Label-free optical microscopy: Photon Absorption Remote Sensing (PARS) and other methods for label-free histopathological imaging of tissues

Abstract

Emerging label-free microscopy methods offer promising new avenues to view cells and tissues in their native environment, minimizing external influences. These label-free techniques are an exciting departure from gold standard methods for visualizing microscopic cellular and tissue structures, which rely on centuries-old chemical staining processes. In current practice, chemical labelling can unavoidably interfere with specimens’ physical and biochemical integrity. As a result, samples are effectively consumed by staining with only a single stain set normally applied to each sample. This limitation is especially impactful in applications such as clinical oncology and medical histopathology. In these settings, irreversible staining processes can severely limit the diagnostic utility of samples; especially when there is limited sample volume (e.g., brain tumor biopsies). As an alternative, label-free imaging techniques offer a potential avenue to visualize subcellular tissue anatomy while preserving samples in their entirety. Subsequently label-free microscopy methods have significant potential to greatly increase the diagnostic utility of each specimen, thereby enhancing patient outcomes. This thesis focuses on developing new methods for label-free microscopy, specifically emphasizing techniques for label-free histopathology. As a starting point the targeted objective is to develop a label-free analog to chemical hematoxylin and eosin (H&E) staining. This objective is chosen as H&E represents the gold standard contrast applied in effectively every clinical diagnostic case. Subsequent developments in this thesis can be broken into three major sections, which focus on (1) developing label-free microscopy methods for H&E-like imaging, (2) exploring the biomolecular specificity of developed methods to validate the label-free H&E-like contrast, and (3) producing a label-free microscopy architecture capable of meeting the imaging requirements necessary for clinical adoption. The first collection of works explores the development of a range of label-free microscopy methods. These studies establish new variations and combinations of optical absorption and scattering microscopes to visualize microscopic tissue anatomy label-free. These efforts ultimately resulted in the development of a new optical absorption microscopy modality, Photon Absorption Remote Sensing (PARS). This comprehensive technique provides biomolecule-specific visualizations characterizing the dominant photophysical effects caused when photons are absorbed by a biomolecule. As a direct result, novel PARS specific contrasts are developed as the total absorption (TA) and quantum efficiency ratio (QER). These PARS measurements may provide unique views into biomolecules’ excited state dynamics, accessing characteristics related to the quantum yield. By specifically probing specimens’ response to the absorption of deep ultraviolet light, PARS is shown to provide label-free contrast directly reminiscent of gold standard chemical H&E staining methods. As a proof of concept, the initial PARS architecture is applied to capture submicron resolution images of key H&E-like diagnostic markers across a variety of human and animal tissue specimens. The second section of this thesis expands the basis for PARS histopathology by validating PARS capacity to produce H&E-like visualizations. Two main avenues of exploration are pursued in this effort. The first endeavor explores the underlying biomolecular contrast of the PARS measurements. Established statistical methods are applied to develop characteristic PARS profiles for biomolecules. These PARS signatures are then applied to map the abundance of molecules label-free inside complex specimens. As a proof of concept, key diagnostic features including nuclei, red blood cells, and connective tissues are directly characterized and unmixed label-free. Resulting statistical abundance mappings are directly validated against chemically stained ground truth counterparts. The second endeavor introduces an end-to-end pipeline which uses deep learning-based image-to-image transforms to emulate chemical H&E visualizations from label-free PARS data. Resulting PARS emulated H&E-like visualizations are validated against chemical H&E staining through a clinical concordance study. In this diagnostic validation study, statistical analysis is applied to determine if pathologists produce the same diagnoses on both PARS and chemical H&E images. In this preliminary test, the PARS-based virtual staining method achieves > 90% concordance with very high statistical confidence (Kappa > 0.7) across all measured diagnostic tests. The final thesis section develops a new PARS architecture which achieves pragmatic imaging performance, nearing the requirements for clinical diagnostic settings. The presented system features a hybrid opto-mechanical scanning architecture which allows for high-speed MHz rate imaging. This results in imaging speeds which are more than an order of magnitude faster than earlier PARS embodiments developed in the PhotoMedicine Labs (at the University of Waterloo). This work simultaneously develops an end-to-end control system and imaging workflow which enables fully automated PARS imaging of whole specimens. Deep learning methods are applied to the resulting PARS images to produce virtual H&E-like visualizations. Qualitative and quantitative methods are applied to validate the imaging performance across a range of human and animal tissue samples. Results indicate the PARS virtual H&E images are largely indistinguishable from chemically H&E-stained ground truth images. Notably, the presented system forms the basis for a commercially available clinically ready prototype for label-free PARS histopathology imaging. In total, the findings presented across this thesis encompass the development of a new variation of microscopy technique (PARS). This method provides unique views into the absorption and scattering characteristics of specimens opening a new avenue of label-free contrast. For the presented histopathology application, PARS can provide powerful H&E-like images which may circumvent key challenges of chemical staining. In clinical histopathology, this method could enhance the diagnostic utility of tissue specimens directly improving patient outcomes. Beyond histopathology, the principles of PARS may be directly applicable to a wide range of imaging applications spanning material science, biological research, and clinical diagnostics. Overall, the methods developed in this thesis lays the groundwork for new label-free optical absorption microscopy techniques, which are already achieving real-world commercial and clinical success in histopathology applications.