Multiphoton microspectroscopy in living plant cells

Abstract

Microspectroscopic measurements in plant cells are complicated by the presence of dense cellular structures such as the cell wall that causes severe light scattering. In addition, the low penetration depth of the excitation light limits the fluorescence signal originating from deeper cell layers in thick multi-cellular plant preparations when single-photon excitation (SPE) is applied. However, two-photon excitation (TPE) can overcome these problems. We report on two-photon microscopy studies of Histone 2B-YFP, a nuclear-expressed protein involved in chromatin packaging. In contrast to SPE, TPE allows imaging throughout the whole root. Therefore by using TPE it was also possible to visualize the root quiescent centers using SCARECROW-EGFP localized in the middle of the root. The interactions between various members of the <i>Arabidopsis thaliana</i> embryogenesis receptor kinase family (AtSERK) have been studied by monitoring Forster resonance energy transfer (FRET) between AtSERK-ECFP and -EYFP fusion proteins using fluorescence lifetime imaging microscopy (FLIM) of the two-photon excited ECFP component.