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 (Cu(2+) ) in vivo. For this purpose, we employed a green fluorescent protein (GFP) form able to change its fluorescence lifetime upon Cu(2+) binding. We demonstrate that this technique is selective for Cu(2+) . 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 Cu(2+) . Cu(2+) 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 Cu(2+) distribution in vivo is demonstrated in prokaryote and eukaryote cells.