Wide-field Polarimetric Second Harmonic Generation Microscopy for Histology Imaging

Nonlinear optical microscopy provides several contrast mechanisms including second harmonic generation (SHG), third harmonic generation (THG) and multiphoton excitation fluorescence (MPEF) that can contribute with additional information to hematoxylin and eosin (H&E) stained tissue histopathology, which is used as a gold standard for cancer diagnostics. Large area imaging is required for practical applications of nonlinear microscopy in whole-slide digital pathology. Nonlinear optical histopathology encompasses multiscale imaging starting from the ultrastructural (nanoscopic) scale, where polarization of SHG is applied to deduce the structural changes of extracellular matrix (ECM) collagen. The distribution of ultrastructural parameters on the microscopic scale provides the information, for example, about the tissue changes at the tumor margins. The hundreds of microns to millimeters scale allows to investigate the spread of induced changes in the tissue providing information about the tumor invasiveness and advancement that can aid in tumor staging and prognostics of the disease. In this thesis, we developed a novel polarimetric wide-field SHG microscopy technique that allows for whole-slide imaging of histopathology samples. We optimized imaging conditions of wide-field microscopy by investigating photobleaching effects of SHG and MPEF signals induced with wide-field illumination in unstained and H&E stained histology tissue sections. A theoretical and experimental description of MPEF and SHG bleaching in H\&E stained samples is performed, providing the basis for choosing optimal laser settings for wide-field microscopy. Moreover, we developed the SHG Stokes polarimetry method (SP-SHG) for quick calculation of ultrastructural parameters required for large area imaging. Generation of polarization states is achieved using liquid crystal variable retarders. SP-SHG is performed on large area images (700 um x 700 um) and ultrastructural parameters are calculated from the polarimetric parameters without data fitting, that shortens the post-processing time of polarimetric data by more than an order of magnitude. Wide-field polarimetric SHG microscopy was used for imaging tendon tissue, as well as histopathology samples from kidney, liver, lung and breast tissue. Changes in the collagen structure are demonstrated for normal and breast tumor tissue. The thesis demonstrates that the novel wide-field polarimetric SHG microscopy imaging method can be readily applied for nonlinear whole-slide digital pathology investigations.

Department dc:contributor.department

Physics

Year dc:date.issued

2023

Author dc:creator

• Uribe Castano, Leonardo Jose

Advisor dc:contributor.advisor

• Barzda, Virginijus V

Handle dc:identifier.uri

http://hdl.handle.net/1807/127863

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/127863