Abstract
The 4pi principle first described by Stefan Hell and Ernst Stelzer in 1992 is still one of the best strategies for increasing the axial optical resolution of a fluorescent sample. The commercial version of this microscope was not very successful due to the tedious procedure needed to align the two opposing laser beams. The system is superb in revealing small cellular structures (<1 µm) in crowded 3D environments. Also, larger structures like prophase chromosomes or synaptonemal complexes with small distances between the objects can be segmented in 3D. Heterogeneous refractive indices in the sample put some limitations on the technique. The possibility of using the two lenses as two different microscopes can reveal structures in small objects with completely different refractive indices like gas bubbles.
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