Multimodal 3D photoacoustic remote sensing and confocal fluorescence microscopy imaging.

Significance: Complementary absorption and fluorescence contrast could prove useful for a wide range of biomedical applications. However, current absorption-based photoacoustic microscopy systems require the ultrasound transducers to physically touch the samples, thereby increasing contamination and limiting strong optical focusing in reflection mode.

Aim: We sought to develop an all-optical system for imaging cells and tissues using the three combined imaging modalities: photoacoustic remote sensing (PARS), epifluorescence, and confocal laser scanning microscopy (CLSM).

Approach: A PARS subsystem with ultraviolet excitation was used to obtain label-free absorption-contrast images of nucleic acids in ex vivo tissue samples. Co-integrated epifluorescence and CLSM subsystems were used to verify the 2D and 3D nuclei distribution.

Results: Complementary absorption and fluorescence contrast were demonstrated in phantom imaging experiments and subsequent cell and tissue imaging experiments. Lateral and axial resolution of ultraviolet-PARS (UV-PARS) is shown to be 0.39 and 1.6  μm, respectively, with 266-nm light. CLSM lateral and axial resolution was measured as 0.97 and 2.0  μm, respectively. This resolution is sufficient to image individual cell layers with fine optical sectioning. UV-PARS images of cell nuclei are validated in thick tissue using CLSM.

Conclusions: Multimodal absorption and fluorescence contrast are obtained with a non-contact all-optical microscopy system for the first time and utilized to obtain images of cells and tissues with subcellular resolution.