Bringing optical nanoscopy to life - Super-resolution microscopy of living cells

Abstract

Microscopy is possibly the best tool we have to peer into the microscopic world to enhance our understanding of the usually invisible, but highly complex and vital events every moment taking place inside living cells. Microscopy is brilliant, but also has its physical constraints and technical limitations. Technical advances have in the last decade pushed optical microscopy past physical limits previously thought unbreakable by the introduction of super-resolution optical microscopy techniques, also referred to as optical nanoscopy. This thesis is about bringing the recent advances in super-resolution optical microscopy to applications in living cells. It is a part of the UiT Tematiske Satsinger program, aiming to strengthen interdisciplinary research and collaboration between traditionally separate fields of science. Three imaging modalities with good prospects for the future of live-cell nanoscopy are covered: structured illumination microscopy (SIM), fluorescence fluctuation based super-resolution microscopy (FF-SRM), and photonic chip-based total internal reflection fluorescence microscopy (c-TIRFM). Results: SIM was found suitable for up to four-color volumetric and widefield super-resolution imaging of living cells, but yet following fast, multicolor subcellular dynamics remains extremely challenging mainly due to technical constraints from the necessary light dose and acquisition time. FF-SRM was found, for most current applications in bio-imaging, underdeveloped. While there seems to be a huge yet unharnessed potential for FF-SRM in future live-cell imaging applications, the tested techniques were found too simplistic and unrealistic in their basic sample assumptions. We developed an FF-SRM reconstruction software with improved computational speed and ease of use. Although large challenges were encountered, the FF-SRM method MUSICAL was employed with success in combination with machine learning for the analysis of nanoscale motion patterns of subcellular vesicles. The reduction of background signal achieved by using TIRFM is widely exploited in super-resolution microscopy. The recently developed c-TIRFM, allowing for extreme fields-of-view compared to traditional implementations of TIRFM, was adapted for live-cell imaging applications. Multimodal imaging of living hippocampal neurons in a custom-made incubation chamber was shown on photonic waveguides. Furthermore, the exploitation of multimodal waveguide illumination patterns for super-resolution imaging via musical image reconstruction was demonstrated.