Single molecule localization microscopy (SMLM) surpasses the diffraction limit by sequentially imaging individual fluorescent molecules. By incorporating 4Pi detection to capture self-interference from the fluorescence emitters via two objective lenses, 4Pi-SMLM achieves isotropic sub 5 nm resolution in three dimensions (3D). This thesis details the design, construction, and evaluation of a Cryo-4Pi-SMLM system, aiming to enable subcellular 3D fluorescence imaging in native states with isotropic localization precision under cryogenic conditions, providing a 3D correlative light and electron microscopy solution...

Total internal reflection fluorescence (TIRF) microscopy is an important imaging tool for the investigation of biological structures, especially the study on cellular events near the plasma membrane. Imaging at cryogenic temperatures not only enables observing structures in a near-native and fixed state but also suppresses irreversible photo-bleaching rates, resulting in increased photo-stability of fluorophores. Traditional TIRF microscopes produce an evanescent field based on high numerical aperture immersion objective lenses with high magnification, which results in a limited field of view and is incompatible with cryogenic conditions. Here, we present a waveguide-based TIRF microscope, which is able to generate a uniform evanescent field using high refractive index waveguides on photonic chips and to obtain cellular observation at cryogenic temperatures. Our method provides an inexpensive way to achieve total-internal-reflection fluorescence imaging under cryogenic conditions.