Correlative two-color two-photon (2C2P) excitation STED microscopy

We present a two-color two-photon stimulated emission depletion microscopy technique (2C2P-STED) that correlates a confocal image with a super-resolved image employing the inherent self-referencing mechanism of nonlinear excitation. The novel approach overcomes the substantial challenge posed by two different imaging modalities in laser-scanning fluorescence microscopy for colocalization on the nanometer scale. Demonstrating the principle of 2C2P-STED, we show for the first time super-resolved images of the gram-positive bacteria Streptococcus pneumoniae TIGR4 pilus type-1. A signal-to-noise ratio (SNR) greater than 10 was achieved in 2C2P excitation mode and approximately 70 nm details were resolved in 2P-STED.