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

Saturated excitation (SAX) microscopy offers high-depth discrimination predominantly due to nonlinearity 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 confirmed 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.