Patent Description:
Zinc homeostasis, including labile Zn<NUM>+ fluctuation, is closely associated with many physiological processes and disease pathologies. As the newly proposed "second messenger," labile Zn<NUM>+ is involved in both inter- and intracellular signal regulation and transmission, and new phenomena for labile Zn<NUM>+-associated signal transduction, such as zinc sparks and zinc waves, have been observed using fluorescence imaging. Except for various organelles having different Zn<NUM>+ levels and dynamics, many physiological processes involving multiple organelles are also associated with Zn<NUM>+ fluctuation. In particular, a growing body of evidence suggests that Zn<NUM>+ is pivotal to autophagy and autophagy can prompt significant changes in intracellular Zn<NUM>+. As a dynamic process for cells to degrade and recycle proteins and senescent organelles to overcome stressful conditions, autophagy is associated with aging, neurodegenerative diseases, diabetes, and fatty liver disease, and is a potential target for diagnosis and treatment. It is therefore important to determine Zn<NUM>+ signaling relationships among organelles during autophagy and other cellular processes.

<NPL> discloses the in vitro and in vivo imaging application of a <NUM>,<NUM>-naphthalimide-derived Zn<NUM>+ fluorescent sensor with nuclear envelope penetrability.

As scientists have begun to better understand the role of labile zinc in key cellular functions, a need has emerged for improved methods for detecting and visualizing labile zinc in biological materials.

Accordingly, provided herein are improved methods for detecting, imaging, and tracking labile zinc in biological materials, including living cells and organoids. The presently disclosed methods permit nanometer-level detection of labile zinc and correlation with morphological features of organelles to visualize the distribution of labile zinc in the biological material. Advantageously, the present methods do not require organelle-specific targeting molecules or co-localization with additional dyes.

In an aspect of the invention, there is provided a method for detecting labile zinc (Zn<NUM>+) in a biological material is provided, the method comprising: (a) contacting the biological material with a composition comprising NapBu-BPEA; and (b) imaging the biological material via molecular fluorescence imaging to detect the labile zinc in the biological material.

Also described is a method for morphology-correlated detection of labile zinc localization in a subcellular organelle of a living cell is provided, the method comprising: (a) contacting the cell with a composition comprising NapBu-BPEA; (b) imaging the cell via super-resolution imaging to visualize a distribution of labile zinc in the cell; and (c) correlating the distribution of labile zinc in the cell with at least one morphological feature of the subcellular organelle to determine labile zinc localization in the subcellular organelle.

Also described is a method of tracking a change in labile zinc localization in a biological material is provided, the method comprising: (a) contacting the biological material with a composition comprising NapBu-BPEA; (b) imaging the biological material via super-resolution imaging to detect the labile zinc in the biological material; (c) repeating step (b) at determined time intervals and comparing obtained super-resolution imaging data to track the change in labile zinc localization in the biological material over time.

These and other objects, features, embodiments, and advantages will become apparent to those of ordinary skill in the art from a reading of the following detailed description and the appended claims.

The details of embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document.

While the following terms are believed to be well understood in the art, definitions are set forth to facilitate explanation of the presently-disclosed subject matter. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently-disclosed subject matter belongs.

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about. " Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

As used herein, the term "about," when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some examples ±<NUM>%, in some examples ±<NUM>%, in some examples ±<NUM>%, in some examples ±<NUM>%, in some examples <NUM>%, and in some examples ±<NUM>% from the specified amount, as such variations are appropriate to perform the disclosed method.

As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.

Zinc is a key micronutrient for mammals and plays a role in a variety of cellular pathways. Labile zinc functions in cells as a signaling molecule and as a component of many proteins and enzymes. "Labile zinc," as used herein, refers to intracellular Zn<NUM>+. Zn<NUM>+ is the most abundant intracellular metal ion found in the cytosol, vesicles, nucleus, and organelles of mammalian cells.

"Biological material," as used herein, refers to living cells, tissues, organoids, and organisms. In embodiments, the biological material is mammalian. In specific embodiments, biological materials for use in the disclosed methods are selected from the group consisting of living cells, organoids, and combinations thereof.

"NapBu-BPEA" refers to a fluorescent probe having the following structure:
<CHM>
<NUM>,<NUM>-(bis(pyridin-<NUM>-ylmethyl)aminoethyl)amino-N-n-butyl-<NUM>,<NUM>-naphthalimide. NapBu-BPEA comprises a N,N'-bis(pyridin-<NUM>-ylmethyl)ethane-<NUM>,<NUM>-diamine (BPEA) moiety bound to <NUM>,<NUM>-naphthalimide (Nap) and modified with a butyl group (Bu).

"Molecular fluorescence imaging" refers to imaging techniques that leverage the optical emissions of molecules that have been excited to higher energy levels by absorption of electromagnetic radiation. Generally, molecular fluorescence imaging uses fluorophore-labeled dyes or proteins, which may be used for either direct or indirect detection of a target. Molecular fluorescence imaging has application in observing the dynamics of gene expression, protein expression, and molecular interactions in living cells. Various molecular fluorescence techniques are known in the art and suitable for use in the present methods.

"Super-resolution imaging" refers to a class of techniques that permits imaging of subcellular structures with a spatial resolution beyond the diffraction limit of conventional light microscopy. See <NPL>). Various super-resolution imaging techniques are known in the art and include, but are not limited to, structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, ground state depletion (GSD) microscopy, photo-activated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and the like. In specific embodiments of the present disclosure, the super-resolution imaging technique employed is SIM.

"Structured illumination microscopy" (SIM) provides enhanced spatial resolution and involves illuminating a sample with patterned light and using software to analyze the information in Moiré fringes outside the normal range of observation. Reconstruction software deciphers the images at about <NUM>-fold higher resolution than the diffraction limit, or ~<NUM>. SIM is suitable for imaging thicker sections, for 3D imaging, and for live-cell imaging. See <NPL>).

The present disclosure is directed to novel methods of imaging labile zinc in biological materials with high spatial resolution at the nanometer scale. These methods open new avenues for the study of molecular mechanisms in biological materials, including living cells and organoids.

For dynamic tracking and quantification of labile zinc, molecular fluorescence imaging is a reliable method for rapidly tracking biological materials in situ, particularly due to its high sensitivity, selectivity, rapid response, and noninvasiveness. The presently disclosed methods employ molecular fluorescence imaging to track the temporal and spatial distribution of zinc in cells, subcellular organelles, and organoids. Because subcellular organelles are the units that perform various physiological functions in cells, it is pivotal to detect changes in zinc levels in organelles during autophagy. However, due to the limitations of fluorescent imaging resolution, detecting zinc dynamics in organelles has remained a challenge.

Although optical microscopy presents several advantages in detecting the intracellular content of bioanalytes, its resolution of approximately <NUM> limits its applicability. In recent years, the development of super-resolution imaging technology has overcome the shortcomings of the diffraction limit of traditional optical technology and been used to perform imaging once deemed impossible. Structured illumination microscopy (SIM), having a spatial resolution of approximately <NUM>, is a particularly favorable option for live cell imaging.

Because small molecular probes offer numerous advantages - large-scale synthesis, simple labeling, tunable wavelengths, and good repeatability, to name a few - small molecular zinc fluorescent probes have become reliable candidates for super-resolution imaging. However, constructing such probes continues to present several setbacks, including frequent photobleaching and low fluorescence quantum yields. For the purpose of biological imaging, naphthalimide (Nap) fluorophores afford a simple structure and high molar extinction coefficients that make them exceptional as probes. Exhibiting a large degree of conjugation, strong rigidity, and, in turn, a low probability of nonradiative transition, the molecular structure of Nap fluorophores affords good photostability and facilitates high fluorescence quantum yields. Nap fluorophores have a lower imaging background signal and favor the 3D super-resolution imaging of large objects. Moreover, the multiple derivative sites of their structure is conducive to introduce organelle-targeting groups to realize the detection of biological species at the level of organelles. For that reason, a variety of specific subcellular organelle-targeting probes have been developed, through modified targeting groups. However, adding targeting groups requires multiple modification sites, which limits the pool of suitable agents. Moreover, because some targeting groups cannot achieve the desired targeting effect, they may alter the imaging performance and the probe's specificity. Thus, it is desirable to develop a method for detecting subcellular organelles using fluorescent probes without the need to introduce organelle-targeting groups.

Based on past successes in the super-resolution imaging of the dynamics of subcellular organelles, it is currently possible to differentiate typical subcellular organelles by morphological features, including rod-shaped mitochondria with cristae, punctate lysosomes, and reticular endoplasmic reticulum. The presently disclosed methods combine the fluorescent distribution of NapBu-BPEA with the morphological features of organelles revealed by super-resolution imaging to provide morphology-correlated detection (MCoD) and determine the localized level of biological species of subcellular organelles, without the use of targeting groups.

NapBu-BPEA has been developed for the super-resolution imaging of labile zinc with a spatial resolution peaking at about <NUM>. By correlating the super-resolution MCoD with the change in ion levels indicated by NapBu-BPEA, the present methods achieve pan-cell zinc detection for individual subcellular organelles via the use of only one probe. Further, NapBu-BPEA has been used to image organoids, which provides a practical basis for studying Zn-related chemical biology in complex biological systems.

NapBu-BPEA is a Zn<NUM>+-selective, reversible, turn-on response fluorescence sensor with a low detection limit and strong binding ability that can be applied to map whole intracellular organelles in living cells except nuclei. By using NapBu-BPEA, it was observed that CCCP-induced damaged autophagy increased intracellular Zn<NUM>+, which would transform into autophagosomes. NapBu-BPEA permits monitoring of the accumulation and fluctuation of intracellular concentrations of labile zinc under different conditions of induced autophagy.

Accordingly, in one embodiment, a method for detecting labile zinc (Zn<NUM>+) in a biological material is provided, the method comprising: (a) contacting the biological material with a composition comprising NapBu-BPEA; and (b) imaging the biological material via molecular fluorescence imaging to detect the labile zinc in the biological material. In specific embodiments, the molecular fluorescence imaging technique comprises super-resolution imaging. Various super-resolution imaging methods are known in the art and suitable for use in the disclosed methods. In embodiments, the super-resolution imaging is selected from the group consisting of structured illumination microscopy (SIM), and stimulated emission depletion (STED) microscopy. Other examples of super-resolution imaging techniques include ground state depletion (GSD) microscopy, photo-activated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM). In a very specific embodiment, the super-resolution imaging technique is structured illumination microscopy (SIM). Advantageously, SIM provides an enhanced spatial resolution peak of about <NUM>. In embodiments, the molecular fluorescence imaging produces imaging data captured digitally or photographically.

In embodiments, detecting the labile zinc in a biological material comprises visualizing a distribution of labile zinc in the biological material. In embodiments, the method further comprises correlating the distribution of labile zinc in the biological material with a morphological feature of a subcellular organelle to determine labile zinc localization in the subcellular organelle. In a specific embodiment, the morphological feature of the subcellular organelle is selected from the group consisting of shape, structure, size, and combinations thereof.

The presently disclosed methods are suitable for imaging a variety of biological materials. In embodiments, the biological materials comprise organoids, living cells, or subcellular organelles. In embodiments, the biological material is in situ or in vivo.

Advantageously, the presently disclosed methods employ the fluorophore NapBu-BPEA, which does not comprise a specific subcellular organelle-targeting group. Moreover, in embodiments, the presently disclosed methods do not require co-localization with a second dye to correlate the distribution of labile zinc in the biological material with a morphological feature of a subcellular structure, such as an organelle.

In embodiments of the disclosed methods, the NapBu-BPEA binds Zn<NUM>+ in a <NUM>:<NUM> ratio. In embodiments, the NapBu-BPEA binds Zn<NUM>+ reversibly.

Optionally, the methods disclosed herein further comprise quantifying the labile zinc in the biological material based on the obtained imaging data. Such quantification may be accomplished using methods known in the art.

Also encompassed is a method for morphology-correlated detection of labile zinc (Zn<NUM>+) localization in a subcellular organelle of a living cell is provided, the method comprising: (a) contacting the cell with a composition comprising NapBu-BPEA; (b) imaging the cell via super-resolution imaging to visualize a distribution of labile zinc in the cell; and (c) correlating the distribution of labile zinc in the cell with at least one morphological feature of the subcellular organelle to determine labile zinc localization in the subcellular organelle. In further examples, the method additionally comprises (d) repeating steps (b) and (c) at determined time intervals and comparing obtained super-resolution imaging data obtained at the determined time intervals to track a change in labile zinc localization in the subcellular organelle over time.

Determined time intervals may be selected by the skilled artisan. In examples, the time intervals may be measured by days, hours, minutes, or seconds. In examples, the time intervals range in duration from about <NUM> second to about <NUM> hour. That is, in examples, the biological material is imaged at time intervals ranging from about <NUM> second to about <NUM> hour. For example, in a specific example, a biological material may be imaged according to the present methods every <NUM> minute, every <NUM> minutes, every <NUM> minutes, every <NUM> minutes, every <NUM> minutes, and so on, for a duration of time selected by the skilled person. In examples, the time interval can be any duration of time suitable to image changes in Zn<NUM>+ in a biological material.

In examples, the super-resolution imaging is selected from the group consisting of structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, ground state depletion (GSD) microscopy, photo-activated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM). In a very specific embodiment, the super-resolution imaging technique is structured illumination microscopy (SIM). Advantageously, SIM provides an enhanced spatial resolution peak of about <NUM>.

The presently disclosed methods are suitable for imaging a variety of biological materials. In embodiments, the biological material comprises an organoid, a living cell, or a subcellular organelle. In embodiments, the biological material is in situ or in vivo.

Advantageously, the presently disclosed methods employ the fluorophore NapBu-BPEA, which does not comprise a specific subcellular organelle-targeting group. Moreover, in embodiments, the presently disclosed methods do not require co-localization with a second dye to correlate the distribution of labile zinc in the biological material with a morphological feature of a subcellular structure, such as an organelle. In embodiments, the morphological feature of the subcellular organelle is selected from the group consisting of shape, structure, size, and combinations thereof. In specific embodiments, identifiable morphological features include, but are not limited to, the rod-like shape of mitochondria; the cristae (folded inner membranes) of mitochondria; the punctate appearance of lysosomes; and the reticular appearance of the endoplasmic reticulum, comprising a network of interconnected flattened membrane sacs and/or tubules. Such morphological features are readily distinguishable by the methods disclosed herein.

In still another example, a method of tracking a change in labile zinc (Zn<NUM>+) localization in a biological material, the method comprising: (a) contacting the biological material with a composition comprising NapBu-BPEA; (b) imaging the biological material via super-resolution imaging to detect the labile zinc in the biological material; and (c) repeating step (b) at determined time intervals and comparing obtained super-resolution imaging data to track the change in labile zinc localization in the biological material over time.

In examples, the super-resolution imaging is selected from the group consisting of structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, ground state depletion (GSD) microscopy, photo-activated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM). In a very specific example, the super-resolution imaging technique is structured illumination microscopy (SIM). Advantageously, SIM provides an enhanced spatial resolution peak of about <NUM>.

In examples, the biological material is a cell or an organoid. In embodiments, the NapBu-BPEA does not comprise a specific subcellular organelle-targeting group.

Although an organelle-specific second fluorophore is not required to achieve nanometer-level imaging sufficient to distinguish organelles by the present methods, in examples, the skilled artisan may wish to pair NapBu-BPEA with a second fluorophore targeted to one or more specific organelles. Accordingly, in examples, the methods set forth herein may include contacting the biological sample with a second fluorophore and imaging the biological material to visualize both NapBu-BPEA and the second fluorophore. Suitable second fluorophores include, but are not limited to, MitoTracker™ Green FM (MTG), MitoTracker™ DeepRed FM (MTDR), LysoTracker™ Red DND-<NUM> (LTR), ER-Tracker™ Red, Hoechst <NUM>, and the like.

The following examples are given by way of illustration are not intended to limit the scope of the disclosure.

All solvents and reagents are of analytical grade and used without further purification. <NUM>-bromo-<NUM>, <NUM>-naphthalic anhydride, n-butylamine, ethylenediamine, picolyl chloride and Ru(bpy)<NUM><NUM>+ were purchased from Energy Chemical Inc. (Shanghai, China). KCl, CaCl<NUM>, MgCl<NUM>, NaCl, FeSO<NUM>, FeCl<NUM>, Zn(NO<NUM>)<NUM>, NiCl<NUM>, CdCl<NUM>, MnCl<NUM>, BaCl<NUM>, CrCl<NUM>, Al<NUM>(SO<NUM>)<NUM> and Pb(NO<NUM>)<NUM> were purchased from Sinopharm Chemical Reagent (Nanjing, China). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA) and <NUM>-[<NUM>-(<NUM>-hydroxyethyl)-<NUM>-piperazinyl]ethanesulfonic acid (HEPES) were purchased from Sigma (Shanghai, China). Pyrithione sodium salt, N, N, N', N'-Tetrakis (<NUM>-pyridylmethyl) ethylenediamine were obtained from Fisher Scientific Inc. (Ohio, USA). MitoTracker™ Green FM (MTG), MitoTracker™ DeepRed FM, LysoTracker™ Red DND-<NUM> (LTR), ER-Tracker™ Red and Hoechst <NUM> were purchased from Invitrogen (Ohio, USA). Autophagosome Detection dye (DAPRed) and Cytoxicity LDH Assay Kit-WST were purchased from Dojindo (Washington, USA). The cell culture medium, Dulbecco's Modified Eagle Medium (DMEM) and Earle's Balanced Salt Solution (EBSS, calcium, magnesium, phenol red) were purchased from Gibco (Ohio, USA).

The <NUM>H NMR and <NUM>C NMR spectra were determined with a <NUM> Bruker spectrometer with TMS as internal standard. High-Resolution Mass spectrometric data were recorded on an Agilent <NUM> Q-TOF mass spectrometer. The UV-Vis and fluorescence spectra were performed on PerkinElmer Lambda <NUM> spectrophotometer and Horiba FM-<NUM> fluorophotometer. The cell imaging was carried out by Nikon N-SIM system.

The stock solution of NapBu-BPEA was prepared with DMSO of HPLC pure grade to make the concentration as <NUM>, and frozen after packing at -<NUM>. The sample solutions were diluted to the final concentration as <NUM> with <NUM> HEPES buffer (<NUM>, <NUM> KNO<NUM>, <NUM>% DMSO, pH <NUM>) in quartz cuvettes with <NUM> path lengths. The fluorescent spectra were recorded upon the excitation of <NUM>.

The Zn<NUM>+ titration spectra were recorded by adding in aliquots Zn<NUM>+ solution from <NUM> to <NUM> into the NapBu-BPEA solution (<NUM>). The detection limit of NapBu-BPEA was determined by collecting the fluorescence spectra of NapBu-BPEA for <NUM> times to obtain the background noise (σ). The probe's fluorescent sensing selectivity of NapBu-BPEA was recorded after adding metal cation (<NUM> eq K+, Na+, Ca<NUM>+,Mg<NUM>+; <NUM> eq Cd<NUM>+, Ni<NUM>+, Cr<NUM>+, Pb<NUM>+, Al<NUM>+, Co<NUM>+, Fe<NUM>+, Fe<NUM>+, Ba<NUM>+, Mn<NUM>+) to the NapBu-BPEA solution. The fluorescence quantum yields were determined by using Ru(bpy)<NUM><NUM>+ (Φ = <NUM>) in DMSO/HEPES (v:v, <NUM>:<NUM>) (λex = <NUM>) as the reference. To determine binding constant, various amounts of Zn(NO<NUM>)<NUM> (<NUM>~<NUM>) were added to NapBu-BPEA solution buffered with DMSO/HEPES (<NUM>, pH <NUM>, <NUM> KNO<NUM>) containing <NUM> EGTA. The pH stability was measured by recording the fluorescence spectra of NapBu-BPEA solution in the presence of Zn<NUM>+ at different pH adjusted with KOH and HCl.

Wild-type, FIP200 and ATG13 KO HeLa cell lines were gifted from Dr. Jun-Lin Guan's lab (University of Cincinnati). Cells were cultured in DMEM supplemented <NUM>% FBS and <NUM> U/mL penicillin-streptomycin (Gibco) in <NUM>% CO<NUM> incubator at <NUM>.

Human induced pluripotent stem cells (iPSCs) were differentiated into foregut using previously described method. In brief, hiPSCs were detached by Accutase (Thermo Fisher Scientific Inc. , MA, USA) and were seeded on Laminin coated tissue culture plate with <NUM>,<NUM> cells/cm<NUM>. Medium was changed to RPMI <NUM> medium (Life Technologies) containing <NUM> ng/mL Activin A (R&D Systems) and <NUM> ng/mL bone morphogenetic protein <NUM> (BMP4; R&D Systems) at day <NUM>, <NUM> ng/mL Activin A and <NUM>% fetal calf serum (FCS; Thermo Fisher Scientific Inc. ) at day <NUM>, and <NUM> ng/mL Activin A and <NUM>% FCS at day <NUM>. On day <NUM>-<NUM>, cells were cultured in Advanced DMEM/F12 (Thermo Fisher Scientific Inc. ) with B27 (Life Technologies) and N2 (Gibco, CA, USA) containing <NUM> ng/mL fibroblast growth factor (FGF4; R&D Systems) and <NUM> CHIR99021 (Stemgent, MA, USA). Cells were maintained at <NUM> in <NUM>% CO<NUM> with <NUM>% air and the medium was replaced every day. The foregut cells were detached by Accutase and then centrifuged at <NUM> rpm for <NUM>. Cells were resuspended in Matrigel (Corning, In. , NY, USA). A total of <NUM>,<NUM> cells were embedded in <NUM>µL Matrigel drop on the dishes in organoid formation media with <NUM> factors for <NUM> days. After organoid formation, the media was switched to liver specification media for <NUM> days. After the liver specification step, organoids were harvested from Matrigel by scratching and pipetting. Then organoids were re-embedded in Matrigel on the Ultra-low attached plate (Corning) in liver maturation media for <NUM> days. Cultures for HLO induction were maintained at <NUM> in <NUM>% CO<NUM> with <NUM>% air and the medium was added every <NUM> days.

The SIM images were acquired using a Nikon N-SIM system. The blue imaging channel for Hoechst <NUM> with emission bandwidth at <NUM>-<NUM> upon excitation at <NUM>, the green imaging channel for NapBu-BPEA with emission bandwidth at <NUM>-<NUM> upon excitation at <NUM>, the red imaging channel for ER-Tracker Red, LysoTracker Red and DAPRed with emission bandwidth at <NUM>-<NUM> upon excitation at <NUM>, the magenta imaging channel for MitoTracker Deep Red with emission bandwidth at <NUM>-<NUM> upon excitation at <NUM> were utilized. The imaging data analysis and thermal map construction were performed via analysis with ImageJ.

The co-localization experiments were performed with a dual-channel mode. HeLa cells were stained by NapBu-BPEA (<NUM>, <NUM>) and then incubated with Mito-marker Deep Red (<NUM>, <NUM>), Lysotracker Red (<NUM>, <NUM>), ER-Tracker Red (<NUM>, <NUM>), and Hoechst <NUM> (<NUM>µg/mL, <NUM>), respectively. The Pearson's correlation coefficient was calculated using Cellprofiler with co-localization module.

The intracellular Zn<NUM>+ level in autophagy was imaged in HeLa cells. Prior to CCCP (<NUM>, <NUM>) or EBSS treatment (<NUM>, <NUM>), the cells were stained with DAPRed (<NUM>, <NUM>). The cells were finally stained by NapBu-BPEA (<NUM>, <NUM>) before SIM imaging.

The fresh organoids were transferred into petri dish. After <NUM> CCCP treatment for <NUM>, the organoids were incubated with <NUM> NapBu-BPEA for <NUM>. Then the organoids were imaged with z stack at different depths.

The suspension of HeLa cells diluted with <NUM>µL DMEM was plated into <NUM>-well plate. The inoculated cells were pre-cultured overnight in <NUM>-well plate and replaced with a new <NUM>µL DMEM. <NUM>µL DMEM containing different concentrations of NapBu-BPEA was added and cultured in CO<NUM> incubator at <NUM> for <NUM>. After <NUM>µL lysis buffer was added to the high contrast wells, <NUM> was cultured in the CO<NUM> incubator at <NUM>. After <NUM>µL working solution was added to each well, it was cultured for <NUM> under dark and room temperature. After <NUM>µL stop solution was added to each well, the absorbance of <NUM> was determined immediately by a microplate reader (Thermomax, Molecular Devices).

All data were analyzed and statistically calculated using Microsoft Excel <NUM> software (Microsoft, Redmond, WA). The results are expressed as mean ± standard deviation (SD) unless otherwise stated. The statistical differences between the experimental groups were analyzed by double-tailed Student's t-test. When p < <NUM>, it was considered to have statistical significance. All statistical graphs were performed using Origin <NUM> (OriginLab Corporation, MA, USA).

Each experiment was repeated at least three times independently with similar results. All images shown are representative results from biological replicates.

The Zn<NUM>+ fluorescent probe NapBu-BPEA was rationally grafted from a Nap fluorescent platform onto a N,N'-bis(pyridin-<NUM>-ylmethyl)ethane-<NUM>,<NUM>-diamine (BPEA) moiety, which is often used as a Zn<NUM>+ chelator (<FIG>). Nap fluorophore was selected due to its favorable optical stability, high extinction coefficient, and high fluorescence quantum yields. Using ChemDraw's predictive algorithms, the lipophilic parameter (logP) of NapBu-BPEA was predicted to be <NUM>, which is significantly greater than that of another Zn<NUM>+ probe, Naph-BPEA (i.e., <NUM>). The intermediate compounds and target compound were characterized by <NUM>H spectra, <NUM>C spectra, and high-resolution mass spectrometry (<FIG>).

Synthesis of <NUM>-bromo-N-n-butyl-<NUM>, <NUM>-naphthalimide (<NUM>): n-butylamine (<NUM>, <NUM> mmol) was added to <NUM>-bromo-<NUM>,<NUM>-naphthalic anhydride (<NUM>, <NUM> mmol) dissolved in ethanol (<NUM>) and mixed in <NUM> three-necked flask. Then the mixture was stirred and refluxed at <NUM> under N<NUM> for <NUM>. After the reaction finished and cooled to room temperature, the mixture was filtered and washed with ethanol (<NUM> × <NUM>). The residue was collected and dried as light yellow power in <NUM>% yield (<NUM>). <NUM>H NMR (<NUM>, Chloroform-d) δ <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>).

Synthesis of N-n-butyl-<NUM>-(aminoethylene) amino-<NUM>, <NUM>-naphthalimide (<NUM>): <NUM> (<NUM> mmol) compound <NUM> and <NUM> (<NUM> mmol) ethylenediamine were added in a <NUM> three-necked flask. After continuous stirring at <NUM> for <NUM>, the mixture was cooled and poured into <NUM> of ice water. Then the precipitate was collected by filtration, washed with water, and dried to <NUM>% yield (<NUM>). <NUM>H NMR (<NUM>, Chloroform-d) δ <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>).

Synthesis of <NUM>,<NUM>-(bis(pyridin-<NUM>-ylmethyl)aminoethyl)amino-N-n-butyl-<NUM>,<NUM>-naphthalimide (NapBu-BPEA): <NUM> (<NUM> mmol) compound <NUM> were dissolved in <NUM> dry ethanol. The mixture was stirred and refluxed for <NUM> hours under N<NUM>. The reaction process was monitored by TLC. The solvent was evaporated under reduced pressure, once the reaction was completed. The crude product was purified by silica gel column chromatography (CH<NUM>Cl<NUM>: MeOH = <NUM>:<NUM>) to obtain a yellow solid in <NUM>% yield (<NUM>). <NUM>H NMR (<NUM>, Methanol-d<NUM>) δ <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (ddd, J = <NUM>, <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (tt, J= <NUM>, <NUM>, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>). <NUM>C NMR (<NUM>, Methanol-d<NUM>) δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. HR-MS(positive mode): Calcd. <NUM>, Found.

To gauge the spectroscopic sensing behavior of NapBu-BPEA, photophysical properties were tested in a solution of dimethyl sulfoxide (DMSO) and <NUM>-(<NUM>-hydroxyethyl)-<NUM>-piperazineethanesulfonic acid (HEPES) (<NUM>, <NUM> KNO<NUM>, <NUM>:<NUM>, pH <NUM>). As shown in <FIG>, the optical spectra of NapBu-BPEA exhibited a maximum emission peak at <NUM> and maximum absorption peak at <NUM>. The relative fluorescence quantum yield was <NUM>, and the molar extinction coefficient was <NUM> × <NUM><NUM> M-<NUM>cm-<NUM>. When <NUM> eq Zn<NUM>+ was added, the optimum emission peak blue-shifted to <NUM>, with a fluorescence quantum yield of <NUM>, and the maximum absorption peak blue-shifted to <NUM>, with a molar extinction coefficient of <NUM> × <NUM><NUM> M-<NUM>cm-<NUM>.

Next, NapBu-BPEA was titrated with Zn<NUM>+. As shown in <FIG>, owing to the limitations of photoinduced electron transfer (PET), NapBu-BPEA demonstrated an enhanced emission response and an increased concentration of Zn<NUM>+, which rose by <NUM>-fold at <NUM>, accompanied by a slight degree of blue shift. When the concentration of Zn<NUM>+ totaled <NUM>, the fluorescence intensity achieved saturation (<FIG>). UV-vis titration also illustrated a similar phenomenon. As shown by the Job's plot curve in <FIG>, the fluorescence intensity peaked when [Zn<NUM>+]:[NapBu-BPEA] equaled <NUM>:<NUM>, which demonstrates that NapBu-BPEA can bind to Zn<NUM>+ in a <NUM>:<NUM> ratio. The limit of detection was calculated based on the <NUM>σ/slope method as <NUM>. High-resolution mass spectrometry was performed with NapBu-BPEA solution containing <NUM> eq Zn<NUM>+, which revealed a peak of <NUM> belonging to [NapBu-BPEA+Zn+Cl]+.

Along with detecting Zn<NUM>+, NapBu-BPEA was tested for interference from other biologically related metal ions, as shown in <FIG>. The probe did not respond to any other metal ions except Cd<NUM>+. When <NUM> eq Zn<NUM>+ was added, the fluorescence intensity of NapBu-BPEA clearly increased, whereas it changed only slightly in Co<NUM>+ and Ni<NUM>+ solution. However, because the content of Cd<NUM>+, Co<NUM>+, and Ni<NUM>+ in cells was far less than <NUM>, the interference from those metal ions was negligible. These results indicate that NapBu-BPEA has specific fluorescence selectivity for Zn<NUM>+. Further, following the addition of N, N, N', N'-tetrakis <NUM>-pyridylmethyl ethylenediamine (TPEN), a Zn<NUM>+-chelating agent, the NapBu-BPEA fluorescence signal enhanced by Zn<NUM>+ was restored, and an on-off conversion could be observed for five cycles. Taken together, these results indicate that NapBu-BPEA has a favorable reversible response to Zn<NUM>+ (<FIG>). In an analysis of the function of fluorescence intensity combined with labile Zn<NUM>+ concentration (<FIG>), the dissociation constant (Kd) of the complex between Zn<NUM>+ and NapBu-BPEA was determined to be <NUM>. Last, the effect of pH on the fluorescence response of NapBu-BPEA to Zn<NUM>+ was investigated by using pH titration. When bound to Zn<NUM>+, NapBu-BPEA was highly stable at pH <NUM>-<NUM>, which indicates that NapBu-BPEA is suitable for detecting Zn<NUM>+ in a wide range of pH levels.

Next, the use of NapBu-BPEA to detect fluctuations in intracellular Zn<NUM>+ was investigated. First, a water-soluble tetrazolium (WST) kit was used to determine the cytotoxic activity of NapBu-BPEA by measuring the activity of lactate dehydrogenase released into the medium. After incubating HeLa cells with different concentrations of NapBu-BPEA for <NUM>, it was observed that when the concentration reached <NUM>, the activity of the lactate dehydrogenase was less than <NUM>%, while the cell viability exceeded <NUM>%. Together, this data indicates that NapBu-BPEA has a low cytotoxicity and is highly suitable for cell imaging. To ascertain the incubation time, SIM images of HeLa cells were co-incubated with NapBu-BPEA at various time points, which revealed that the fluorescence signal of NapBu-BPEA entering the cells changed only slightly after <NUM> of incubation. These results suggest that <NUM> is the optimal incubation time for cell imaging. To evaluate the staining ability of NapBu-BPEA, the commercially available lysosomal dye LysoTracker Red (LTR), the commercial mitochondrial dye MitoTracker Deep Red (MTDR), the endoplasmic reticulum dye ER-Tracker Red (ERTR), and the nuclear dye Hoechst <NUM> (Hoechst) were used in a co-localization experiment. Therein, HeLa cells were incubated with NapBu-BPEA and commercial dyes at <NUM>. As shown in <FIG>, a strong green fluorescence signal was observed in the green channel, which was attributed to NapBu-BPEA, whereas the other channels (i.e., blue, red, and magenta) showed fluorescence signals of punctate lysosomes, rod mitochondria, reticular endoplasmic reticulum, and round nuclei. By calculating the co-localization Pearson's coefficient and the magnification of the overlapping images, it was found that NapBu-BPEA had distributed within all organelles except the nucleus. Together, the results indicate that NapBu-BPEA can be used to monitor the level of Zn<NUM>+ in individual organelles throughout entire cells.

The visualization of organelle ultrastructures can illuminate the pathology and diagnosis of intracellular diseases. To study the ultrastructural information of intracellular organelles, NapBu-BPEA was used to perform the ultrastructural imaging of organelles in living cells under 3D-SIM. As shown in <FIG>, confocal and SIM imaging of HeLa cells after <NUM> of incubation revealed that NapBu-BPEA afforded a good super-resolution display of lysosomes, represented by dots, and mitochondria, represented by rods with cristae. Next, the mitochondrial intensity of the confocal and SIM images was analyzed, which supported NapBu-BPEA's ability to clearly distinguish the structure of mitochondrial cristae under SIM. To further clarify the resolution of NapBu-BPEA, a full width and half peak (FWHM) up to <NUM> under SIM was obtained (<FIG>), which overcame the optical diffraction limit and achieved super-resolution imaging. These results indicate that NapBu-BPEA can provide excellent accuracy and resolution under SIM.

At the same time, because NapBu-BPEA's optical stability is an important factor in super-resolution imaging, a long-term continuous laser (<NUM>) stimulation of cells was carried out, capturing images every <NUM> in order to monitor the photostability of NapBu-BPEA and MTDR in cells (<FIG>). Among the results, NapBu-BPEA had stained cells and showed negligible photobleaching within <NUM> of laser stimulation, while after <NUM>, MTDR's fluorescence intensity had decreased to less than <NUM>%. These characteristics enabled NapBu-BPEA to dynamically monitor cellular ultrastructures with long-term super-resolution imaging.

In view of these promising results, a <NUM>:<NUM> mixture of ZnCl<NUM> and pyrithione sodium salt, a cell-permeable Zn<NUM>+ carrier (ZnPT) to transport exogenous Zn<NUM>+ to cells, and a cell-permeable Zn<NUM>+-chelating agent, TPEN, were used to evaluate the reversibility of fluorescence response to Zn<NUM>+ in living cells according to methods described elsewhere. To determine the entry time of exogenous Zn<NUM>+ into cells, NapBu-BPEA was incubated and an initial image was captured, after which <NUM> ZnPT (<NUM> ZnCl<NUM>/<NUM> pyrithione sodium salt) was used to replace the culture medium in dye-free and serum-free DMEM. Images collected in situ every <NUM> ultimately showed that the fluorescence intensity was balanced after exogenous Zn<NUM>+ was incubated with HeLa cells for <NUM>. Moreover, when NapBu-BPEA was added by exogenous Zn<NUM>+, it continued to exhibit fluorescence in the cytoplasm, which differed from when Naph-BPEA fluoresced in the nucleus upon being added to exogenous Zn<NUM>+. The variance may be attributed to the difference in lipophilicity. Before the addition of exogenous Zn<NUM>+, NapBu-BPEA showed an exceptionally weak fluorescence background signal in HeLa cells. When different concentrations of exogenous Zn<NUM>+ were added, however, the fluorescence signal increased significantly and intensified with the increased concentration of ZnPT. These results indicate that NapBu-BPEA can be used in imaging intracellular Zn<NUM>+ in living cells.

To test whether NapBu-BPEA responds reversibly to Zn<NUM>+ in cells, TPEN was used to chelate Zn<NUM>+ in cells. When <NUM> TPEN was added to the cells, the fluorescence signal of NapBu-BPEA-labeled cells attenuated significantly, which indicates that NapBu-BPEA can respond reversibly to intracellular Zn<NUM>+.

Mitophagy, a type of autophagy, is necessary for cells to maintain homeostasis. To study the relationship between autophagy and Zn<NUM>+, carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial damage inducer, was used to induce mitophagy in HeLa cells. When the cells were treated with CCCP and incubated with NapBu-BPEA, the overall fluorescence signal of the CCCP-treated cells improved significantly compared with the signal of untreated cells, and bright spots appearing in the cytoplasm were highly coincident with DAPRed, an autophagy detection reagent. Further, subsequent treatment with TPEN prompted the recovery of enhanced fluorescence signals, which indicates that the enhanced signals during cell damage derived from an increased level of Zn<NUM>+.

Considering that ATG13 and FIP200 are essential proteins for autophagy, to further elucidate the relationship between autophagy and changes in level of Zn<NUM>+ two stable knockout (KO) cell lines were obtained (ATG13 KO HeLa cells and FIP200 KO HeLa cells) via CRISPR/Cas9 gene editing technology. Lacking the proteins necessary for autophagy, these KO cells do not undergo autophagy. Once the two cell lines were treated with CCCP, no significant change in fluorescence intensity occurred between the treated and untreated KO cells. Such outcomes confirmed that when mitophagy occurs, the level of Zn<NUM>+ in cells increases, and when autophagy is blocked, the level of Zn<NUM>+ does not change significantly.

Autophagy is generally divided into two types: selective autophagy (i.e., CCCP-induced mitophagy) and non-selective autophagy (i.e., Earle's Balanced Salt Solution (EBSS)-induced autophagy). Mitochondrial morphology in cells stained with commercial mitochondrial dye (MTG) changed from rod-shaped (<FIG>) to dot-shaped (<FIG>) after treatment with CCCP. Although autophagy occurred when HeLa cells were starved with EBSS for <NUM>, after staining mitochondria with MTG, it was found that the mitochondrial morphology remained stick-shaped, which complicated judging whether starvation autophagy occurred by staining with MTG. Surprisingly, when autophagy was induced by CCCP and EBSS, respectively, and stained with NapBu-BPEA, the fluorescence signal of cells treated with CCCP improved significantly, whereas the fluorescence intensity of cells treated with EBSS decreased (<FIG>). These results indicate that changes in level of Zn<NUM>+ differ in cells subjected to different treatments. The process of autophagy is highly complex and involves a great deal of Zn-related proteins; therefore, the observed upregulation or downregulation of Zn<NUM>+ level may have occurred by way of a more complex, indirect mechanism. While not desiring to be bound by theory, these data show that NapBu-BPEA can detect and distinguish changes in Zn<NUM>+ level under different types of autophagy.

Despite the clear relationship between autophagy and the level of Zn<NUM>+ in entire cells, effective methods of detecting changes in levels of Zn<NUM>+ in various organelles during autophagy remain a challenge. Because NapBu-BPEA is distributed throughout all cytosolic organelles, using super-resolution imaging technology combined with SIM allows distinguishing individual organelles by morphological features (<FIG> and <FIG>). Super-resolution MCoD methods can be used to further observe changes in levels of Zn<NUM>+ in organelles during autophagy. As shown in <FIG>, the different treatments of HeLa cells were first analyzed via SIM imaging and confocal imaging, to obtain a thermal map of the Zn<NUM>+ level via fluorescence intensity analysis. Despite the difficulty of differentiating organelles in blurred confocal images, it is easy to distinguish organelles according to their different morphologies in high-resolution SIM images, as evidenced in the mitochondria in Fig, <NUM>(e1) and <FIG>, the endoplasmic reticulum in Fig. <NUM>(g1) and <FIG>, and autophagosomes in bright spots. These data demonstrate that NapBu-BPEA can be applied in super-resolution imaging to differentiate organelles without the need for co-localization with commercial dyes.

Further, as the thermal map distribution revealed, the distribution of fluorescence intensity in each organelle was heterogeneous. The fluorescence intensity on the mitochondrial cristae exceeded that of the mitochondrial matrix, and the distribution of the fluorescence intensity of the endoplasmic reticulum was uneven. Together, these results indicate that super-resolution imaging allows the observation of the distribution of Zn<NUM>+ in each organelle. By extension, to investigate changes in levels of Zn<NUM>+ in organelles during autophagy, mitochondria or endoplasmic reticula were randomly selected as regions of interest via morphology, as shown in <FIG>, respectively. Shown in <FIG>, the analysis of fluorescence intensity revealed that the level of Zn<NUM>+ in mitochondria had increased by <NUM>-fold during autophagy, whereas that of endoplasmic reticulum had increased by <NUM>-fold. While not desiring to be bound by theory, these results suggest that the level of Zn<NUM>+ in mitochondria changes more than that in ER during autophagy, possibly because CCCP can change the mitochondrial membrane potential. The results confirmed that super-resolution MCoD is useful for monitoring labile Zn<NUM>+ at the subcellular level.

The detection of Zn<NUM>+ at the cellular level offers limited information regarding organelles and tissues. As better in vitro culture systems, organoids can mimic certain key characteristics of organs. With a structure and function similar to those of organs, organoids are self-assembled structures, usually formed by the differentiation of stem cells cultured in vitro. Because organoids can readily mimic the structure and function of real organs compared with in vitro cell models, they afford not only significant advantages in scientific research and drug development, but also provide convenience for the simulation of human organs. For the 3D super-resolution imaging of organoids with SIM, the major obstacle is the presence of too much out-of-focus light, which prevents imaging the grating sufficiently to receive useful information. To reduce fluorescent background from high-excitation laser power, probes with high quantum yields may be useful.

Accordingly, NapBu-BPEA was used to image organoids with methods depicted in <FIG>. Organoids treated with CCCP for <NUM> were incubated with NapBu-BPEA for <NUM> and subjected to SIM imaging at different depths. As shown in <FIG>, NapBu-BPEA is successfully imaged at different depths and is functional to distinguish organically combined pluripotent stem cells. Even for the imaging of organoids, SIM provides a resolution below <NUM> (<FIG>), which indicates that NapBu-BPEA can be employed for super-resolution imaging, which is a useful method of studying and visualizing species in organoids. Moreover, as shown in <FIG>, from the local image of a certain section of an organoid, the endoplasmic reticulum is clearly identified by its morphological features. These results indicate that MCoD can be applied to image organoids as well as cells.

Next, NapBu-BPEA was investigated for its intracellular labile Zn<NUM>+ imaging ability. Imaging experiments were performed in HeLa cells upon exogenous Zn<NUM>+ loading via incubation with zinc pyrithione cmplex (ZnPT), a cell-permeable Zn<NUM>+ carrier.

Intracellular labile Zn<NUM>+ enhancement processes were tracked by recording SIM images every <NUM>. The imaging revealed an instant fluorescence enhancement upon ZnPT (<NUM>) incubation, displaying an almost linear increase in the first <NUM> (<FIG>(a, b)). The stable fluorescence signal after <NUM> indicated that the exogenous Zn<NUM>+ loading process is completed quickly. During the Zn<NUM>+ loading process, in addition to the intensity enhancement, the punctate fluorescence in the cytoplasm was retained and no fluorescence appeared in the nucleus even after <NUM> of ZnPT incubation. This intracellular distribution behavior was clearly different from the redistribution behavior of Naph-BPEA upon Zn<NUM>+ loading, which resulted in the uniform fluorescence in the cytoplasm and nucleus. While not desiring to be bound by theory, it is proposed that the butyl tail of NapBu-BPEA is responsible for the retained multiple organelle accumulation behavior of NapBu-BPEA upon Zn<NUM>+ binding.

Higher ZnPT concentration led to higher fluorescence enhancement in the cytoplasm (<FIG> (c-h)), implying that NapBu-BPEA enables fluorescence tracking of labile Zn<NUM>+ enhancement in cells. Intracellular Zn<NUM>+ scavenging for the Zn<NUM>+-loaded HeLa cells with TPEN (a cell membrane permeable Zn<NUM>+ scavenger) treatment resulted in a distinct drop in cytoplasmic fluorescence. This confirmed that the fluorescence enhancement upon ZnPT incubation was really associated with labile Zn<NUM>+ enhancement, and NapBu-BPEA was able to sense intracellular labile Zn<NUM>+ in a reversible manner. The punctate fluorescence distribution pattern was still retained in this Zn<NUM>+ scavenging process.

With the reversible Zn<NUM>+ imaging ability and the multiple organelle distribution behavior upon Zn<NUM>+ binding, NapBu-BPEA was investigated for its application in simultaneous Zn<NUM>+ tracking in multiple organelles (Zn-STIMO) for autophagic cells. HeLa cells were incubated with carbonyl cyanide m-chlorophenylhydrazone (CCCP, a mitochondrial damage inducer) as a mitophagy model. The cells were co-stained with NapBu-BPEA and DAPRed, an autophagy dye incorporating into the autophagosome during double-membrane formation via structural features and emitting under hydrophobic conditions. As shown in <FIG>, SIM imaging in the DAPRed channel for HeLa cells without CCCP incubation showed no red fluorescence, while the red fluorescence in the cells with <NUM> of CCCP exposure (<NUM>) confirmed the mitophagy induction. SIM imaging in the NapBu-BPEA channel showed that the green fluorescence intensity of NapBu-BPEA in the mitophagic cells was ~<NUM>-fold higher than that in non-autophagic cells (Fig. <NUM>(i)). Subsequent TPEN treatment decreased the fluorescence to a level slightly lower than that of cells without autophagy. This reversible fluorescence response in the green channel indicated that the detected NapBu-BPEA fluorescence change was caused by labile Zn<NUM>+ fluctuation, and that NapBu-BPEA was able to visualize labile Zn<NUM>+ fluctuation in mitophagy. In addition, the dynamic labile Zn<NUM>+ tracking in cells exposure to CCCP (<NUM>) disclosed that the autophagy induction processes underwent rapid enhancement of labile Zn<NUM>+ in the initial <NUM> of CCCP incubation, followed by and the subsequent slower Zn<NUM>+ enhancement (data not shown), and the temporal profile of the reversible labile Zn<NUM>+ decrease induced by TPEN treatment <NUM> mins post CCCP incubation was also observed.

The Zn<NUM>+ level in cells changes from time to time, so tracking dynamic Zn<NUM>+ in living cells is an important goal. Here, dynamic labile Zn<NUM>+ tracking was performed in the CCCP-induced mitophagy of HeLa cells via Zn-STIMO (<FIG>). The temporal profiles of mean fluorescence in the mitochondria, ER, and autophagosome/autolysosome (Aps/Als) revealed that the distinct labile Zn<NUM>+ enhancement appeared <NUM> later than CCCP incubation, and the Aps/Als displayed the more distinct enhancement of labile Zn<NUM>+ than the mitochondria and ERs (<FIG>). This dynamic tracking of labile Zn<NUM>+ demonstrated the capability of Zn-STIMO via SIM imaging to detect labile Zn<NUM>+ fluctuation in different organelles.

The citation of any document is not to be construed as an admission that it is prior art with respect to the present invention.

It is to be further understood that where descriptions of various embodiments use the term "comprising," and/or "including" those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of" or "consisting of.

The foregoing description is illustrative of particular embodiments of the invention but is not meant to be a limitation upon the practice thereof.

Claim 1:
A method for detecting labile zinc (Zn<NUM>+) in a biological material, the method comprising:
(a) contacting the biological material with a composition comprising NapBu-BPEA, having the structure
<CHM>
and
(b) imaging the biological material via molecular fluorescence imaging to detect the labile zinc in the biological material.