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Polzer, Christoph; Ness, Stefan; Mohseni, Mojtaba; Kellerer, Thomas; Hilleringmann, Markus; Rädler, Joachim O. and Hellerer, Thomas (2019): Correlative two-color two-photon (2C2P) excitation STED microscopy. In: Biomedical Optics Express, Vol. 10, No. 9: pp. 4516-4530

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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 (SINIR) greater than 10 was achieved in 2C2P excitation mode and approximately 70 nm details were resolved in 2P-STED. (C) 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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