In tumor resection surgery, the entire tumor must be removed to prevent local recurrence of cancer. To achieve effective and successful tumor resection surgery, an intraoperative examination is performed for quick decision-making during the surgical process. Examination of frozen sections is a common method, but it has limitations that it requires time-consuming tissue processing procedures which leads to interpretation errors. Photoacoustic microscopy (PAM) with ultraviolet (UV) laser is a promising intraoperative surgical margin assessment method that provides depth-resolved and label-free imaging of cell nuclei without sectioning and staining. Despite these advantages, conventional PAM still has limited imaging speed that does not allow real-time imaging, because it achieves the volumetric images by raster scanning using 2-axis step motors. To overcome the limitation, we developed a high-speed reflection-mode OR-PAM based on a UV scanner. Using the scanner module, it took 180 seconds to acquire one volumetric data over 1 × 1 mm2. In an in-vitro test, the measured lateral and axial resolution were 1.2 μm and 65.1 μm, respectively. We performed ex-vivo experiments on paraffin sections of tissues after deparaffinization that had been excised from a kidney, liver, colon-cancer and a liver-cancer patient. We could find structures in tumorous tissues distinguishable from normal tissues in 4 × 8 mm2 which is clinically meaningful FOV. We could also identify single nucleus in UV-PAM images, and match it with the corresponding nucleus in microscopic images.
Photoacoustic microscopy (PAM), an emerging biomedical imaging technology, has demonstrated the label-free imaging capability to visualize biomolecules with the aid of superior optical contrast in them. Especially employing ultraviolet (UV) laser at the wavelength of 266 nm, we have developed an UV-PAM. Unlike conventional histology methods such as frozen and formalin-fixed paraffin-embedded (FFPE) sections, UV-PAM can illustrate cell nuclei by utilizing superior light absorption of DNA/RNA without time consuming procedures. In-vitro experiments were conducted to evaluate the spatial resolutions of the developed system. The measured lateral resolution was 1.3 μm, and axial resolution was 62.2 μm. Then we performed ex-vivo experiments using frozen sections of mouse brain to demonstrate the imaging capability of UVPAM as a rapid histology tool. Oxidative stress induced by kainic acid (KA) was monitored using UV-PAM, which is considered as a significant cause for epileptic neuronal brain damage. We have shown the apoptotic feature resulted from the KA-induced hippocampal cell death in a mouse brain section. In contrast to the brain section of the control mouse model, the substantial nuclear marginalization of hippocampal cell death was illustrated in the vulnerable neurons of the CA1 and CA3 regions on the KA-treated mouse with PA imaging. In addition, the PA histologic results were evidenced by the corresponding HE stained images on both the control and the KA-treated mouse, showing similar hippocampal cell death. The PA histologic results could also provide its potential application for use in the monitoring of the morphological changes observed in astrocytes including hypotrophy, hyperplasia, and neoplasia. Further, it might be a beneficial histologic tool for treatment monitoring of neurodegenerative diseases such as acute traumatic brain injury and neuroprotective effects of treatments on the diseases.
A Frozen section examination is the conventional intraoperative histology method widely used in cancer surgery for tumor margin assessment. However, this method is necessary to perform complicated process including sectioning and staining, which require approximately 15 minutes. Particularly with the ultraviolet (UV) laser (266 nm), photoacoustic microscopy (PAM) showed the capability to visualize cell nuclei without the time consuming procedures by utilizing superior optical contrast of DNA/RNA at this wavelength, which can be a potential alternative of the frozen section. However, previously developed UV-PAM is limited to be applied in intraoperative scenarios because it has suffered from slow imaging speed because of 2D mechanical scanning with linear stepper motors. To overcome this limitation, we developed a fast UV-PAM system based on a 2-axis waterproof microelectromechanical systems (MEMS) scanner with the specially fabricated optical components for UV light. This MEMS scanner enables to scan 3 × 3 mm2 range and acquire 400 × 400 pixels image within 20 seconds. The measured spatial and axial resolutions of the developed system are 2.2 and 39 μm, respectively. Finally, we acquired the histology-like PA image of the mouse kidney with characteristic tubular structures of kidney epithelial cells. In the mouse brain, distinct microstructures such as hippocampus and dentate gyrus were differentiated with the validation of frozen section sample.
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