Probing live samples in second-harmonic generation microscopy using specific markers and fluorescent proteins

Abstract

In an effort to complement cellular two-photon excited fluorescence (TPEF) microscopy with structural information from second-harmonic generation (SHG) imaging, we investigated the applicability of fluorescent proteins for SHG imaging. In the first stage, the first hyperpolarizability β, a measure for the second-order nonlinear optical properties of a molecule, was determined for several fluorescent proteins. In a second stage, an established HeLa cell line expressing a membrane protein labeled with a fluorescent protein, was adapted and imaged using simultaneous TPEF and SHG microscopy. The contour of stretched cells observed in these experiments was proven to be originating in microtubules instead of the fluorescent proteins.