Chromophore selective multi-wavelength photoacoustic remote sensing of unstained human tissues.

Identifying positive surgical margins after resection of cancer often triggers re-excision and adjuvant treatments. Incomplete initial resections result in poorer patient outcomes, psychological and financial stress to the patient and increased healthcare costs. Surgical margins are typically assessed post-operatively using time consuming and expensive slide-based histopathology tissue analysis. Currently, a real-time non-contact virtual histology-like intraoperative margin assessment tool is not available. To address this need, we have developed a non-contact multi-wavelength reflection-mode, photoacoustic remote sensing (PARS) microscope demonstrating chromophore selective contrast in human tissues. We show the capabilities of multi-wavelength PARS microscopy utilizing both 266 nm and 532 nm excitation wavelengths and a 1310 nm detection wavelength. Cell nuclei and hemoglobin were visualized at the cellular scale without the addition of exogenous contrast agents. These works provide a critical step towards a virtual histology tool to provide intraoperative histology-like information in living tissue.

All-optical Reflection-mode Microscopic Histology of Unstained Human Tissues.

Surgical oncologists depend heavily on visual field acuity during cancer resection surgeries for in-situ margin assessment. Clinicians must wait up to two weeks for results from a pathology lab to confirm a post-operative diagnosis, potentially resulting in subsequent treatments. Currently, there are no clinical tools that can visualize diagnostically pertinent tissue information in-situ. Here, we present the first microscopy capable of non-contact label-free visualization of human cellular morphology in a reflection-mode apparatus. This is possible with the recently reported imaging modality called photoacoustic remote sensing microscopy which enables non-contact detection of optical absorption contrast. By taking advantage of the 266-nanometer optical absorption peak of DNA, photoacoustic remote sensing is efficacious in recovering qualitatively similar nuclear information in comparison to that provided by the hematoxylin stain in the gold-standard hematoxylin and eosin (H&E) prepared samples. A photoacoustic remote sensing system was employed utilizing a 266-nanometer pulsed excitation beam to induce photoacoustic pressures within the sample resulting in refractive index modulation of the optical absorber. A 1310-nanometer continuous-wave interrogation beam detects these perturbed regions as back reflected intensity variations due to the changes in the local optical properties. Using this technique, clinically useful histologic images of human tissue samples including breast cancer (invasive ductal carcinoma), tonsil, gastrointestinal, and pancreatic tissue images were formed. These were qualitatively comparable to standard H&E prepared samples.