Monitoring intracellular Zn2+ using fluorescent sensors: Facts and artifacts

Abstract

Fluorescent zinc sensors are powerful tools to monitor changes in intracellular free or loosely bound Zn2+ ([Zn2+]i). If molecular weight is considered, these sensors can be categorized as low molecular weight probes or genetically encoded proteins. Based on the nature of the signal emitted by these sensors, they can be divided into two groups: (1) nonratiometric sensors whose fluorescence intensity reports [Zn2+] and (2) ratiometric sensors, with [Zn2+] being represented by a ratio of fluorescence measured at two wavelengths. Interpretation of the data obtained using nonratiometric intensity-based sensors is often complicated because their signal is affected not only by [Zn2+] but also by several [Zn2+]-unrelated phenomena; for example, sensor concentration, light path length (i.e., thickness of a cell or organelle), or photobleaching. Signal ratioing compensates for such [Zn2+]i-unrelated effects and for this reason ratiometric sensors are preferable tools to monitor [Zn2+]i. Nevertheless, to interpret these data, critical analysis is necessary. In particular, calibration of the data in terms of [Zn2+]i is problematic if the intracellular concentration of a sensor approaches or exceeds its Zn2+ Kd, which is often the case. Also, highly concentrated intracellular Zn2+ sensors, by chelating Zn2+, directly affect [Zn2+]i. Further, plasma membrane-permeable Zn2+ sensors may act as Zn2+ ionophores. The purpose of this chapter is to sensitize the reader to these issues and, if possible, to suggest some remedies.