Visualization of Cu2+uptake and release in plant cells by fluorescence lifetime imaging microscopy

A principal objective in life sciences is the visualization of biochemical processes. Fluorescence-based techniques are widely used to demonstrate transport of relevant substances across cellular membranes. In this paper we report a novel noninvasive, real-time fluorescence lifetime imaging microscopy method for visualizing uptake and release of divalent copper ions (Cu2+) in vivo. For this purpose, we employed a green fluorescent protein (GFP) form able to change its fluorescence lifetime upon Cu2+ binding. We demonstrate that this technique is selective for Cu2+. We show the reversible decrease of the fluorescence lifetime of GFP from 2.2 to 1.6 ns in Escherichia coli and from 1.8 to 1.3 ns in root cells of Arabidopsis after the addition of Cu2+. Cu2+ uptake of epidermal tobacco cells leads to a drop of the GFP lifetime from 2.5 to 2.2 ns. In summary, the spatially resolved visualization of Cu2+ distribution in vivo is demonstrated in prokaryote and eukaryote cells.