Biological imaging beyond the diffraction limit by saturated excitation (SAX) microscopy

We present an alternative high-resolution fluorescence imaging technique, saturated excitation (SAX) microscopy, for observations of biological samples. In the technique, we saturate the population of fluorescence molecules at the excited state with high excitation intensity. Under this condition, the fluorescence intensity is no longer proportional to the excitation intensity and the relation of the fluorescence and excitation intensity shows strong nonlinearity. In the centre of laser focus, the nonlinear responses induced by the saturation appear notably because of higher excitation intensity. By detecting fluorescence signals from the saturated area, we can push the spatial resolution beyond the diffraction barrier in three dimensions. SAX microscopy can be realized with a simple optics, where a laser intensity modulation sisytem and a lock-in amplifier are simply added to a conventional confocal microscope system. Using the SAX microscope, we demonstrated high-resolution imaging of a biological sample by observing mitochondria in HeLa cells. We also examined the nonlinear response of commercially available dyes under saturated excitation conditions.