by using two different excitation wavelengths. The internal connections between different subcellular structures are revealed by quantitatively comparing the spatial distributions of microtubules/CCP and microtubules/mitochondria in a single cell. Current results suggest that the proposed OR-PAM may serve as an efficient tool for subcellular and cytophysiological studies.