Open-top Bessel beam two-photon light sheet microscopy for three-dimensional pathology.

Nondestructive pathology based on three-dimensional (3D) optical microscopy holds promise as a complement to traditional destructive hematoxylin and eosin (H&E) stained slide-based pathology by providing cellular information in high throughput manner. However, conventional techniques provided superficial information only due to shallow imaging depths. Herein, we developed open-top two-photon light sheet microscopy (OT-TP-LSM) for intraoperative 3D pathology. An extended depth of field two-photon excitation light sheet was generated by scanning a nondiffractive Bessel beam, and selective planar imaging was conducted with cameras at 400 frames/s max during the lateral translation of tissue specimens. Intrinsic second harmonic generation was collected for additional extracellular matrix (ECM) visualization. OT-TP-LSM was tested in various human cancer specimens including skin, pancreas, and prostate. High imaging depths were achieved owing to long excitation wavelengths and long wavelength fluorophores. 3D visualization of both cells and ECM enhanced the ability of cancer detection. Furthermore, an unsupervised deep learning network was employed for the style transfer of OT-TP-LSM images to virtual H&E images. The virtual H&E images exhibited comparable histological characteristics to real ones. OT-TP-LSM may have the potential for histopathological examination in surgical and biopsy applications by rapidly providing 3D information.

High-contrast visualization of human skin cancers with combined reflectance confocal and moxifloxacin-based two-photon microscopy: An ex vivo study.

BACKGROUND AND OBJECTIVES: Precise determination of cancer margin during skin cancer surgery is crucial for complete resection and further clinical prognosis. Although reflection confocal microscopy (RCM) has been used for perioperative guiding, its reflection contrast has limitations in detecting cancer cells in the dermis. We previously developed combined reflection confocal (RC) and moxifloxacin-based two-photon (MB-TP) microscopy for sensitive cancer detection by using multiple contrast mechanisms. In this study, the performance of combined microscopy was characterized in various skin cancer specimens and compared with standard methods. MATERIALS AND METHODS: Seven human skin specimens in total including two normal ones, three basal cell carcinomas (BCCs), and two squamous cell carcinomas (SCCs) were collected and imaged in fresh condition. Moxifloxacin ophthalmic solution was topically instilled for cell labeling for 3-5 minutes, then mosaic imaging with the combined microscopy was conducted. The imaged specimens were imaged again after exogenous nuclear labeling for comparison and then processed for standard hematoxylin and eosin histology. RESULTS: Combined RC and MB-TP microscopy visualized both cell and extracellular matrix structures of the skin specimens with multiple contrasts of reflection, moxifloxacin fluorescence, autofluorescence, and second harmonic generation. It distinguished normal cell structures in the skin dermis such as hair follicles, sebaceous and eccrine glands from BCC nests, and SCCs based on cell organization. Normal cell structures had organized cell arrangements for their functions, while cancer cell structures had dense and disorganized cell arrangements. Cellular features found by combined microscopy images were confirmed by both TP microscopy with nuclear labeling and histological examination. CONCLUSIONS: The imaging results showed the potential of combined microscopy for sensitive cancer detection and in vivo guiding of skin cancer surgery.

Fast and sensitive delineation of brain tumor with clinically compatible moxifloxacin labeling and confocal microscopy.

Delineation of brain tumor margins during surgery is critical to maximize tumor removal while preserving normal brain tissue to obtain optimal clinical outcomes. Although various imaging methods have been developed, they have limitations to be used in clinical practice. We developed a high-speed cellular imaging method by using clinically compatible moxifloxacin and confocal microscopy for sensitive brain tumor detection and delineation. Moxifloxacin is a Food and Drug Administration (FDA) approved antibiotic and was used as a cell labeling agent through topical administration. Its strong fluorescence at short visible excitation wavelengths allowed video-rate cellular imaging. Moxifloxacin-based confocal microscopy (MBCM) was characterized in normal mouse brain specimens and visualized their cytoarchitecture clearly. Then, MBCM was applied to both brain tumor murine models and two malignant human brain tumors of glioblastoma and metastatic cancer. MBCM detected tumors in all the specimens by visualizing dense and irregular cell distributions, and tumor margins were easily delineated based on the cytoarchitecture. An image analysis method was developed for automated detection and delineation. MBCM demonstrated sensitive delineation of brain tumors through cytoarchitecture visualization and would have potentials for human applications, such as a surgery-guiding method for tumor removal.