Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters.

Saturated excitation (SAX) microscopy offers high-depth discrimination predominantly due to nonlinear- ity in the fluorescence response induced by the SAX. Calculation of the optical transfer functions and the edge responses for SAX microscopy revealed the contrast improvement of high-spatial frequency components in the sample structure and the effective reduction of background signals from the out-of-focus planes. Experimental observations of the edge response and x-z cross-sectional images of stained HeLa cells agreed well with theoretical investigations. We applied SAX microscopy to the imaging of three-dimensional cultured cell clusters and con- firmed the resolution improvement at a depth of 40 μm. This study shows the potential of SAX microscopy for super-resolution imaging of deep parts of biological specimens. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including