Increasing the Lateral Resolution in Confocal Fluorescence and Bio-luminescence Microscopes

Improving the spatial resolution in optical microscopes is a challenging task for many applications. In fluorescence confocal microscopy, the resolution is given by the Detection Efficiency Function (DEF) resulting from the product of the Excitation Efficiency Function (EEF) by the Collection Efficiency Function (CEF). The focalisation of the pump laser beam by a high numerical aperture objective lens defines the excitation efficiency volume whereas the 3D image of the pinhole represents the collection efficiency volume. Many works aimed to reduce the excitation volume, but only few studies have been carried out in order to improve the CEF. 4Pi type C microscope [1,2] has shown to be a powerful solution for reducing considerably the axial dimension of the DEF. This result is obtained by superposing two coherent illumination beams [1-3] and by adding coherently the two fluorescence wavefronts emitted on the both sides of the luminescent sample [4]. In order to increase the lateral resolution of 4Pi type C microscopes, we have proposed to add an image inversion system in one arm of the microscope [5]. This system, referred as 4Pi’ microscope, leads to a reduction of the CEF by using the “spatial incoherence” of fluorescent samples. 4Pi’ microscopes can operate in one or two photon excitation modes [6] and even without any excitation in case of bioor chemi-luminescence. In order to use the same concept, we propose to couple classical confocal microscopes to a particular interferometer [7]. We show that, in this case, the lateral resolution is improved by a factor of 1.5. Moreover this configuration brings several other advantages that we will describe during the conference.