High-Resolution Fluorescence Imaging by Saturated Excitation (SAX): Its Principle and Imaging Properties in Biology

Abstract

The spatial resolution in optical microscopy is limited by the diffraction limit of light. Here we report the use of nonlinear fluorescence response emerging under saturated excitation (SAX) conditions to improve the spatial resolution of confocal fl uorescence microscopy beyond the diffraction limit. Since the nonlinear fl uorescence response is localized in the excitation focus, it gives the information of sample structures in a region smaller than the focal volume. To detect the nonlinear fl uorescence signals from fl uorescence probes, the sample is excited by an intensity-modulated laser light, and the fl uorescence intensity is demodulated at harmonic frequencies of the laser modulation. SAX microscopy also possesses high background signal rejection capability because the saturated excitation of fl uorescence probes is spatially confined in the excitation focus. Using a SAX microscope, we demonstrate fl uorescence imaging of a single fl uorescent nanodiamond and stained HeLa cell with a spatial resolution beyond the diffraction limit.