Far-field localization microscopy such as STORM or PLAM has pushed fluorescence microscopy to the nanoscale which enables us to build a refined understanding of the intracellular organization of cells with near-molecular resolution. In this thesis, I will review our recent efforts on building up a localization microscope with the ability of conducting 3D and multicolor imaging in 20nm resolution using regular organic fluorophore or photoactive fluorescent proteins. However, precise localization requires high signal to background ratio and is difficult to realize for deep-cell imaging due to unwanted background from out-of-focus layers. Another super-resolution method SOFI is capable of reducing background but with a lower resolution. We combine SOFI and Localization Microscopy (...[ Read more ]

Far-field localization microscopy such as STORM or PLAM has pushed fluorescence microscopy to the nanoscale which enables us to build a refined understanding of the intracellular organization of cells with near-molecular resolution. In this thesis, I will review our recent efforts on building up a localization microscope with the ability of conducting 3D and multicolor imaging in 20nm resolution using regular organic fluorophore or photoactive fluorescent proteins. However, precise localization requires high signal to background ratio and is difficult to realize for deep-cell imaging due to unwanted background from out-of-focus layers. Another super-resolution method SOFI is capable of reducing background but with a lower resolution. We combine SOFI and Localization Microscopy (LM) to achieve super-resolution for deep cell imaging. With this method we successfully obtained super-resolution images of multivesicular bodies (MVBs) in tobacco BY-2 cells, reaching 15 μm above cell bottom where strong background occurs. Also we demonstrated a series of well-sectioned images of mitochondria at different depths in a thick mammalian cell. The combination SOFI+LM can perform widefield 3D super-resolution sectioning by localization microscopy without confocal scanning even for thick samples with high background.