Optical nanoscopy of tissue sections
ForfatterVillegas Hernandez, Luis Enrique
The study of organ tissues, namely, histology, is essential for clinical medicine, as it allows the identification of a wide range of diseases. Traditionally, two major techniques, optical and electron microscopy, have been used for morphological observation of tissue sections. Optical microscopy (OM) allows for fast-acquisition high-throughput imaging, at the expense of diffraction-limited resolution (~200nm), whereas electron microscopy (EM) provides with sub-diffraction resolution at low throughput. Routine histological studies require visualization of large field-of-view (FOV) at high throughput. Pathologies such as nephrotic syndrome, brain neoplasms, neuromuscular disorders, to name just a few, require sub-diffraction resolution, placing EM as the primary technique for its clinical diagnose. In recent years, a new discipline of super-resolution microscopy (SRM), also referred to as optical nanoscopy, has emerged, which achieves sub-diffraction resolution at a faster throughput than EM, allowing for visualization of biological processes at nanoscopic scale. To date, benefits of SRM have been exploited mainly on cellular studies, yet they remain practically unexplored for histological analysis. This master project aimed to image, for the first time, histological samples using two super-resolution optical systems at the UiT – The Arctic University of Norway, namely, the commercial OMX microscope, and the waveguide chip-based microscope setup. Formalin-fixed paraffin-embedded (FFPE) and cryo-preserved tissue sections from human and non-human origin were fluorescently labeled and imaged on the OMX using super-resolution structure illumination microscopy (SIM) and diffraction-limited deconvolution microscopy (DV). Similarly, histological samples were imaged on the waveguide chip-based microscope setup using diffraction-limited total internal reflection fluorescent (TIRF) microscopy. Furthermore, a correlative light-light microscopy has been performed, to compare the three previously mentioned microscopy techniques. SIM images of ultra-thin cryo-sections proved the ability of this technique to resolve structures within 120nm distance and exhibited better contrast than diffraction-limited DV images, allowing for visualization of sub-cellular structures present in the tissue. Contrarily, SIM images of FFPE sections led to reconstruction artifacts due to tissue autofluorescence, impairing the sub-diffraction resolution, yet allowing for better contrast enhancement as compared to the DV images. The results from the waveguide chip-based setup validated the feasibility of this platform for TIRF imaging of tissue sections and demonstrated the ability of this technique for high-throughput large-FOV imaging. Notably, the waveguide chip proved to be an optimal substrate for imaging cryo-sections on the OMX, providing a flat reflective-surface that allowed in-focus tile mosaic images and observation of subcellular features that were otherwise not discernible using coverslips or glass slides as substrates. The findings of this study open a pioneering research path for the implementation of SIM in clinical studies that were traditionally governed by costly low-throughput techniques such as EM. Importantly, the results from the waveguide chip-based microscope obtained in this study set the foundations for further development of a high-throughput super-resolution imaging platform for routine diagnosis of histological sections.
ForlagUiT Norges arktiske universitet
UiT The Arctic University of Norway
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