4Pi live cell imaging

One distinctive advantage of light microscopy over electron microscopy is it ability to selectively image sub-cellular details three-dimensionally in living cells. Live cell imaging is the domain of light microscopy. In this thesis a live cell imaging setup relying on 4Pi fluorescence microscopy is presented. To speed up the imaging process and record cellular processes of the order of a few seconds a new method of multifoci-generation via the stacking of birefringent crystals is implemented. The axial optical sectioning ability of the system is ~120 nm and the recording of an average-sized 3D data stack is accomplished in less than 10 seconds. The microscope’s applicability to live cell imaging is verified by recording both mitochondrial fusion and fission events in live yeast and the movement of PML bodies in live human U2OS cells. To push the limits of applicability of the system further, second harmonic generation is tested as an alternative label-free coherent contrast mechanism and binary amplitude filters are successfully applied. Second harmonic generation turns out to be an imaging technique confined to specific applications. With the implementation of amplitude filters 4Pi microscopy of aqueous tissue becomes convenient and broadly applicable