Illuminating mobile zinc with fluorescence from cuvettes to live cells and tissues

Abstract

With the aid of chemoselective sensors, fluorescence microscopy has emerged as an indispensable tool to visualize the distribution and dynamics of various biologically important molecules in live specimens. Motivated by our interest in understanding the chemistry and biology of mobile zinc underlying its physiological and pathological roles, over the past decade, our laboratory has developed an extensive library of zinc fluorescence probes. In this chapter, we provide essential information about our sensor toolbox in order to assist investigators interested to apply our constructs to study various aspects of mobile zinc biology. We illustrate their use with several examples of imaging both exogenous and endogenous mobile zinc in live cells and tissues using various versions of fluorescence microscopy, including confocal and two-photon microscopy.

Figure 1

Fluorescence spectra of 0.5 µM ZP1 with incrementally higher concentrations of zinc. Spectra were acquired in pH 7.0 buffer (50 mM PIPES, 100 mM KCl) at 25 °C; λ_ex = 507 nm. The first 10 spectra correspond to free zinc concentration between 0 and 24 nM; the eleventh spectrum was obtained with ~25 µM free zinc. Inset: integrated emission (rectangles) was analyzed by least-squares fitting (solid line) to give a dissociation constant of 0.7 nM. Reprinted with permission from Walkup et al, 2000. Copyright 2000 American Chemical Society.

Figure 2

Fluorescence imaging of live HeLa cells treated with 5 µM ZP1 (left to right: DIC image, nuclear stain Hoechst 33258, and ZP1). Addition of 50 µM zinc/pyrithione resulted in fluorescence enhancement, which was reversed by treatment with 100 µM TPEN. Scale bar = 25 µm.

Figure 3

Fluorescence imaging of live HeLa cells incubated with a trappable probe, QZ2E. (a) DIC image, (b) nuclear stain Hoechst 33258, (c) QZ2E, (d) green fluorescence signals 5 min after addition of 100 µM 1:1 Zn/pyrithione, and (e–h) green signals during periodic washing taken at 10 min-intervals. Scale bar = 25 µm. Reprinted with permission from McQuade and Lippard, 2010. Copyright 2010 American Chemical Society.

Figure 4

Confocal fluorescence images of live dentate gyrus neurons stained with ZP3 or ZS5. (A) ZP3 allowed for visualization of endogenous mobile zinc; (B) ZS5 enabled imaging the dynamics of zinc mobilization following SNOC-triggered nitrosative stress. Signals in both experiments were reversed by TPEN addition. Scale bar in B = 25 µm. Adapted with permission from (A) Chang et al, 2004a and (B) Nolan et al, 2006. Copyright (A) 2004 Elsevier Science Ltd and (B) 2006 American Chemical Society.

Figure 5

Two-photon and confocal fluorescence images of acute mouse hippocampal slices stained with ZP3. Images acquired with two-photon microscopy show (a) the entire slice section of a hippocampus, (b) the stratum lucidum layer, and (c) individual giant mossy fiber boutons. Confocal microscopy produced similar zinc-evoked fluorescence signals after ZP3 staining (d), which diminished upon addition of TPEN (e). Scale bars = (a) 800 µm, (b) 200 µm, or (c) 10 µm. Adapted with permission from (a–c) Chang et al, 2004b and (d–e) Chang et al, 2004a. Copyright (a–c) 2004 American Chemical Society and (d–e) 2004 Elsevier Science Ltd.

Scheme 1

Structures of selected zinc-selective fluorescence probes. Among them ZP1 and ZP4 are commercially available.

Scheme 2

Structures of dual-function MRI/fluorescence probes.