Multispectral photoacoustic remote sensing microscopy using 532nm and 266nm excitation wavelengths

Photoacoustic remote sensing (PARS) is a non-contact imaging modality that is based on the optical absorption contrast of endogenous molecules. PARS has shown promise in vascular imaging, blood oxygenation estimation, and virtual biopsy without the need for exogenous labels. Here we demonstrate simultaneous imaging of cell nuclei and blood using UV and visible excitation wavelengths. This is important for decoupling blood signals from cell nuclei signals in removed tissue and resection beds. A 532nm fiber laser is split with one light path frequency doubled using a CLBO crystal to 266nm. These two wavelength lasers are co-aligned and co-focused with a 1310nm interrogation beam and using a reflective objective to image microvasculature and cell nuclei with intrinsic optical absorptions at 532nm and 266nm, respectively. These images are taken serially and co-registered with lateral resolutions of 1.2μm and 0.44μm respectively. Co-alignment using multiple wavelengths is demonstrated using carbon fiber phantoms. We imaged both paraffin embedded tissue and in vivo mouse ear. Cell nuclei in sectioned tissues were clearly visualized with a SNR of 42dB while hemoglobin demonstrated an SNR of 39dB. In vivo cell nuclei and vasculature images produced an SNR up to 40dB and 35dB, respectively.