Techniques for measuring cellular zinc
Abstract
The development and improvement of fluorescent Zn2+ sensors and Zn2+ imaging techniques have increased our insight into this biologically important ion. Application of these tools has identified an intracellular labile Zn2+ pool and cultivated further interest in defining the distribution and dynamics of labile Zn2+. The study of Zn2+ in live cells in real time using sensors is a powerful way to answer complex biological questions. In this review, we highlight newly engineered Zn2+ sensors, methods to test whether the sensors are accessing labile Zn2+, and recent studies that point to the challenges of using such sensors. Elemental mapping techniques can complement and strengthen data collected with sensors. Both mass spectrometry-based and X-ray fluorescence-based techniques yield highly specific, sensitive, and spatially resolved snapshots of metal distribution in cells. The study of Zn2+ has already led to new insight into all phases of life from fertilization of the egg to life-threatening cancers. In order to continue building new knowledge about Zn2+ biology it remains important to critically assess the available toolset for this endeavor.
Introduction
Zinc is an essential metal, and the proper balance of zinc is critical to the health of organisms [1]. At the molecular level, the coordination of zinc ions (Zn2+) to individual proteins and enzymes either to stabilize protein structure or to create a catalytic center has been well characterized [2]. Further, bioinformatics studies predict that 10% of human proteins require Zn2+ for their structure and function [3], indicating that Zn2+ is necessary for the proper function of thousands of proteins. Work at the cellular level strives to connect our molecular understanding of Zn2+ biology to observations correlating organismal Zn2+ status and health. Mammalian cells maintain a total concentration of Zn2+ in the hundreds of micromolar [4]. While most of this Zn2+ is bound to proteins and inaccessible, a pool of labile Zn2+, which is non-protein bound and complexed to a variety of small molecule ligands [5], has been detected in the cytosol with a concentration in the hundreds of picomolar. Furthermore, there is growing evidence of a labile Zn2+ pool in organelles [4, 6]. The concentration of Zn2+ in cells and across organelles is maintained by a complex set of 24 Zn2+ transporters [7, 8]. This pool of labile Zn2+ is available to bind to newly synthesized proteins, but the importance of Zn2+ to organismal and proteomic stability and the cellular energy allocated to the transportation of Zn2+ has led to the hypothesis that Zn2+ may also serve as a signal [9].
In order to gain insight into the biology of Zn2+ at a fundamental level it is important to understand both the distribution and dynamics of accessible Zn2+ in cells. Research into the distribution of Zn2+ generally strives to generate a detailed quantitative map of where Zn2+ is located in order to define possible sources and sinks of labile Zn2+ and identify whether there is a heterogeneous distribution of total Zn2+. To rigorously assign Zn2+ to a specific organelle, experimental approaches must be high enough resolution to unambiguously distinguish organelle structures (such as electron microscopy techniques) or the probe being used to measure Zn2+ must be restricted to a specific organelle. Fluorescent probes and elemental mapping techniques (see below) applied can both be used to develop such a Zn2+ map. As detailed in this review, the two approaches provide complementary information on different types of samples: live cells in the case of probes, and fixed samples or fixed time points in the case of elemental mapping techniques. On the other hand, research into the dynamics of Zn2+ must be carried out in live cells using fluorescent sensors in order to obtain temporal information about Zn2+ fluxes. Ideally, such tools will be sensitive to small changes in Zn2+ concentrations. For measuring both distribution and dynamics, tools must respond specifically and selectively to labile Zn2+.
A growing number of fluorescent small molecule probes and protein-based sensors are being developed to measure both the dynamics and subcellular distribution of labile Zn2+ in live cells. A wide range of sensors has been developed with diverse characteristics including: targeting to different organelles, signal detection at various wavelengths, and binding to Zn2+ with altered affinities. The application of probes and sensors in live cell imaging experiments requires many controls to evaluate whether the sensor is selectively measuring labile Zn2+, targeted only to the cellular area of interest, perturbs Zn2+ homeostasis and regulation, and validate that the signal changes are sensitive only to fluctuations in Zn2+ [6]. Thus, it is both wise and prudent to employ complementary imaging methods on fixed samples or fixed time points to corroborate and further define the cellular distribution of zinc, without adding probes to cells. These data can strengthen and confirm interesting observations gathered through the development and use of Zn2+ sensors and probes.
In the past several years, several elemental mapping methods including mass-spectrometry-based and X-ray fluorescence-based techniques have greatly improved in sensitivity and spatial resolution, allowing for the study of trace metal distribution at the cellular and subcellular levels [10–13]. By analyzing the significance of heterogeneous metal distributions at high resolution and quantifying biologically relevant changes in these distributions, researchers have gained insight into physiology, pharmacology, toxicology, pathology, and other disciplines. Besides providing a snapshot of total elemental distribution, mass-spectrometry approaches are capable of resolving individual isotopes of Zn2+ and other metals, and X-ray techniques can be used to determine chemical speciation. The information obtained from these approaches can complement studies using tools for measuring labile Zn2+ pools in live samples.
Probes and Sensors
Several types of systems have been developed to study Zn2+ in cells by fluorescence microscopy. We refer the reader to several recent reviews that provide comprehensive coverage of these probes and sensors [4, 6, 14]. In this review, we will briefly summarize the types of sensors available for Zn2+ detection, and move on to a discussion of the experimental challenges associated with using probes and important controls that should be carried out to minimize misinterpretation of data. Many of the available tools fall under two general classes of probes: small molecule probes and protein sensors based on Förster Resonance Energy Transfer (FRET) [6, 14, 15]. The strengths and weaknesses of these two classes of probes, as well as summary of commonly used probes are highlighted in Figure 1. Small molecule probes usually increase in fluorescence upon chelation of Zn2+. The strengths of small molecule probes are that they can be cell permeable and therefore are easy to apply to cells, they are bright and yield a high fluorescence signal over background autofluorescence of cells, and can be made to fluoresce at various wavelengths. Small molecule probes have also been adapted to give ratiometric signals that allow for normalization for changes in fluorescence that are not due to chelation of Zn2+ [16, 17]. FRET-based protein sensors have also been developed to measure Zn2+ in cells. These sensors consist of two fluorescent proteins (FPs) and a Zn2+ coordinating site that is designed to change the relative orientation and distance of the FPs upon binding leading to a change in FRET signal from the donor FP to the acceptor FP. The ratiometric nature of these sensors allows for correction for protein concentration, sample thickness, and movement. The sensors are genetically encoded, can be targeted to organelles, and, through mutation, can be tuned to bind Zn2+ with a variety of affinities. Different colored FRET based sensors have been derived from the many available FPs, increasing flexibility in experimental protocols.
Panel A) Comparison of properties of small molecule and FRET based protein sensors. Panel B) Small molecule probes typically contain a fluorophore attached to a Zn2+ chelating moiety (FluoZin-3 is shown as an example). FRET-based protein probes typically contain two fluorescent proteins separated by a Zn2+-binding domain. Small molecule probes tend to increase in fluorescence intensity upon binding Zn2+, whereas FRET-based probes are characterized by an increase in intensity at one wavelength and decrease at another (Sample fluorescence emission spectra are depicted with arrows indicating the change upon Zn2+ addition.) Panel C) Most commonly used probes and sensors. Companies and labs that provide these sensors for general use are included in parentheses.
Beyond these two general classes of probes, other platforms have been developed that rely on different strategies to detect and report Zn2+. Hybrid sensors combine a synthetic portion with a genetically encoded portion. The Lippard lab has used SNAP-tag to genetically target the small molecule sensor ZinPyr-1 [18]. In a similar vein, the Fierke group created sensors that combine the Zn2+ binding enzyme, carbonic anhydrase, with a small molecular fluorophore and a FP [19]. These systems aim to combine the advantages of both small molecule and protein-based systems: the modularity and brightness of the small molecules with the targetability and ratiometric signals of protein based systems. A new DNA based probe for Zn2+ has recently been developed [20]. This system relies on a photocaged DNAzyme, which can be activated with light and cleaves in the presence of Zn2+ separating a fluorophore, fluorescein, from a quencher, dabcyl. This process creates a molecular beacon for Zn2+. Further development of this platform might allow for a new class of Zn2+ sensing molecules. It is useful to note the diversity of strategies for sensor design, as different sensors have different strengths and application of complementary sensor platforms can strengthen cell-based studies. Below we turn to recent work that points to the need for careful controls in the application of these molecules and the importance of understanding of the underlying chemistry that allows these molecules to detect Zn2+. We also refer the reader to an excellent recent review that discusses the complex solution chemistry and speciation of zinc ions in biological environments [5]
Progress and challenges in applying FRET-based protein sensors to measure Zn2+
FRET-based sensors have been applied to measure the concentration of Zn2+ in the cytosol and organelles in a variety of cell lines. Figure 2 outlines the in situ calibration that is carried out in order to use ratiometric sensors to convert the FRET ratio to a normalized parameter for comparing relative levels of labile Zn2+ in different cells or under different conditions. Briefly, once cells are expressing the sensor, the ‘resting’ FRET ratio or the signal of the sensor before manipulation of Zn2+, is measured. Subsequently, the FRET ratios of the apo-sensor and the fully saturated sensor must be measured in order to determine the fractional saturation of the sensor at rest in each individual cell. To measure the FRET ratio of the apo sensor, the Zn2+ concentration is lowered to its extreme by adding excess cell permeable Zn2+ chelators, N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) or Tris(2-pyridylmethyl)amine (TPA). To measure the FRET ratio of the Zn2+-saturated sensor, the Zn2+ concentration is elevated by adding excess concentrations of Zn2+ combined with either a cell permeabilizing agent (digitonin or saponin) or an ionophore (pyrithione) [21]. The ratio of the maximum signal of the sensor over the minimum signal of the sensor is called the dynamic range (DR).
Diagrams of Zn2+ calibrations: Panel A. depicts a sensor that increases in FRET ratio when Zn2+ binds. Sensors with this mechanism include the Zif, Zap, and eZinCh families. Panel B. depicts a sensor that decreases in FRET ratio when Zn2+ binds. The eCALWY family reports Zn2+ abundance by this mechanism. The equations used to determine the resting concentration of zinc are given where n equals the hill coefficient and KD is the dissociation constant determined by titration of the sensor in vitro [25–31]. These equations assume that the probes form a 1:1 complex, that the probes are at a low enough concentration in cells for the fluorescence intensity to be linearly proportional to the concentration, and that the probes behave similarly in cells and in vitro [22].
The in situ calibration is typically used to calculate the fractional saturation of the sensor in individual cells, and can be used along with the KD value to estimate the concentration of Zn2+ in a particular location. The fractional saturation of the sensor provides a relative comparison of Zn2+ in different cells or under different conditions, where higher saturation suggests higher levels of labile Zn2+ and lower saturation suggests lower levels. Estimation of the Zn2+ concentration requires further assumptions and data processing, including accurate measurement of the sensor KD. The dissociation constants of these sensors are usually determined through in vitro titration of the sensors with known amounts of Zn2+, and fitting of the resulting data using a ratio-based approach [22]. This approach assumes that the donor flourophore signal decreases at the same rate that the acceptor flourophore signal increases during FRET. Recently, Pomorski et al. have shown that many ratiometric sensors do not conform to this assumption and have developed a more rigorous intensity-calibrated approach for fitting the titration data that should be applied to determining the apparent binding constants of ratiometric sensors [23]. It is worth noting that the reported KD values of many FRET sensors will likely need to be adjusted using the approach of Pomorski et al. In this review, we report the currently published KD values for consistency. It has also been suggested that the isosbestic wavelength of FRET sensors can be used as an internal standard to determine the binding affinities of sensors; this approach decreases the dynamic range by using the intensity change at a single wavelength [24]. Finally, some attempts have been made to measure in situ KDs, but this is challenging due to the complexity of the cellular environment.
Four different FRET-based protein sensor platforms, Zif, Zap, eCALWY, and eZinCh, have been applied using the above procedure to measure the concentration of Zn2+ in the cytosol of a variety of cell types. Interestingly, these sensors have very different modes of Zn2+ sensing. The ZifCY and ZapCY sensors, developed by the Palmer Lab, connect two FPs, truncated ECFP and Citrine, through zinc finger zinc-binding domains (ZBDs) that are unstructured in the unbound state with a sensor architecture of: donor FP – ZBD – acceptor FP [25–27]. Coordination of Zn2+ to the zinc-binding domain structures the linker between the two FPs leading to an increase in FRET signal. Mutation of the zinc binding domain of the ZapCY proteins led to the development two sensors with different apparent zinc disassociation constants at pH 7.4: ZapCY1, KD’ = 2.5 pM, and ZapCY2, KD’ = 811 pM [26]. The FPs have also been exchanged in the ZapCY platform to give a range of colors of sensors [28]. The Merkx Lab has developed two sensor platforms, the eCALWY and eZinCh sensors. In the eCALWY sensors, the FPs were engineered by making two mutations in the FPs (S208F and V224L) creating intermolecular contacts between the two FPs [29]. Two copper binding proteins, Atox1 and WD4 were modified to decrease binding of copper and linked between the two FPs, yielding a Zn2+-specific sensor with the following sensor architecture: donor FP – Atox1 – linker – WD4 – acceptor FP. Coordination of Zn2+ by the two copper binding domains breaks the contacts between the FPs lowering the FRET signal. In the final platform, eZinCh, the FPs have been engineered to create a Zn2+ binding site between the two FPs using two mutations (A206H and S208C) in each FP. These mutated FP are linked by an unstructured, flexible linker with the overall architecture: donor FP – linker – acceptor FP [30]. When Zn2+ is coordinated to the sensor there is an increase in the FRET ratio. Members of both the eCALWY and eZinCh platforms have been developed to bind zinc with a variety of binding constants and have a variety of colors of FPs [14, 30–32]. Although these platforms have very different modes of sensing, they converge on a labile Zn2+ concentration in the hundreds of picomolar in the cytosol of many cell types.
There are a few concerns that should be addressed in order to ensure the sensors are functioning well: the sensor should be localized to the area of the cell of interest, the concentration of the sensor should be such that it does not perturb the labile Zn2+ pool, the apparent disassociation constant of the sensor for Zn2+ should be close to the concentration of labile Zn2+ in the cellular compartment of interest, and the binding modality should be such that the sensor doesn’t complex Zn2+ bound to proteins [6, 33]. A few key experiments suggest that genetically encoded FRET-based protein sensors are measuring the native labile Zn2+ pool in the cytosol. The lessons learned from measuring Zn2+ concentrations in the cytosol may help to inform protocols for measuring Zn2+ in other organelles.
Qin and coworkers critically compared FluoZin-3 AM and ZapCY2 to demonstrate that ZapCY2 was measuring the labile Zn2+ pool [34]. First, the localization of the sensors was compared. By incorporating a nuclear exclusion signaling sequence, ZapCY2 can be localized only to the cytosol and not to other organelles. In comparison, the small molecule probe, FluoZin-3 AM, co-localizes with a Golgi marker as well as localizing in the cytosol. To ensure that the concentration of ZapCY2 was not perturbing the Zn2+ pool, the concentration of Zn2+ measured by both ZapCY2 and FluoZin-3 AM was measured as a function of sensor concentration. While raising the extracellular concentration of FluoZin-3 AM from 0–10 µM appeared to deplete the Zn2+ pool, the fractional saturation of ZapCY2 remained constant across a range of intracellular sensor concentrations from ~0–80 µM demonstrating that ZapCY2 does not detectably deplete the Zn2+ pool. Depletion of the Zn2+ pool by FluoZin-3 AM may be caused by the high concentration of the probe that accumulates inside the cell as hydrolysis of AM-ester probes traps the probes intracellularly. By this process, the intracellular concentration of these probes reaches hundreds of micromolar which may allow the probes to interfere with the homeostasis of the hundreds of picomolar concentrations of labile Zn2+ [35]. In contrast to the dye that is added acutely to cells and measured a few hours later, the long-term expression of the genetically-encoded sensors over the course of days at a relatively low concentration (~10 µM) may allow these sensors to become part of the cellular Zn2+ buffer and therefore non-perturbing to the labile Zn2+ pool.
Another important concern to address when using sensors to quantify Zn2+ is whether the sensor is truly accessing the labile pool. The Zn2+ coordination environment is flexible in both geometry and number of ligands [5]. Consequently, probes have the potential to form ternary complexes by binding to Zn2+ that is already coordinated to a protein. The propensity of ternary complex formation is likely much lower for FRET-based sensors than small molecular probes, due to the steric bulk of FPs and protein-based zinc binding domains compared to small chelates, however this has yet to be tested. Sensors with low KD values or present at high concentrations could also shift the native equilibrium of Zn2+ thereby perturbing the labile pool. In either scenario the sensor would access Zn2+ from outside of the labile pool. If this were the case then sensors of various affinities would be able to access different pools of Zn2+ and measurement of Zn2+ with sensors possessing different affinities for Zn2+ would give rise to different results for the amount of Zn2+. Vinkenborg and co-workers tested this hypothesis by applying a panel of eCALWY variants to the cytosol of INS-1 cells with a range of apparent Zn2+ disassociation constants (KD’) from 2 pM to 2.9 nM at pH 7.4 [29]. A plot of the fractional saturation of each variant against the disassociation constants of the variants gives a sigmoidal shape and a consistent cytosolic concentration of ~ 400 pM. Similar experiments were carried out by Qin et al in HeLa cells, yielding a cytosolic concentration of ~ 180 pM [34]. These experiments provide evidence that both sensor platforms are accessing the same pool of labile Zn2+ regardless of the affinity of the sensor.
These kinds of experiments strengthen the measurements collected through the application of sensors. As more sensors are developed and more measurements are made in a variety of cell types and in many organelles it is important to think critically about the various aspects of the sensors that could be affecting the measurements. Next we will examine measurements of Zn2+ concentrations using FRET-based sensors in the endoplasmic reticulum (ER), Golgi apparatus, and mitochondria where measurement of Zn2+ with different classes of probes has been less consistent. The variability in these measurements points to a need for critical examination of the fundamental chemistry of these tools, and to an interesting area of zinc biology as the distribution and dynamics of Zn2+ in the organelles continues to be studied.
There are a number of challenges associated with measuring Zn2+ in organelles, including ensuring that the sensor is properly localized, measuring Zn2+ in a small crowded environment with an uncertain Zn2+ buffering capacity, and measuring Zn2+ in different chemical environments (pH, redox state, etc). Table 1 summarizes the estimates of organelle Zn2+ obtained using three different sensor platforms and reveals the variability in estimates of Zn2+ in organelles. ZapCY1 and ZifCY1 were the first FRET-based genetically encoded sensors to be targeted to the ER and the Golgi in HeLa cells. Colocalization with canonical markers of the ER and Golgi was used to demonstrate localization to the targeted organelle [26]. Treatment of the cells with TPEN resulted in a small decrease in the high affinity sensor, ZapCY1 (KD’= 2.5 pM, pH 7.4), and no response in the low affinity sensor, ZifCY1 (KD’= 1 µM, pH 7.4), suggesting that Zn2+ concentration in the organelles was lower than the detection limit of ZifCY1. In situ calibrations suggested that ZapCY1 (dynamic range in the ER = ~2) was 26% saturated in the ER and 18% saturated in the Golgi. Using these data and the in viro KD’, the concentrations of labile Zn2+ in the ER and the Golgi were estimated to be 0.9 ± 0.1 pM and 0.6 ± 0.1 pM, respectively. When ZapCY1 and ZifCY1 were targeted to the mitochondria each sensor reported a very different concentration of labile Zn2+ in the organelle [27]. Because of the very low dynamic range of ZifCY1 in the mitochondria, this sensor was re-engineered by replacing the YFP with circularly-permuted Venus to increase the dynamic range. The circular permutation of Venus changes the orientation of the fluorophore with respect to the CFP, altering the FRET efficiency between the two FPs. This resulted in the development of a high dynamic range (DR=2.5), but lower affinity sensor, mito-ZifCV1.173. Importantly, Zif sensors that incorporated other versions of circularly-permuted Venus had very low dynamic ranges and appear more saturated with Zn2+ in comparison to the high dynamic range sensors (~40% vs ~10%) although all the sensors had the same Zif1 binding domain and probably similar affinities for Zn2+. This observation highlights the need for high dynamic range sensors (above ~1.2 based on these data) for accurate measurement of Zn2+ concentration. Because the environment in the mitochondrial matrix is crowded and has a variable pH, the KD’ of ZapCY1 was determined in situ to be 1.6 pM. This value matched the in vitro KD measured at the same pH. ZapCY1 was saturated 16 ± 10 % in the mitochondria (dynamic range = ~2), and, using these data and the in situ KD’ for the sensor, the concentration of Zn2+ in the mitochondria of HeLa cells was estimated to be about 0.14 pM. Although the range of sensor concentration was limited in the organelles, perturbation the labile Zn2+ concentration at increasing levels of sensor expression was not detected [26, 27].
Table 1
Summary of measurements of labile Zn2+ made in organelles with protein based sensors.
| Sensor | Dynamic Range in Organelle | Percent Saturation | in vitro KD' (pM) | pH of KD' measurement | Estimated Zn2+ concentration (pM) |
|---|---|---|---|---|---|
| ER-CALWY-4 [29, 36] | 1.25 – 1.4 * | > 80% * | 630 | 7.1 | 1600 – >5000 |
| ER-CALWY-4 [29, 30] | 1.4 * | 33% | 630 | 7.1 | 390 ± 170 |
| ER-CALWY-6 [29, 36] | 1.1 * | 0 – 50 % | 2900 | 7.1 | 5600 – 7200 |
| ER-ZinCh-2 [30] | 1.4 * | ~ 55 % | 1000 ± 100 | 7.1 | 800 ± 600 |
| ER-ZapCY1 [26] | 2.16 ± 0.06 | 26% | 2.5 | 7.4 | 0.9 ± 0.1 |
| Golgi-ZapCY1 [26] | 2.09 ± 0.07 | 18% | 2.5 | 7.4 | 0.6 ± 0.1 |
| mito-CALWY-4 [36] | 1.2 – 1.55 * | ~ 80 % | 60 | 7.8 | 180 – 300 |
| mito-CALWY-4 [30, 36] | 1.6 * | ~ 66 % | 60 | 7.8 | 42 ± 28 |
| mito-ZinCh-2 [30] | 1.9 * | 23 ± 6 % | 10 | 7.8 | 3.3 ± 1.2 |
| mito-ZapCY1 [27] | 3.22 ± 0.57 | 8.7 ± 5.8 % | 1.6 | 8 (in situ) | 0.14 |
| mito-CA based sensor [19] | 1.66 * | 0.15 ± 0.05 | in isolated mitochondria | 0.15 | |
Starred (*) values were estimated by the authors from published data. Note the KD values are those reported in the relevant references.
The Merkx Lab has also applied their sensors to the mitochondria and ER. Three different sensors were targeted to the ER of a variety of cell lines (eCALWY-4, KD’ = 630 pM; eCALWY-6, KD’ = 2.9 nM; eZinCh-2, KD’ = 1.0 ± 0.1 nM) [30, 36]. The dynamic range of the sensors in the ER varied from 1.1 to 1.4, depending on the sensor and the cell type, yielding a range of Zn2+ concentrations from 800 pM to 7.2 nM. eZinCh-2 and eCALWY-4 were both used to measure the labile Zn2+ concentrations in the mitochondria of HeLa cells where the dynamic range in mitochondria ranged from 1.2 to 1.9 and estimations of Zn2+, determined using the in vitro KD' values, ranged from 3.3 ± 1.2 pM applying eZinCh-2 to 180 ± 300 pM applying e-CALWY-4 [30].
In summary, while it is easy to point to the variability in Zn2+ estimates obtained using genetically encoded sensors and find fault with the FRET-based protein sensors [4], a better understanding the details of why the variability occurs will lead to the development of more robust tools that can be targeted to organelles to uncover the details of Zn2+ biology.
Re-examination of data collected using small molecule probes
Over the last 30 years small molecule probes have been engineered for and applied in a variety of contexts to study Zn2+ biology in cells and tissues. Over this period, progress has been made in designing probes to gain enhanced photophysical properties, to tune the affinities of probes for Zn2+, to be ratiometric, to be reaction-based, and to target different organelles. These improvements have led to the application of probes to complex biological questions and led to important new knowledge about Zn2+ homeostasis and signaling [6]. However, recent work has led to re-examination of data collected using small molecule probes that emphasizes the need to better understand the chemistry of these probes and to develop clear controls for verifying the reactivity and properties of probes in cells. Studies carried out by the Petering Lab have pointed out the subtleties of the interaction of small molecule probes, Newport Green, TSQ, and Zinquin with Zn2+ in the cell [37, 38]. Briefly, it was always assumed that these sensors substituted for weakly bound ligands in cells, coordinating labile Zn2+, and increasing in fluorescence intensity. However, examination of the dyes under a variety of conditions shows that these probes have multiple interactions with both labile and bound Zn2+ that cause detectable fluorescence increases. Although papers have claimed to use Newport Green to image native Zn2+ dynamics, the dye has a micromolar affinity for Zn2+, and it is therefore difficult to find an application where it detects changes in the labile Zn2+ pool, given that estimates of cytosolic Zn2+ are in the hundreds of picomolar range. TSQ and Zinquin were both found to form fluorescent ternary sensor-Zn-protein adducts, meaning that the probes access and report Zn2+ that is tightly bound to proteins in addition to labile Zn2+. This observation is interesting in light of recent work done by the Merkx Lab that re-examined data that was collected in fixed cells using Zinquin with live cells and genetically encoded FRET sensors. Work with Zinquin suggested that Zn2+ was mobilized from ER stores by activation of the Zn2+ transporter Zip7 in the presence of EGF and ionomycin in Tamoxifen-resistant MCF-7 cells. Through application of eCALWY-4 and eZinCh-2 in the cytosol and ER, no such dynamics of Zn2+ were seen [31]. The application of Zn2+ sensors allows for the start of interrogation of the biology of Zn2+, but studies like these are excellent reminders that other imaging techniques can lend increased rigor to the data obtained with sensors.
Total elemental imaging and mapping
Numerous analytical techniques are available for visual mapping of elemental distribution in biological samples. Here we will discuss several approaches that can be divided into two classes: mass spectrometry-based and X-ray fluorescence-based imaging techniques. A schematic of the differences between these techniques is outlined in Figure 3. Due to space limitations, this review will cover only a subset of the various methods in these categories, focusing on the most widely used techniques reported in the literature. We will describe the basic properties of each imaging technique and provide a few examples of how they have been applied to the field of Zn2+ biology. An important distinguishing feature of these techniques is that they provide a measure of the total amount of a metal of interest, as opposed to just the labile or accessible metal pool, and most techniques permit measurement of multiple elements at once, allowing for correlation between metals and other abundant biological elements, such as phosphorus. These techniques are traditionally applied to fixed samples of cells, or biological specimens such as plants or seeds at a fixed time point. While these techniques cannot be applied to live cell imaging, they can offer complementary elemental snapshots at multiple time points.
Schematic of mass-spectrometry and X-ray fluorescence-based imaging techniques. A) Mass-spectrometry-based imaging techniques rely on point-by-point sample ablation using a laser or primary ion beam to dispel charged particles or secondary ions that are subsequently analyzed by mass-spectrometry. Ions are separated by their mass-to-charge ratio. B) X-ray fluorescence techniques rely on the photoelectric effect; when an incident beam (X-ray or e−) bombards a sample, electrons are ejected from inner shells and vacancies are filled by outer-shell electrons. In this process, X-rays are released and detected at characteristic energies for each element.
Mass-spectrometry techniques
Mass-spectrometry based techniques involve identification of specific chemicals within a sample by separation of ions according to their mass-to-charge ratio. Early mass-spectrometry applications did not provide spatial resolution, but newer approaches often contain mapping capabilities, thus enabling researchers to define the spatial distribution of metal ions. Mass-spectrometry-based spatial imaging allows for a point by point mass-spectrum distribution map generated by ablation of a sample at thousands of spots [13]. The different types of massspectrometry techniques can be distinguished by their method of ionization. Here we will discuss laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and secondary ion mass spectrometry (SIMS), the major two mass-spectrometry imaging techniques that are used to study the localization of metals in biological samples.
LA-ICP-MS
LA-ICP-MS has been widely applied in the life sciences for analyzing biological samples for metal localization in medical studies, such as tumor growth analysis, and biochemistry of animal model diseases [13, 39]. This method uses high-energy focused lasers to ablate particles from a solid sample; the particles are then carried by gas (Ar or He) into an ICP-MS platform where ionization occurs and charged ions are detected by their mass-to-charge ratio. Although LA-ICP-MS has a relatively low spatial resolution compared to other imaging techniques (1 µm limit), it offers very high sensitivity (ng/g) and multi-elemental imaging capability [11, 40, 41]. Particularly unique to both LA-ICP-MS and SIMS (discussed below) is the ability to detect multiple isotopes of individual elements, allowing for snapshots of dynamic trace element transport and distribution [40].
One notable application of this technology was the use of laser ablation coupled to multi-collector-ICP-MS (LA-MC-ICP-MS) to measure Zn2+ isotope incorporation in rat brain thin sections in an effort to identify the rate of uptake into different regions of the brain [42]. This is particularly important because the mechanisms of Zn2+ acquisition and distribution at the tissue and organismal level are not well understood. Further, alterations in brain Zn2+ homeostasis have been linked to the development of neurological diseases and thus it is critical to understand Zn2+ metabolism in the brain [43]. In this study, animals were injected with solutions enriched in two stable isotopes of Zn2+, 67Zn and 70Zn, at two time points compared to control animals injected with either saline only or a Zn2+ solution of natural isotopic composition [44]. Isotope ratio maps revealed a heterogeneous distribution of Zn2+ isotope ratios in different physiological features of brain tissue (hippocampus, amygdala, cortex, and hypothalamus) suggesting different turnover kinetics in specific regions of the brain. In another study using LA-ICP-MS, researchers analyzed brain tissue sections and found that the 31P/66Zn ratio decreased beyond tumor boundaries, demonstrating that changes in this ratio can be used to identify healthy vs. cancerous tissue [45]. Similarly, a study of liver sections demonstrated that metals can be used as disease biomarkers; Fe and Cu levels are higher in diseased liver tissue, while Zn2+ levels are decreased, and these alterations can be used to predict changes from healthy to diseased liver status [46]. Further examples demonstrating the application of LA-ICP-MS include cerebral imaging metals including Zn2+ in the pathophysiology in the mouse models of Parkinson’s disease and Alzheimer’s disease [47–49].
SIMS
Another analytical mass-spectrometry technique used for imaging elements and their isotopes is SIMS. SIMS uses a focused primary ion beam to dispel particles from a sample surface. The ions that are ejected after this surface bombardment are called secondary ions, which are subsequently analyzed in a mass spectrometer. The primary strength of SIMS is that it can offer excellent spatial resolution, particularly for NanoSIMS, which has a spatial resolution of 50 nm. In general, the width and energy of the beam dictates the achievable resolution; a wide, high intensity beam allows for depth profiling, while a narrower, low intensity beam may only erode the surface, making quantification difficult [11]. Further limitations include: the technique does not provide information about chemical state [11] and SIMS analysis requires samples to be in a vacuum, and hence samples must either be cryo-fixed or must be fixed at low temperatures, dehydrated, and resin-embedded [10]. For such preparations, care must be taken to ensure elemental distribution is not altered as a result of sample preparation. Although SIMS can be used to detect most elements in the periodic table and their different isotopes, a few elements such as Zn2+, Cd, and Mn have poor secondary ion yield and are therefore difficult to detect at standard concentrations (Zhao, 2014, Moore 2012). SIMS has, however been used to successfully determine Zn2+ localization in Poplar leaves from plants grown on Zn2+-contaminated soil [50], suggesting that although SIMS may not be not ideal for detecting low levels of Zn2+, it may be appropriate to study biological samples with elevated Zn2+ levels. Further, in studies where another approach is applied to study Zn2+ localization, SIMS may offer the ability to study localization of other lighter metals of interest such as P and Si that cannot be examined as effectively by other approaches, such as X-ray fluorescence (discussed in detail in the next section of this review) [51]. In cases where X-ray fluorescence and SIMS are used in combination, adjacent thin sections can be analyzed.
X-ray fluorescence techniques
In addition to mass-spectrometry techniques, the use of X-ray fluorescence techniques provide a complementary approach for mapping metal distribution in biological specimens, and offer the additional advantage of obtaining information on multiple elements and chemical speciation of elements of interest. X-ray fluorescence methods rely on the photoelectric effect; when an atom is exposed to high-energy radiation, electrons are ejected from inner shells, leading to core vacancies, which are filled by outer-shell electrons. As outer shell electrons decay, energy is released in the form of fluorescence. Because the energy of the emitted photons is characteristic for each element, and the intensity is proportional to concentration, X-ray fluorescence provides information on the identity and amount of a given element in a sample. Samples can be raster scanned line by line through a focused incident X-ray beam to yield a high resolution, high sensitivity elemental map [10, 11]. X-ray fluorescence techniques are categorized according to the type of incident radiation. Here we will discuss synchrotron X-ray fluorescence (SXRF), which uses X-rays, and electron-probe energy-dispersive spectroscopy (EDS), which uses electrons and is often combined with a scanning electron microscope (SEM). Other techniques beyond the scope of this review include micro-particle-induced X-ray emission (μPIXE), which uses charged atoms such as protons.
Synchrotron X-ray fluorescence
SXRF possesses a number of advantages for mapping metals and other elements in biological samples. Synchrotron facilities have high-intensity photon sources that are more than ten times brighter than those of conventional X-ray tubes [10], thus giving rise to high sensitivity and submicron spatial resolution [13, 52]. As opposed to mass spectrometry-based techniques, SXRF is generally non-destructive and does not require vacuum. Further, the sensitivity of SXRF increases with increasing atomic number, making it well-suited to study trace elements and heavy metals/metalloids, including Zn2+.
A recent application of SXRF fluorescence demonstrates the strengths of this approach for mapping metals and other elements with sub-cellular resolution, where the location and redistribution of metals was monitored during mitosis of mammalian cells. Briefly, washed and dried NIH 3T3 cells attached to Si-nitride windows were analyzed by SXRF and researchers found that Zn2+, Cu, and S remained colocalized throughout mitotic states and that Zn2+ increased during mitosis compared to interphase, suggesting a role for Zn2+ in the cell cycle [53]. Because SXRF imaging has the potential to study metal distribution at the subcellular level in studies such as these, important questions are raised about the significance of variable sub-cellular metal accumulation and speciation. To confirm subcellular localization, SXRF may be performed in combination with organelle localization using fluorescent markers. For a recent review on the application of this concept, see Roudeau et al., 2014 [54].
X-ray absorption spectroscopy
In addition to mapping elemental localization by SXRF, X-ray absorption spectroscopy (XAS) is another synchrotron-based technique that can be used to analyze the chemical speciation of individual elements. This capability is unique to synchrotron-based approaches and is not offered by the previously discussed mass-spectrometry techniques. An XAS analysis involves progressively increasing the incident X-ray beam energy at a specific point in a sample and collecting X-ray fluorescence at each individual energy. The XAS spectrum includes the X-ray absorption near-edge structure (XANES) and the extended X-ray absorption fine structure (EXAFS). XANES covers the energy range from about −50 to +200 eV of the absorption edge and is sensitive to the oxidation states of elements, while EXAFS covers energies from the absorption edge to approximately +800eV and is well-suited to elucidate coordination chemistry, including the identity and number of coordinating atoms and their interatomic distance [10]. Depending on the researcher’s specific needs, these techniques can be applied to several regions of interests in samples using a micro-focused beam or alternatively applied to bulk tissues using a larger mm beam. It is important to note that in order to properly identify speciation within a sample, an XAS spectrum must be compared to known standards and thus researchers may be limited by the availability of appropriate standards.
Synchrotron beam-lines generally allow for collection of both SXRF mapping and XANES or EXAFS allowing researchers to map metal distribution and perform more detailed analysis of the chemical speciation in specific regions of the sample. For example, SXRF analysis of frozen murine macrophage cells identified Zn2+ hotspots, or putative Zn2+ vesicles, called “zincosomes” [55]. These spots were then analyzed by XAS to elucidate chemical speciation information of the zincosomal Zn2+. XANES and EXAFS revealed that Zn2+ was bound to one sulfur atom at a distance of 2.28 Å and two and a half histidine atoms and one oxygen atom at 1.97 Å. For a few recent reviews on the application of X-ray absorption spectroscopy for metal speciation, refer to Gräfe et al. 2014, West et al., 2015, and Zhao, et al. 2014 [10, 56, 57].
EDS
Although Synchrotron-based X-ray fluorescence techniques are powerful, access to Synchrotrons can limit the widespread availability of these techniques. An additional approach for elemental mapping by X-ray fluorescence is electron-probe energy-dispersive spectroscopy (EDS), also called electron probe microanalysis (EPMA), which uses an electron beam to bombard a sample. As with Synchrotron-induced fluorescence, the X-rays emitted following bombardment of a sample with an electron-beam, are characteristic for individual elements and the intensity is proportional to the amount of the element in the sample. EDS is usually used in combination with a scanning electron microscope (SEM), a transmission electron microscope (TEM), or a scanning transmission electron microscope (STEM) to allow for analysis of additional morphological features of the sample. Simultaneous high resolution imaging of subcellular structures in combination with elemental analysis within these structures is particularly useful for studying composition of whole cell samples that are a few micrometers thick [13]. Recently, this technique has been applied by fitting a cryo-compatible STEM with an EDS detector. Researchers demonstrated the ability to spatially map K, Fe, and Zn2+ in red blood cells with STEM-EDS [58]. They found an even distribution of these metals throughout RBCs and because of the combination with STEM, they were also able to study these metal distributions in the context of the flexible RBC morphology. We will discuss an additional example of STEM-EDS in the section entitled “combinatorial imaging approach”.
Perspectives
Each technique highlighted above offers a different degree of sensitivity, specificity, and spatial resolution; the advantages and limitations of each should be carefully considered for individual experimental design. Besides the fundamental pros and cons of each approach, access to equipment may present additional limitations for researchers. For example, a STEM-EDS system may not be available at a researcher’s home university. Further, many of the X-ray fluorescence techniques, such as SXRF, require access to specialized synchrotron facilities. Access to these facilities usually necessitates a prior arrangement of beam time through award of successful written proposal submitted well in advance. Even with awarded time, researchers are allotted a limited period of time and each scan may take upwards of several hours. Researchers may have to compromise resolution of their elemental maps to decrease scanning time in order to study more samples. Because of issues such as these, studies using these techniques are often limited in sample size/biological replicates. Thus, these approaches are better suited for targeted hypothesis-driven experiments, rather than discovery-based screens or high-throughput studies.
Quantitative analysis of zinc distribution using complementary approaches
As emphasized above, different tools permit measurement of different metal pools; small molecule probes and sensors can be used to quantify the subcellular distribution of labile Zn2+, whereas mass-spectrometry and X-ray fluorescence-based techniques can be used to map the distribution of total Zn2+. Each analytical tool offers a unique piece of information such that combination of multiple techniques provides a powerful means to generate a comprehensive picture of Zn2+ distribution.
An elegant example of this combinatorial approach is illustrated by the recent study of Zn2+ fluxes involved in the fertilization mammalian egg. Previous studies found that labile Zn2+ is released from eggs during a meiotic checkpoint in what is referred to as a “Zn2+ spark” [59]. To elucidate the molecular mechanism of these Zn2+ sparks, researchers used a combination of four approaches to resolve zinc distribution and measure Zn2+ concentrations in single cells before and after sparks. Using a synthetic fluorescent Zn2+ probe and an extracellular zinc dye for live-cell imaging at different stages of egg development, researchers that Zn2+-rich compartments are the source of Zn2+ that is exocytosed during Zn2+ sparks. Further, elemental composition of meiosis II (MII) eggs was analyzed at an ultrastructural level with a STEM microscope equipped with EDS detectors, allowing for anatomical and elemental imaging. Thin sections of fixed, resin-embedded MII eggs were analyzed with STEM-EDS. Bright, vesicularlike structures near the ooplasmic membrane were found in Z-contrast STEM images, indicating elements of higher molecular weight. The EDS spectra measured in these regions identified by STEM compared to nearby cytoplasmic regions revealed an increased Zn2+ intensity in the vesicular-like bodies. Authors point out that STEM-EDS is not very quantitative, so they further turned to high-resolution SRXF to confirm their results by mapping thin sections of the MII eggs. In these experiments, they also saw Zn2+-enriched compartments, consistent with both the live-cell imaging and the STEM-EDS analysis. Finally, quantification by highly sensitive SRXF allowed for determination of total Zn2+ concentration in the vesicular stores. Application of four complementary technique allowed the authors to conclude that Zn2+-containing vesicles near the membrane are the source of Zn2+-sparks, and the amount of Zn2+ within these vesicles is consistent with the amount of Zn2+ released.
Conclusions
We have discussed the methods and considerations for imaging labile and total Zn2+ using various tools and approaches. As others have pointed out, there is room for improved communication between the chemists who develop tools such as FRET sensors, experts of particular imaging techniques (i.e. synchrotron beamline scientists), and the biologists who apply them [11, 60]. With the extensive and ongoing development of a vast array of chemical and analytical tools available to study labile and total Zn2+ pools, the scientific community is now well-poised to answer fundamental questions about Zn2+ biology. Understanding the dynamics and distribution of metals is essential for deciphering how particular distribution patterns are set up, which genes control these processes, how they differ between cell-types, and how they are altered in healthy vs. diseased states. To aid in answering these questions, Zn2+ pools can be depleted or elevated, manipulated by pharmacology, or altered by genetics (i.e. knockdowns or overexpression of particular Zn2+ transport/homeostatic genes). By combining these biologically relevant perturbations with the chemical and analytical analysis methods, we will continue to elucidate fundamental aspects of Zn2+ biology.
Acknowledgments
We would like to acknowledge financial support from NIH GM084027 and DP1-GM114863 (to A.E.P.).
Footnotes
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.



