Optical-Resolution Photoacoustic Microscopy for Volumetric and Spectral Analysis of Histological and Immunochemical Samples

Optical-resolution photoacoustic microscopy (OR-PAM) is an imaging modality with superb penetration depth and excellent absorption contrast. Here we demonstrate, for the first time, that this technique can advance quantitative analysis of conventional chromogenic histochemistry. Because OR-PAM can quantify the absorption contrast at different wavelengths, it is feasible to spectrally resolve the specific biomolecules involved in a staining color. Furthermore, the tomographic capability of OR-PAM allows for noninvasive volumetric imaging of a thick sample without microtoming it. By immunostaining the sample with different chromogenic agents, we further demonstrated the ability of OR-PAM to resolve different types of cells in a coculture sample with imaging depths up to 1 mm. Taken together, the integration of OR-PAM with (immuno) histochemistry offers a simple and versatile technique with broad applications in cell biology, pathology, tissue engineering, and related biomedical studies.