THESIS
2014
vii, 34 pages : color illustrations ; 30 cm
Abstract
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 (...[
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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.
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