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Histopathological examination typically involves tissue resection or biopsy, fixation, sectioning, and staining protocols. A non-contact high-resolution photoacoustic remote sensing microscopy system is presented which is capable of depth-resolved imaging of cell nuclei in fixed and fresh tissues without the need for stains or labels. The reflection-mode system is based on a 0.5 numerical aperture reflective objective and enables fast optical scanning using a 600 kHz repetition rate fiber laser to produce histological-like images with 0.39 µm resolution and with close agreement to traditional H&E and fluorescence staining.

Histological evaluation of tissues is currently a lengthy process that typically precludes intraoperative margin assessment. While numerous approaches have aimed to address the need for intraoperative virtual histology, none have yet proved sufficiently efficacious. We demonstrate the use of a new all-optical imaging modality, photoacoustic remote sensing (PARS), capable of virtual histopathological imaging, while simultaneously providing visualization of microvasculature in both freshly resected tissues and live animal subjects. We demonstrate high resolutions of 0.44µm and 1.2µm for 266-nm and 532-nm excitation wavelengths, respectively, as well as the characterization of maximum permissible exposure limits for both excitation wavelengths.

Histopathology of lipid-rich tissues is often a difficult endeavor, owing to the limited tissue processing workflows that can appropriately preserve tissue while keeping fatty deposits intact. Here, we present the first usage of near-infrared (NIR) photoacoustic remote sensing (PARS) to achieve imaging contrast from lipids without the need for exogenous stains or labels. In our system, the facile production of 1225 nm excitation pulses is achieved by the stimulated Raman scattering of a 1064 nm source propagating through an optical fiber. PARS-based detection is achieved by monitoring the change in the scattering profile of a co-aligned 1550 nm continuous-wave interrogation beam in response to absorption of the 1225 nm light by lipids. Our non-contact, reflection-mode approach can achieve a FWHM resolution of up to 0.96 µm and signal-to-noise ratios as high as 45 dB from carbon fibers and 9.7 dB from a lipid phantom. NIR-PARS offers a promising approach to image lipid-rich samples with a simplified workflow.

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