Isotropic Single-objective Microscopy: Experiment and Application

Improving the spatial resolution in optical microscopes is a challenging task for many applications. In fluorescence confocal microscopy, the resolution is given by a lenght Detection Efficiency Function (DEF) which looks like a rice grain. 4Pi type C microscope has shown to be a powerful solution for considerably reducing the axial dimension of the DEF [1] and even its lateral dimension [2]. This results are obtained by superposing two coherent illumination beams and by adding coherently the two fluorescence wave-fronts emitted on the both sides of the luminescent sample. This set-up is very interesting but is somehow difficult to be used by non-specialized users since its alignment is not simple. Isotropic single objective (ISO) microscopy [3] is a solution based on the same principle with comparable results. The main advantage of this technique is that ISO works only with one objective which allows an implementation on all confocal systems.