3D super-resolved in vitro multiphoton microscopy by saturation of excitation

We demonstrate a significant resolution enhancement beyond the conventional limit in multiphoton microscopy (MPM) using saturated excitation of fluorescence. Our technique achieves super-resolved imaging by temporally modulating the excitation laser-intensity and demodulating the higher harmonics fr...

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Published inOptics express Vol. 23; no. 17; pp. 22667 - 22675
Main Authors Nguyen, Anh Dung, Duport, François, Bouwens, Arno, Vanholsbeeck, Frédérique, Egrise, Dominique, Van Simaeys, Gaetan, Emplit, Philippe, Goldman, Serge, Gorza, Simon-Pierre
Format Journal Article
LanguageEnglish
Published United States 24.08.2015
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Summary:We demonstrate a significant resolution enhancement beyond the conventional limit in multiphoton microscopy (MPM) using saturated excitation of fluorescence. Our technique achieves super-resolved imaging by temporally modulating the excitation laser-intensity and demodulating the higher harmonics from the saturated fluorescence signal. The improvement of the lateral and axial resolutions is measured on a sample of fluorescent microspheres. While the third harmonic already provides an enhanced resolution, we show that a further improvement can be obtained with an appropriate linear combination of the demodulated harmonics. Finally, we present in vitro imaging of fluorescent microspheres incorporated in HeLa cells to show that this technique performs well in biological samples.
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content type line 23
ISSN:1094-4087
DOI:10.1364/OE.23.022667