A Comprehensive Overview of Zinc Ion Fluorescent Probes: From TSQ / Zinquin to ZnAF / Zinpyr-1 – Mechanistic Comparison and Aladdin Selection Guide

Introduction

Zinc (Zn) is the second most abundant transition metal in the human body. As a catalytic, structural, and regulatory ion, it participates in a wide range of biological processes, including immune responses, oxidative stress, apoptosis, aging, and neuronal signal transduction. Intracellular “free/exchangeable” Zn²⁺ is recognized as an important signaling species, and disruption of its homeostasis is closely linked to neurological disorders such as Alzheimer’s disease, Parkinson’s disease, and epilepsy.

Fluorescence imaging provides a highly sensitive, spatially resolved approach for detecting Zn²⁺ in live cells and tissues. In this article, we systematically review several widely used classes of zinc ion fluorescent probes—TSQ / Zinquin, the ZnAF series, and Zinpyr-1. We compare them in terms of structural principles, spectral properties, cell permeability, and application scenarios, and, in combination with the current Aladdin product portfolio, propose a practical guide for probe selection.

Design Concepts and Classification Dimensions of Zinc Ion Fluorescent Probes

A typical Zn²⁺ fluorescent probe consists of two components:

1. Fluorophore: e.g., quinoline, fluorescein, or difluorofluorescein, which determines the excitation/emission wavelengths, quantum yield, and photostability.
2. Chelating recognition unit (chelator): commonly DPA (di(2-picolyl)amine), TPEN-type structures, etc., which form complexes with Zn²⁺ and modulate the fluorescence signal via PET (photoinduced electron transfer) or ICT (intramolecular charge transfer) mechanisms.

From a practical standpoint, the following dimensions are key when selecting a Zn²⁺ probe:

1.Emission color / spectrum: blue (quinoline-based) vs. green (fluorescein-based), and whether it is compatible with existing channels and other dyes.

2.Cell permeability:

• Free acid / unprotected forms: typically cell-impermeant, suitable for extracellular or solution-based assays;
• DA / AM / ester forms: typically cell-permeant, suitable for loading into live cells.

3.Affinity (Kd) and dynamic range: high affinity in the nM range vs. moderate affinity in the hundreds of nM range, which determines whether the probe is better suited for detecting “trace zinc” or “zinc-rich pools.”

4.Application scenarios: live cells, brain slices, plant roots, serum/buffer solutions, or specific studies such as apoptosis and neuronal organelle targeting.

Quinoline-Based Blue-Emitting Probes: TSQ and the Zinquin Family

2.1 TSQ: The Classic Membrane-Permeable Zinc Probe

TSQ (N-(6-methoxy-8-quinolyl)-4-methylbenzenesulfonamide, CAS 109628-27-5) is one of the earliest and most widely used fluorescent probes for zinc ions:

• Structure: quinoline ring + p-toluenesulfonamide.
• Membrane permeability: readily crosses cell membranes and enters cells and tissues.
• Fluorescence response: upon complexation with Zn²⁺, it shows strong blue-green fluorescence with excitation at 334 nm and emission at 495 nm.
• Selectivity: high selectivity for Zn²⁺; maintains a robust fluorescence response even in the presence of Ca²⁺ and Mg²⁺, making it particularly suitable for labeling zinc-rich regions in neural tissue (e.g., mossy fibers in the hippocampus).

Advantages

• A classic probe with extensive literature, especially in the neuroscience field.
• Blue emission, which facilitates combination with green/red fluorescent proteins.

Limitations

• Requires UV/near-UV excitation, imposing somewhat higher demands on both samples and microscope hardware.
• Coordination behavior is relatively complex (e.g., possible formation of Zn(TSQ)₂ and other complexes), so more extensive calibration is needed for rigorous quantitative applications.

Typical applications:

Neural tissue sections, zinc-rich neurons, and classic staining of zinc protein distributions.

2.2 Zinquin: An Upgraded TSQ Analogue

Zinquin and its derivatives can be regarded as “second-generation” TSQ analogues, optimized in terms of spectral properties and cell permeability.

(1) Zinquin ethyl ester – Cell-permeable ethyl ester

• CAS: 181530-09-6; quinoline scaffold with an ethyl ester protecting group.
• After entering cells, the ester is cleaved by intracellular esterases to generate the Zinquin free acid, which becomes negatively charged and is retained intracellularly, preventing efflux.
• Forms 1:1 or 2:1 complexes with Zn²⁺, with Kd values of approximately 370 nM and 850 nM, respectively, suitable for detecting medium to high concentrations of labile Zn²⁺.
• Spectral properties: Ex ≈ 368 nm, Em ≈ 490 nm; blue emission compatible with DAPI/UV excitation channels.

(2) Zinquin AM – AM ester with high loading efficiency

• AM (acetoxymethyl) ester form, more soluble in organic solvents and suitable for preparing high-concentration stock solutions.
• Typical spectral parameters: Ex ~ 355–360 nm, Em ~ 491 nm.
• Typical loading conditions: 5–40 μM, 37 °C incubation for 15–30 min; commonly used in confocal imaging and flow cytometry.

Advantages

• Compared with TSQ, the Zinquin series offers improved intracellular localization and retention.
• Multiple protected forms (ethyl ester, AM ester, free acid) provide flexibility to combine “intracellular vs. extracellular” applications as needed.

Limitations

• Still requires UV/near-UV excitation.
• Moderate affinity; well suited for studying “zinc redistribution” or “zinc mobilization” (e.g., Zn²⁺ release during apoptosis), but not necessarily ideal for detecting extremely low basal Zn²⁺ levels.

Typical applications:

Zinc mobilization associated with apoptosis, subcellular zinc distribution in neurons, and multicolor imaging schemes where Zn²⁺ is assigned to the blue channel.

Note: In cells, TSQ/Zinquin primarily report on the functional pool of exchangeable Zn²⁺ and Zn-binding proteins, rather than the absolute concentration of “free Zn²⁺” in a strict sense. This distinction should be carefully considered when interpreting results and designing quantitative experiments.

3. Fluorescein-Based Green-Emitting Probes: ZnAF-2 / ZnAF-2F and Zinpyr-1

Fluorescein-based Zn²⁺ probes are characterized by visible-light excitation, high sensitivity, and PET-based turn-on mechanisms, making them highly suitable for modern confocal microscopy, flow cytometry, and high-content imaging platforms.

3.1 ZnAF-2 / ZnAF-2 DA: High-Affinity Cell-Impermeant / Cell-Permeant Pair

According to the design by Nagano and co-workers, ZnAF-2 links a DPA/TPEN-related 2-pyridylmethylamine chelating motif to a fluorescein scaffold:

1. ZnAF-2 (Cell Impermeant)

(a) Exists in the free acid form and crosses membranes poorly, making it more suitable for:

• Extracellular Zn²⁺ and receptor-facing Zn²⁺;
• Quantitative detection of Zn²⁺ in purified protein/buffer systems.

(b) Kd ≈ 2.7 nM; fluorescence increases by ~50-fold upon 1:1 complexation with Zn²⁺; Ex/Em ≈ 492/515 nm.

2. ZnAF-2 DA (Cell Permeant)

(a) Diacetate (DA) ester form, freely membrane-permeable.

(b) After entering live cells or brain slices, it is hydrolyzed by intracellular esterases to ZnAF-2, which is then trapped inside the cell.

(c) Ex/Em are likewise 492/515 nm, with green emission and low background.

Applications:

1. ZnAF-2: In vitro enzyme assays, Zn²⁺ bound to proteins, and dynamics of extracellular Zn²⁺.
2. ZnAF-2 DA: High-sensitivity imaging of Zn²⁺ in live cells, brain slices, and tissue sections.

3.2 ZnAF-2F / ZnAF-2F DA: Difluorofluorescein-Based Upgrades

Building on ZnAF-2, the ZnAF-2F series introduces a difluorofluorescein core to improve photostability and fluorescence efficiency.

1. ZnAF-2F (Cell Impermeant)

(a) CAS 443302-09-8; Ex/Em ≈ 492/515 nm.

(b) At pH 7.4, the basal quantum yield is very low (~0.006), while binding to Zn²⁺ can enhance fluorescence by up to ~60-fold; Kd is in the nM range.

2. ZnAF-2F DA (Cell Permeant)

(a) DA ester form with good membrane permeability; once inside the cell, it is hydrolyzed to ZnAF-2F and retained intracellularly.

(b) Well suited for monitoring changes in labile Zn²⁺ in neurons and hippocampal slices.

Compared with ZnAF-2, the ZnAF-2F series offers superior signal-to-noise ratio, lower background fluorescence, and improved pH stability, making it particularly suitable for demanding in vivo imaging and quantitative studies.

3.3 Zinpyr-1: A High-Affinity, Golgi-Preferential Zn²⁺ Probe

Zinpyr-1 (ZP-1) is another important fluorescein-based Zn²⁺ probe:

• Structure: dichlorofluorescein core with two DPA chelating arms.
• Affinity: Kd ≈ 0.7 ± 0.1 nM, higher affinity than the ZnAF-2 series and suitable for detecting lower levels of labile Zn²⁺.
• As Zn²⁺ concentration increases, the excitation maximum blue-shifts from ~515 nm to ~507 nm; emission is typically collected using a 513–558 nm bandpass filter (standard FITC channel).
• In live cells, it primarily stains the Golgi apparatus and Golgi-related vesicles, enabling studies of intracellular Zn²⁺ storage and trafficking.

Applications:

• Mapping the fine spatial distribution of intracellular exchangeable Zn²⁺.
• High-affinity, low-background imaging in live cells and plant roots.

4. How to Select an Appropriate Probe Based on Experimental Needs?

Below is a practical selection guide that matches experimental scenarios with spectral/channel requirements and recommended Aladdin products.

Experimental Scenario / Requirement	Spectral / Channel Requirements	Recommended Probe (Aladdin Product)	Key Selection Notes and Description
Live-cell / brain slice imaging; visible-light excitation, green channel (FITC)	Excitation with visible light (~488–495 nm); emission collected in the FITC channel (~510–530 nm).	Z275826 ZnAF-2 DA, Cell-Permeant Zinc Ion Fluorescent Probe	DA ester form with good membrane permeability; hydrolyzed by intracellular esterases to release ZnAF-2, which is retained inside cells. nM-range Kd, high affinity, low background; suitable for real-time, dynamic monitoring of intracellular Zn²⁺ changes.
		Z1452630 ZnAF-2F DA, Cell-Permeant Zinc Ion Fluorescent Probe	Difluorofluorescein-based analogue with lower background and higher signal-to-noise ratio; ideal for high-quality live-cell/brain slice experiments (e.g., neuronal and synaptic Zn²⁺ studies).
		Z276239 Zinpyr-1, Cell-Permeant Zinc Ion Fluorescent Probe	Kd ≈ 0.7 nM with higher affinity, suitable for systems with low Zn²⁺ background; preferentially localizes to the Golgi apparatus and related vesicles in live cells, making it useful for studying intracellular Zn²⁺ storage and transport.
Neural tissue sections and classic “zinc-rich” brain regions (mossy fibers, cortex, etc.), blue-channel imaging	Requires UV / near-UV excitation and blue emission; green/red channels reserved for GFP / mCherry, etc.	T334118 TSQ Zinc Ion Fluorescent Probe	Classic quinoline-based Zn²⁺ probe with good membrane permeability and extensive literature support; commonly used for staining zinc-rich neuronal regions such as hippocampal mossy fibers; considered a “traditional standard” in zinc neurobiology.
		Z274867 Zinquin ethyl ester Zinc Ion Fluorescent Probe	Quinoline ethyl ester form that readily enters cells and is hydrolyzed by esterases to become trapped intracellularly; suitable for Zn²⁺ imaging in cultured neurons and brain slices.
		Z1451896 Zinquin AM Zinc Ion Fluorescent Probe	AM ester form with higher loading efficiency; ideal for live-cell/neuronal imaging requiring high loading efficiency and shorter incubation times.
Zn²⁺ “release” and “redistribution” during apoptosis / oxidative stress	Focus on Zn²⁺ mobilization and marked local increases in concentration; blue channel or UV excitation acceptable.	Z274867 Zinquin ethyl ester Zinc Ion Fluorescent Probe	Moderate affinity combined with blue emission; well suited to visualizing changes in zinc-rich pools mobilized from protein-binding sites during apoptosis or oxidative stress, with intuitive changes in signal intensity.
		Z1451896 Zinquin AM Zinc Ion Fluorescent Probe	Suitable for cell lines requiring high loading efficiency; can be combined with apoptosis markers (e.g., Annexin V) for multiparametric analysis.
		(In combination) N159625 TPEN Metal Chelator	Used as a Zn²⁺ depletion control: TPEN pretreatment should markedly reduce the Zinquin signal, confirming that the fluorescence arises primarily from Zn²⁺.
Accurate quantification of Zn²⁺ in purified proteins, buffer solutions, or extracellular milieu	Response required only in solution; probe entry into cells is undesirable.	ZnAF-2, Cell-Impermeant Zinc Ion Fluorescent Probe	Free acid, cell-impermeant form; nM-range Kd with strong fluorescence enhancement upon Zn²⁺ binding; suitable for generating standard curves and precise quantification using fluorometers or plate readers.
		ZnAF-2F, Cell-Impermeant Zinc Ion Fluorescent Probe	Difluorofluorescein upgrade with extremely low background and higher signal-to-noise ratio; ideal for high-sensitivity Zn²⁺ detection in enzyme assays, metalloprotein studies, and buffer systems.
Multicolor confocal imaging with FITC channel occupied by other probes/proteins	Zn²⁺ signal routed through the blue channel (UV excitation + blue emission).	T334118 TSQ Zinc Ion Fluorescent Probe	UV/near-UV excitation with blue-green emission; can be combined with GFP (green) and mCherry (red), minimizing spectral overlap.
		Z274867 Zinquin ethyl ester / Z1451896 Zinquin AM Zinc Ion Fluorescent Probes	Quinoline-based blue Zn²⁺ probes; ideal as the “blue Zn²⁺ channel” in multicolor imaging, in combination with fluorescent proteins, Ca²⁺ probes, and others.

5. Experimental Considerations and Control Setup

Regardless of which Zn²⁺ probe is used, the following controls are strongly recommended in experimental design:

1. TPEN / EDTA negative controls

• Pre-treat samples with TPEN (a high-affinity Zn²⁺ chelator) to remove labile Zn²⁺. A marked decrease in fluorescence signal confirms that the observed signal indeed arises from Zn²⁺.

2. Exogenous Zn²⁺ positive control

• Add ZnSO₄ (typically in the μM to tens of μM range) together with a Zn²⁺ ionophore (such as pyrithione) for short-term co-incubation. A robust increase in fluorescence can be used to evaluate the dynamic range of the probe.

3. Avoiding heavy metal interference

• Quinoline- and fluorescein-based Zn²⁺ probes can be interfered with by heavy metals such as Hg²⁺ and Cd²⁺, while open-shell metal ions such as Cu²⁺ and Ni²⁺ often cause fluorescence quenching. Buffer formulations should therefore minimize contamination by these metals.

4. Optimization of loading conditions

• For DA/AM/ester-type probes, concentrations should not be too high; in most cases, 1–10 μM is sufficient to obtain good signal.
• Excessive loading times or overly high concentrations may lead to cytotoxicity or probe aggregation/precipitation.

5. Photobleaching and quenching

• All fluorescent probes are subject to photobleaching. Samples should be protected from light as much as possible, and during microscopy, laser power and exposure time should be kept as low as practicable.

6. Product Classification Table for Zinc Ion Fluorescent Probes

Zinc Probes, Chelators, Metal Salts, Ionophores, and Nucleic Acid Dyes

Category	Product Name	CAS	Aladdin Cat. No.	Grade & Purity	Key Features (Structure / Spectra / Performance)	Brief Application Description
Quinoline scaffold · Blue-emitting Zn²⁺ probe	TSQ Zinc Ion Fluorescent Probe (TSQ [N-(6-Methoxy-8-quinolyl)-p-toluenesulfonamide])	109628-27-5	T334118	≥98%	Classic quinoline–p-toluenesulfonamide structure; good membrane permeability; forms complexes with Zn²⁺ that produce blue-green fluorescence, typical Ex/Em ≈ 334/495 nm; high selectivity for Zn²⁺ with strong emission maintained in the presence of Ca²⁺/Mg²⁺.	Suitable for imaging zinc in neural tissue sections, zinc-rich brain regions (e.g., mossy fibers), and intracellular zinc-binding proteins; commonly used for classic “zinc-rich synapse” staining and compatible with multicolor imaging (e.g., with DAPI/GFP).
Quinoline scaffold · Blue-emitting Zn²⁺ probe	Zinquin ethyl ester Zinc Ion Fluorescent Probe	181530-09-6	Z274867	≥99%	Upgraded TSQ analogue; quinoline ethyl ester structure with high lipophilicity and membrane permeability; once inside cells, the ethyl ester is hydrolyzed by esterases to yield negatively charged Zinquin free acid, which is retained intracellularly; typical Ex/Em ≈ 355–368/490–491 nm; moderate affinity suitable for detecting medium to high concentrations of labile Zn²⁺.	Used for fluorescence imaging of labile Zn²⁺ in live cells, including zinc mobilization and changes in zinc homeostasis during apoptosis; compatible with confocal microscopy, flow cytometry, and spectrofluorometric measurements.
Quinoline scaffold · Blue-emitting Zn²⁺ probe	Zinquin AM Zinc Ion Fluorescent Probe (Zinquin AM ester)	181530-16-5	Z1451896	–	AM ester form of Zinquin; highly lipophilic with excellent loading efficiency; intracellular esterases cleave the AM groups to release negatively charged Zinquin, giving stable signals and intracellular retention; molecular weight ~458.48; Ex/Em similar to Zinquin (~355–360/490 nm).	Suitable for monitoring overall cellular zinc status (cellular Zn²⁺ status) in live cells; applicable to various cell lines for confocal and flow cytometric analysis; commonly used to assess Zn²⁺ dynamics.
Fluorescein scaffold · Green-emitting Zn²⁺ probe	ZnAF-2, Cell-Impermeant Zinc Ion Fluorescent Probe	321859-11-4	–	–	Fluorescein conjugated to a TPEN-type chelating structure; free acid, cell-impermeant form; high affinity for Zn²⁺ with Kd ≈ 2.7 nM; 1:1 binding to Zn²⁺ results in ~50-fold fluorescence enhancement; Ex/Em ≈ 492/515 nm; negligible affinity for Ca²⁺, Mg²⁺, Na⁺, K⁺, giving extremely low background.	Suitable for high-sensitivity quantitative detection of Zn²⁺ in buffer solutions, protein systems, or extracellular environments; also useful for method development and free Zn²⁺ measurements in tissue/cell supernatants.
Fluorescein scaffold · Green-emitting Zn²⁺ probe	ZnAF-2 DA, Cell-Permeant Zinc Ion Fluorescent Probe	357339-96-9	Z275826	≥90%	Diacetate (DA) ester form of ZnAF-2; membrane-permeable and hydrolyzed by esterases in cells/tissues to ZnAF-2, which is subsequently retained intracellularly; Ex/Em ≈ 492/515 nm, green emission; maintains the high affinity and low background characteristics of ZnAF-2.	Used for high-sensitivity imaging of labile Zn²⁺ in live cells, brain slices, and tissue sections; suitable for confocal microscopy, high-content imaging, and flow cytometric measurement of intracellular Zn²⁺ levels.
Fluorescein / difluorofluorescein scaffold · Green-emitting Zn²⁺ probe	ZnAF-2F, Cell-Impermeant Zinc Ion Fluorescent Probe	443302-09-8	–	–	Difluorofluorescein-based upgrade of ZnAF-2; cell-impermeant; extremely low basal quantum yield at pH 7.4 (~0.006); fluorescence intensity can increase by ~60-fold upon Zn²⁺ binding; Kd in the nM range; typical Ex/Em ≈ 492/517 nm.	Ideal for in vitro Zn²⁺ quantification with high signal-to-noise requirements (protein assays, enzyme reactions, buffer systems); particularly suitable as a probe for method development and standard curve generation.
Fluorescein / difluorofluorescein scaffold · Green-emitting Zn²⁺ probe	ZnAF-2F DA, Cell-Permeant Zinc Ion Fluorescent Probe	443302-10-1	Z1452630	–	DA ester form of ZnAF-2F with good membrane permeability; intracellular esterases convert it to ZnAF-2F, which is retained for extended periods; extremely low background under physiological conditions, with Zn²⁺ binding enhancing fluorescence by up to ~60-fold; Ex/Em ≈ 492/515 nm; sensitive to subtle changes in Zn²⁺ within physiological ranges.	Well suited for dynamic monitoring of Zn²⁺ in cultured cells and hippocampal slices; typical applications include neuronal activity, synaptic Zn²⁺ release, and high-end imaging of metal homeostasis disturbances.
Fluorescein scaffold · Green-emitting Zn²⁺ probe	Zinpyr-1, Cell-Permeant Zinc Ion Fluorescent Probe	288574-78-7	Z276239	≥95%	Dichlorofluorescein core with dual DPA chelating arms; good membrane permeability; the first Zn²⁺ binding site shows high affinity with Kd ≈ 0.7 ± 0.1 nM; as Zn²⁺ concentration increases, the excitation maximum shifts from ~515 nm to ~507 nm; emission is typically collected with a 513–558 nm bandpass filter; in live cells, it primarily stains the Golgi apparatus and related vesicles.	Suitable for high-resolution localization and imaging of low-background exchangeable Zn²⁺ in live cells and plant roots; particularly useful for studying Zn²⁺ storage and transport in the Golgi/vesicles and for quantitative/semi-quantitative analysis of free Zn²⁺ levels.
Metal chelator · Zn²⁺ control reagent	TPEN Metal Ion Chelator (N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine)	16858-02-9	N159625	≥97%	Prototypical high-affinity chelator for transition metals, with highest affinity for Zn²⁺ (Zn²⁺ > Fe²⁺ > Mn²⁺) and very low affinity for Ca²⁺, Mg²⁺, Na⁺, K⁺; membrane-permeable and lipophilic, able to cross artificial and biological membranes.	Commonly used as a negative control/clearing agent in Zn²⁺ probe experiments: samples are pretreated to remove labile Zn²⁺ and validate the Zn²⁺ specificity of probe signals; widely used in cell biology to model Zn²⁺ deficiency, study Zn²⁺-dependent signaling pathways, oxidative stress, and apoptosis, and to investigate metal dyshomeostasis in disease models (e.g., neurodegenerative diseases, tumors).
Metal chelator · General multivalent metal chelator	EDTA (Ethylenediaminetetraacetic acid)	60-00-4	E112489	Cell culture grade ≥99%	Polycarboxylate multidentate ligand capable of chelating a wide range of divalent and trivalent metal ions (especially Ca²⁺ and Mg²⁺); highly water-soluble and one of the most commonly used general-purpose metal chelators in laboratories.	EDTA has high affinity for many divalent and trivalent metals (including Zn²⁺), but is poorly membrane-permeable and lacks Zn²⁺ selectivity. In cell-based experiments it typically provides a relatively “mild” general chelation condition, and is often used in comparison with TPEN as an auxiliary negative control or to remove metal impurities from solutions.
Metal salt · Exogenous Zn²⁺ source	Zinc sulfate heptahydrate (ZnSO₄·7H₂O)	7446-20-0	Z657123	Animal component-free, low endotoxin, cell culture grade ≥99%	Commonly used inorganic zinc salt with good water solubility and easy preparation of standard solutions; dissociates to provide Zn²⁺ and is non-fluorescent.	Used as an exogenous Zn²⁺ positive control: co-incubation with an ionophore (e.g., pyrithione) transiently elevates intracellular Zn²⁺ levels, allowing evaluation of the sensitivity and dynamic range of Zn²⁺ fluorescent probes.
Metal ion carrier (Zn²⁺ ionophore)	Pyrithione (2-Mercaptopyridine-N-oxide)	1121-31-9	M474482	≥99%	Classic Zn²⁺ ionophore, used as the free acid or sodium salt; forms lipophilic complexes with Zn²⁺ that promote Zn²⁺ transport across cell membranes.	Commonly used together with ZnSO₄ in fluorescent probe experiments to construct Zn²⁺ loading positive controls: rapidly increases intracellular exchangeable Zn²⁺ and verifies whether the probe can effectively “light up.”
Metal ion carrier (Zn²⁺ ionophore)	Sodium pyrithione	3811-73-2	M111101	≥96%	Sodium salt of pyrithione with good water solubility; likewise forms membrane-permeable complexes with Zn²⁺.	Used in combination with ZnSO₄ for rapid Zn²⁺ loading in cells or tissues; a widely used “Zn²⁺ + ionophore” combination in Zn²⁺ fluorescence imaging literature.
Nucleic acid dye · Blue-channel nuclear stain	DAPI (4',6-Diamidino-2-phenylindole, dihydrochloride)	28718-90-3	D106471	≥98%	Classic blue fluorescent nucleic acid dye; fluorescence increases ~20-fold upon binding to AT-rich regions of double-stranded DNA; typical Ex/Em ≈ 358/461 nm (405 nm excitation is also common).	Used as a nuclear stain/counterstain: in multicolor imaging, often combined with green or red fluorescent probes (e.g., ZnAF/Zinpyr-1, GFP, mCherry); can serve as a nuclear reference when imaging Zn²⁺ probes.
Nucleic acid dye · Red-channel live/dead stain	Propidium iodide (PI)	25535-16-4	P266304	≥98% (HPLC)	Classic red fluorescent nucleic acid dye; cell-impermeant and only enters cells with compromised membranes or dead cells; fluorescence increases strongly upon intercalation into DNA/RNA; typical Ex/Em ≈ 535/617 nm.	Used together with Zn²⁺ probes to monitor cell viability: for example, co-applied with Zinpyr-1 in flow cytometry or microscopy to simultaneously assess Zn²⁺ signals and distinguish live/dead cells; also widely used in cell cycle analysis and late-stage apoptosis detection.

7. References

1. Vallee BL, Falchuk KH. The biochemical basis of zinc physiology. Physiological Reviews. 1993;73(1):79–118.

2. Rink L, Haase H. Zinc homeostasis and immunity. Trends in Immunology. 2007;28(1):1–4.

3. Haase H, Rink L. Zinc signals and immunity. International Journal of Molecular Sciences. 2017;18(10):2222.

4. Wang B, Fang T, Chen H. Zinc and central nervous system disorders. Nutrients. 2023;15(9):2140.

5. Frederickson CJ. Neurobiology of zinc and zinc-containing neurons. International Review of Neurobiology. 1989;31:145–238.

6. Meeusen JW, Tomasiewicz H, Nowakowski A, Petering DH. TSQ (6-methoxy-8-p-toluenesulfonamido-quinoline), a common fluorescent sensor for cellular zinc, images zinc proteins. Inorganic Chemistry. 2011;50(16):7563–7573.

7. Huang X, Cuajungco MP, Atwood CS, et al. Flux of intracellular labile zinc during apoptosis (gene-directed cell death) revealed by a specific chemical probe, Zinquin. Chemistry & Biology. 1994;1(3):153–161.

8. Hirano T, Kikuchi K, Urano Y, Nagano T. Improvement and biological applications of fluorescent probes for zinc, ZnAFs. Journal of the American Chemical Society. 2002;124(29):6555–6562.

9. Maruyama S, Kikuchi K, Hirano T, Urano Y, Nagano T. A novel, cell-permeable, fluorescent probe for ratiometric imaging of zinc ion. Journal of the American Chemical Society. 2002;124(40):10650–10651.

10. Burdette SC, Walkup GK, Spingler B, Tsien RY, Lippard SJ. Fluorescent sensors for Zn²⁺ based on a fluorescein platform: synthesis, properties and intracellular distribution. Journal of the American Chemical Society. 2001;123(32):7831–7841.

11. Goldsmith CR, Lippard SJ. Analogues of Zinpyr-1 provide insight into the mechanism of zinc sensing. Inorganic Chemistry. 2006;45(16):6474–6478.

12. Sinclair SA, Sherson SM, Jarvis R, Camakaris J, Cobbett CS. The use of the zinc-fluorophore, Zinpyr-1, in the study of zinc homeostasis in Arabidopsis roots. New Phytologist. 2007;174(1):39–45.

13. Alker W, Schwerdtle T, Schomburg L, Haase H. A Zinpyr-1-based fluorimetric microassay for free zinc in human serum. International Journal of Molecular Sciences. 2019;20(16):4006.

14. Rana U, Srivastava AK, Petering DH. Zinc binding ligands and cellular zinc trafficking: apo-metallothionein, glutathione and TPEN. Journal of Inorganic Biochemistry. 2008;102(3):489–499.

15. Sinclair SA, Sherson SM, Jarvis R, Camakaris J, Cobbett CS. Zinc-fluorophore Zinpyr-1 as a tool to examine zinc localization in plant tissues. New Phytologist. 2007;174(1):39–45.

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