Saturated excitation of fluorescent proteins for sub diffraction-limited imaging of living cells in three dimensions

Abstract

We report, for the first time, the saturated excitation (SAX) of fluorescent proteins for sub diffraction-limited imaging of living cells in three-dimensions. To achieve saturation, a bright yellow and green fluorescent protein (Venus and EGFP) that exhibits a strong nonlinear fluorescence response to the high excitation intensity at the laser focus is used. Harmonic demodulation of the fluorescence signal produced by a modulated excitation light extracts the nonlinear fluorescence signals. After constructing the image from the nonlinear components, we obtain fluorescence images of living cells with spatial resolution beyond the diffraction limit. We also applied linear decon-volution to SAX microscopy and found it effective in further enhancing the contrast of small intracellular structures in the SAX image, confirming the expansion of the optical transfer function in SAX microscopy. © 2013 The Author(s) Published by the Royal Society. All rights reserved.