Structured light illumination for extended resolution in fluorescence microscopy

During the last two decades fluorescence microscopy has become a powerful experimental tool in modern biology. Resolution of optical microscopes is limited by the diffraction nature of light and amounts to approximately 200 mn for point objects imaged with green light and high-NA objectives. Recently, several successful attempts have been made to break the resolution limit of microscopes. One of them is the so-called harmonic excitation light microscopy. 2D structured illumination produced by four interfering laser beams improves the lateral resolution by a factor of 2 to reach 100nm. Structured illumination extends optical resolution since spatial frequencies beyond the classical cut-off frequency are brought into the passband of the optical microscope by frequency mixing. The extended passband is reconstructed computationally from several images acquired with shifted illumination patterns. Here we discuss an extension towards high resolution imaging of thick specimens by combining 2D structured illumination with deconvolution techniques. (c) 2004 Elsevier Ltd. All rights reserved.