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

Abstract 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.