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

TL;DR

This paper introduces a super-resolution multiphoton microscopy technique that surpasses traditional resolution limits by saturating fluorescence excitation and demodulating higher harmonics, enabling detailed imaging of biological samples.

Contribution

The authors develop a novel method using saturated excitation and harmonic demodulation to achieve super-resolved multiphoton microscopy beyond conventional limits.

Findings

Enhanced lateral and axial resolution demonstrated on fluorescent microspheres

Further resolution improvement with harmonic combination

Effective in vitro imaging of fluorescent microspheres in HeLa cells

Abstract

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.